WO2018137173A1 - 提高植物对赤霉素抑制剂敏感性的方法及其应用 - Google Patents
提高植物对赤霉素抑制剂敏感性的方法及其应用 Download PDFInfo
- Publication number
- WO2018137173A1 WO2018137173A1 PCT/CN2017/072607 CN2017072607W WO2018137173A1 WO 2018137173 A1 WO2018137173 A1 WO 2018137173A1 CN 2017072607 W CN2017072607 W CN 2017072607W WO 2018137173 A1 WO2018137173 A1 WO 2018137173A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- plant
- protein
- hrp
- transgenic plant
- gene
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8291—Hormone-influenced development
- C12N15/8297—Gibberellins; GA3
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Definitions
- the lower part of the light can inhibit the growth of the main stem of the cotton plant, shorten the internodes of the cotton plant, and compact the plant type, thereby preventing the cotton plant from prospering and delaying its closure period.
- DPC can also increase the root vigor of cotton plants (Tian Xiaoli et al., 2006), while DPC can also increase the stability of cell membranes and increase the resistance of cotton plants (Shao Li, 2004).
- DPC digital personal computer
- the application of DPC in cotton on the whole country accounts for more than 80% of the planting area. It can regulate cotton under the conditions of growing environment in the field. It can reduce the growth of the main stem of cotton and reduce the plant height by shortening the internode and reducing the number of internodes. Its compact plant type effectively controls the growth of cotton and shapes the ideal plant type to optimize its economic traits (He Zhongpei et al., 1991; Reddy et al., 1992).
- DPC can inhibit the growth of tomato plants, increase the content of chlorophyll and soluble sugar in seedlings, reduce the relative conductivity, and significantly increase the yield of large fruit tomatoes (Mao Xiujie et al., 1999; Wang Mei et al., 2012).
- DPC gramineous crops such as maize was not significant.
- the treatment of maize Zhengdan 958 with 1000mg/L DPC showed no significant difference between plant height and stem diameter and control (Chen, 2012).
- the technical problem to be solved by the present invention is how to increase the sensitivity of gibberellin inhibitors in plants.
- the method for improving the sensitivity of a plant to a gibberellin inhibitor provided by the present invention comprising A1) or A2):
- A1 increasing the content of a protein in a recipient plant or enhancing the activity of said protein in said recipient plant to obtain a transgenic plant
- the transgenic plant has increased sensitivity to the gibberellin inhibitor compared to the recipient plant;
- the protein is named Plant Height Associated Protein (HRP) and is a protein of the following a) or b) or c):
- the amino acid sequence of position 1-821 of sequence 1 is the amino acid sequence of the plant height-related protein HRP.
- the plant height-associated protein HRP gene represented by nucleotides 1-2463 of SEQ ID NO: 2; the amino acid sequence of 824-996 of SEQ ID NO: 1 is the amino acid sequence of GFP, and the nucleotides of nucleotides 2470-2720 of SEQ ID NO: 2 The DNA molecule is shown to be encoded.
- a label as shown in Table 1 may be attached to the amino terminus or carboxy terminus of the protein shown in SEQ ID NO: 1 in the Sequence Listing.
- the HRP in the above b) can be artificially synthesized, or the encoded gene can be synthesized first, and then obtained by biological expression.
- the gene encoding the HRP in the above b) may be one or more bases by deleting a codon of one or several amino acid residues in the DNA sequence shown in positions 1-2463 of SEQ ID NO: 2 in the sequence listing. A missense mutation of the base pair, and/or a coding sequence for the tag shown in Table 1 attached at its 5' end and/or 3' end.
- the HRP in the above d) can be artificially synthesized, or the encoded gene can be synthesized first, and then obtained by biological expression.
- the gene encoding the HRP in the above d) may be a codon that lacks one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2 in the sequence listing, and/or a missense mutation of one or several base pairs. And/or the coding sequence of the tag shown in Table 1 is attached at its 5' end and/or 3' end.
- the method further comprises knocking out a CPS gene in the recipient plant.
- the CPS gene may be a DNA molecule represented by SEQ ID NO: 4 in the Sequence Listing.
- the knockout may be a mutation of AGCTGAAGCGGATCCCAAG of the CPS gene to AGCTGAAGCGGATCTCCAAG.
- the gene encoding the HRP is a gene represented by 1) or 2) or 3) or 4) or 5) or 6):
- the nucleotide sequence is a cDNA molecule or a DNA molecule of nucleotides 1-2463 of SEQ ID NO: 2;
- nucleotide sequence is the cDNA molecule or DNA molecule of SEQ ID NO: 2 in the sequence listing;
- the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
- nucleotide sequence of SEQ ID NO: 2 encodes the amino acid sequence shown in SEQ ID NO: 1.
- One of ordinary skill in the art can readily mutate the HRP-encoding nucleotide sequences of the present invention using known methods, such as directed evolution and point mutation methods.
- Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the HRP isolated from the present invention are nucleotide sequences derived from the present invention as long as they encode HRP and have HRP function. And equivalent to the sequence of the invention.
- identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 75% or more, or 85% or more, or 90% or more of the nucleotide sequence of the protein represented by amino acid 1-821 of the coding sequence 1 or sequence 1 of the present invention. A nucleotide sequence that is high, or 95% or more identical. Identity can be evaluated using the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
- the stringent conditions are: in a solution of 2 ⁇ SSC, 0.1% SDS, hybridized at 68 ° C and washed twice for 5 min each time, in a solution of 0.5 ⁇ SSC, 0.1% SDS, Hybridization and washing at 68 ° C for 2 times, each time 15 min; or, 0.1 ⁇ SSPE (or 0.1 ⁇ SSC), 0.1% SDS solution, hybridization and washing at 65 ° C.
- the above 75% or more of the identity may be 80%, 85%, 90% or 95% or more.
- the HRP gene can be first modified as follows and then introduced into the recipient seed plant to achieve a better expression effect:
- ligation with a suitable transcription terminator can also increase the expression efficiency of the gene of the invention; for example, tml derived from CaMV, E9 derived from rbcS; any available terminator known to function in plants can be The gene of the present invention is ligated;
- enhancer sequences such as intron sequences (eg, from Adhl and bronzel) and viral leader sequences (eg, from TMV, MCMV, and AMV).
- the HRP gene recombinant expression vector can be introduced into plant cells by conventional biotechnological methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).
- the transgenic plant is understood to include not only the first generation transgenic plants obtained by transforming the HRP gene into the plant of interest, but also its progeny.
- the gene can be propagated in the species, and the gene can be transferred to other varieties of the same species, including commercial varieties, by conventional breeding techniques.
- the transgenic plants include seeds, callus, whole plants, and cells.
- the present invention also provides the following M1 or M2 products:
- M2 the biological material related to the protein, is any one of the following B1) to B20):
- B2 an expression cassette comprising the nucleic acid molecule of B1);
- B3 a recombinant vector comprising the nucleic acid molecule of B1);
- B4 a recombinant vector comprising the expression cassette of B2)
- B5 a recombinant microorganism comprising the nucleic acid molecule of B1);
- B7 a recombinant microorganism comprising the recombinant vector of B3);
- B9 a transgenic plant cell line comprising said nucleic acid molecule of B1);
- B11 a transgenic plant cell line comprising the recombinant vector of B3);
- B12 a transgenic plant cell line comprising the recombinant vector of B4)
- B13 a transgenic plant tissue comprising the nucleic acid molecule of B1);
- B14 a transgenic plant tissue comprising the expression cassette of B2)
- B15 a transgenic plant tissue comprising the recombinant vector of B3);
- B16 a transgenic plant tissue comprising the recombinant vector of B4)
- B17 a transgenic plant organ comprising said nucleic acid molecule of B1);
- B18 a transgenic plant organ comprising the expression cassette of B2)
- B19 a transgenic plant organ comprising the recombinant vector of B3);
- B20 A transgenic plant organ comprising the recombinant vector of B4).
- the expression cassette (HRP gene expression cassette) containing the HRP-encoding nucleic acid molecule described in B2) refers to a DNA capable of expressing HRP in a host cell, and the DNA may include not only a promoter that initiates transcription of the HRP gene, but also a promoter. Terminators that terminate transcription of the HRP gene can also be included. Further, the expression cassette may further comprise an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters.
- promoters include, but are not limited to, constitutive promoter of cauliflower mosaic virus 35S: a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester); tomatoes Protease inhibitor II promoter (PIN2) or LAP promoter (both induced by methyl jasmonate); heat shock promoter (U.S. Patent 5,187,267); tetracycline-inducible promoter (U.S.
- Patent 5,057,422 seed specificity Promoters, such as the millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), seed storage protein-specific promoters (eg, promoters of Bean globulin, napin, oleosin, and soybean beta conglycin (Beachy et al. (1985) ) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety.
- Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminator (see, for example, Odell et al. (I 985 ) Nature 313: 810; Rosenberg et al. (1987) Gene, 56: 125; Guerineau et al. (1991) Mol. Gen. Genet, 262: 141; 1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5: 141; Mogen et al.
- NOS terminator Agrobacterium nopaline synthase terminator
- CaMV 35S terminator cauliflower mosaic virus CaMV 35S terminator
- tml terminator tml terminator
- pea rbcS E9 terminator nopaline and octopine Synth
- a recombinant vector containing the HRP gene expression cassette can be constructed using an existing expression vector.
- the plant expression vector includes a dual Agrobacterium vector and a vector which can be used for plant microprojectile bombardment and the like. For example, pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA).
- the plant expression vector may further comprise a 3' untranslated region of the foreign gene, ie, comprising a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression.
- the polyadenylation signal can direct polyadenylation to the 3' end of the mRNA precursor, such as Agrobacterium tumefaciens-induced (Ti) plasmid genes (such as nopaline synthase gene Nos), plant genes (such as soybean).
- the untranslated regions transcribed at the 3' end of the storage protein gene all have similar functions.
- an enhancer including a translation enhancer or a transcription enhancer, may be used, and these enhancer regions may be an ATG start codon or a contiguous region start codon, etc., but are required to be encoded.
- the reading frames of the sequence are identical to ensure proper translation of the entire sequence.
- the sources of the translational control signals and initiation codons are broad and may be natural or synthetic.
- Translation start The region can be from a transcriptional initiation region or a structural gene.
- the plant expression vector used can be processed, such as a gene encoding a color-changing enzyme or luminescent compound (GUS gene, luciferase) which can be expressed in plants.
- marker genes for antibiotics such as the nptII gene conferring resistance to kanamycin and related antibiotics, the bar gene conferring resistance to the herbicide phosphinothricin, and the hph gene conferring antibiotic resistance to hygromycin
- the dhfr gene that confers resistance to methotrexate, confers glyphosate-resistant EPSPS gene) or chemical-resistant marker gene (such as anti-tuberant gene) and provides mannose-6-capable mannose-capable mannose-- Phosphoisomerase gene. From the safety of transgenic plants, the transformed plants can be directly screened by adversity without any selectable marker genes.
- the vector may be a plasmid, a cosmid, a phage or a viral vector.
- the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.
- the gene encoding the HRP (i.e., the DNA molecule shown at positions 1-2463 of SEQ ID NO: 2) is introduced into Agrobacterium tumefaciens GV3101 by a recombinant vector containing an expression cassette for the gene encoding HRP.
- the recombinant vector is a recombinant vector pSuper1300-HRP obtained by replacing a DNA fragment between the Xba I and Kpn I recognition sequences of the vector pSuper1300 with the DNA molecule shown in positions 1-2463 of the sequence 2, the pSuper1300-HRP and the The difference between pSuper1300 is only that the DNA fragment between the Xba I and Kpn I recognition sequences of the pSuper1300-HRP is replaced with the DNA molecule shown by nucleotides 1-2463 of the sequence 2.
- the recombinant vector pSuper1300-HRP expresses the protein shown in SEQ ID NO: 1.
- the nucleic acid molecule of B1) may be the gene represented by the above 1) or 2) or 3) or 4) or 5) or 6).
- the present invention also provides any of the following N1-N4 applications:
- the plant is a transgenic plant.
- the use of N4 comprises the step of introducing a gene encoding the HRP into a recipient plant to obtain a transgenic plant; the transgenic plant has an increased plant height compared to the recipient plant.
- N1 or N2 include S1) and S2):
- the coding sequence of the HRP-encoding gene is the DNA molecule of SEQ ID NO: 2 in the Sequence Listing.
- the HRP-encoding gene i.e., the DNA molecule represented by nucleotides 1-821 of SEQ ID NO: 2
- a HRP gene recombinant expression vector containing an HRP gene expression cassette is introduced into a plant of interest through a HRP gene recombinant expression vector containing an HRP gene expression cassette.
- the HRP gene can be first modified as follows and then introduced into the recipient seed plant to achieve a better expression effect:
- the amino acid sequence of the HRP gene of the present invention can be changed while changing its codon to conform to plant preference.
- promoters may include constitutive, inducible, temporal regulation, developmental regulation, chemical regulation, tissue-preferred and tissue-specific promoters
- the choice of promoter will vary with the time and space required for expression, and also depends on the target species; for example, a tissue or organ that specifically expresses the promoter, depending on when the receptor is required to develop; although the source is proven
- Many promoters of dicotyledonous plants are functional in monocotyledonous plants, and vice versa, but ideally dicotyledonous promoters are selected for expression in dicotyledons, and promoters of monocotyledons are used for Expression in monocots;
- enhancer sequences such as intron sequences (eg, from Adhl and bronzel) and viral leader sequences (eg, from TMV, MCMV, and AMV).
- the HRP gene recombinant expression vector can be introduced into plant cells by conventional biotechnological methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).
- the transgenic plant is understood to include not only the first generation of transgenic plants obtained by transforming the HRP gene into the plant of interest, but also its progeny.
- the gene can be propagated in the species, and the gene can be transferred to other varieties of the same species, including commercial varieties, by conventional breeding techniques.
- the transgenic plants include seeds, callus, whole plants, and cells.
- the gibberellin inhibitor may be a gibberellin synthesis inhibitor such as a gibberellin biosynthesis inhibitor.
- the gibberellin inhibitor may specifically be a defatted DPC.
- the specific reduction can be a product of Jiangsu Runze Agrochemical Co., Ltd., and the product number is HG/T2856-1997.
- the plant may be a dicot or a monocot.
- the dicot can be Cruciferous plants, such as Arabidopsis thaliana.
- the monocot may specifically be corn.
- the increase in sensitivity of the gibberellin inhibitor is manifested in that the plant plant height reduction rate of the transgenic plants is higher than that of the recipient plants under the same increase in the concentration of the same gibberellin inhibitor.
- the plant height of the 30 mg/L ketamine-treated transgenic HRP gene plant was 33.92% lower than that of the 0 mg/L ketamine-treated transgenic HRP gene plant, and 30 mg/L ketamine treatment.
- the plant height of the recipient plant was 13.60% lower than the plant height of the 0 mg/L ketamine-treated recipient plant.
- Figure 1 shows the plant height of transgenic HRP and ZmCPS gene Arabidopsis before DPC treatment.
- A is the plant height of ZmCPS::ga1-1
- B is the plant height of HRP::ga1-1
- C is the plant height of ZmCPS::ga1-5
- D is the plant height of HRP::ga1-5.
- Figure 2 shows the plant height of differently treated Arabidopsis thaliana.
- A is the plant height of ZmCPS::ga1-1 treated with different concentrations of DPC
- B is the plant height of HRP::ga1-1 treated with different concentrations of DPC
- C is ZmCPS treated with different concentrations of DPC::ga1 Plant height of -5
- D is the plant height of different concentrations of DPC-treated HRP::ga1-5.
- Figure 3 is the plant height of HRP::ga1-1 and HRP::ga1-5 treated with different concentrations of DPC.
- A is the plant height of HRP::ga1-1 treated with different concentrations of DPC;
- B is the plant height of HRP::ga1-5 treated with different concentrations of DPC.
- Figure 4 shows the plant phenotype of ZmCPSKO and WT.
- Figure 5 is a sensitivity analysis of DPC for zmcps-ko plants transfected with HRP.
- the vector pSuper1300 in the following examples is the literature (Lipid transfer protein 3as a target of MYB96mediates freezing and drought stress in Arabidopsis, Journal of Experimental Botany, Guo et al., Vol. 64, No. 6, pp. 1755-1767, The pSuper1300::GFP in 2013) is available to the public from the applicant, and the biological material is only used for the repeated experiments of the present invention and cannot be used for other purposes.
- Agrobacterium tumefaciens GV3101 in the following examples is the literature (A Plasma Membrane Receptor Kinase, GHR1, Mediates Abscisic Acid- and Hydrogen Peroxide-Regulated Stomatal Movement in Arabidopsis, Hua et al., The Plant Cell, Vol. 24: 2546). Agrobacterium strain GV3101 in –2561, June 2012), which is available to the public from the applicant for use in the relevant experiments of the present invention and may not be used for other purposes.
- the Arabidopsis mutants ga1-1 and ga1-5 in the following examples are the Ohio State University of Arabidopsis Product Resource Center products.
- the 0 mg/L aqueous DPC solution in the following examples was ultrapure water
- the 30 mg/L aqueous DPC solution in the following examples was a solution obtained by adding 30 mg of DPC to 1 L of ultrapure water, 500 mg in the following examples.
- the /L DPC aqueous solution was a solution obtained by adding 500 mg of DPC to 1 L of ultrapure water.
- the growth conditions of Arabidopsis thaliana were as follows: a culture period in which the photoperiod was 16 h light/8 h dark, the light intensity was 60 ⁇ mol/m 2 /s, the humidity was 60%-70%, and the temperature was 22 °C.
- the ketamine in the following examples is a product of Jiangsu Runze Agrochemical Co., Ltd., the product number is HG/T2856-1997, and the hydrazine is a gibberellin biosynthesis inhibitor.
- Example 1 Controlling plant height of Arabidopsis thaliana using a kit of plants regulating plant height
- kits for regulating plant height consisting of a plant height-related protein and N,N-dimethylpiperidinium chloride (DPC, also known as 1,1-dimethylpiperidine chloride); A highly related protein, designated HRP, whose amino acid sequence is position 1-821 of sequence 1 in the sequence listing.
- HRP plant height associated protein
- the HRP gene is derived from G. arboreum stone line No. 1 (SXY1) (Li et al., Genome sequence of the cultivated cotton Gossypium arboreum, Nature GenNetics VOLUME 46NUMBER 6JUNE 2014).
- the plant height-related protein HRP gene is a DNA molecule shown in SEQ ID NO: 2 in the Sequence Listing.
- the fragment between the Xba I and Kpn I recognition sequences of the vector pSuper1300 was replaced with the DNA molecule represented by nucleotides 1-2463 of the sequence 2 (ie, the HRP gene) to obtain the recombinant vector pSuper1300-HRP, pSuper1300-HRP and pSuper1300.
- the only difference is that the DNA fragment between the Xba I and Kpn I recognition sequences of pSuper1300-HRP is replaced with the DNA molecule shown by nucleotides 1-2463 of SEQ ID NO:2.
- the recombinant vector pSuper1300-HRP expresses the fusion protein of the plant height-associated protein HRP and GFP shown in SEQ ID NO: 1.
- amino acid sequence of position 1-821 of sequence 1 is the amino acid sequence of the plant height-related protein HRP, which is encoded by the plant height-related protein HRP gene represented by nucleotides 1-2463 of SEQ ID NO: 2;
- the amino acid sequence of 996 amino acid is GFP, which is encoded by the DNA molecule indicated by nucleotides 2470-2720 of SEQ ID NO:2.
- the fragment between the Spe I and Kpn I recognition sequences of the vector pSuper1300 was replaced with the DNA molecule shown in positions 1-2565 of the sequence 3 to obtain the recombinant vector pSuper1300-ZmCPS, and the pSuper1300-ZmCPS differed from the pSuper1300 only in that the pSuper1300-ZmCPS was The DNA fragment between the Spe I and Kpn I recognition sequences was replaced with the DNA molecule shown in SEQ ID NO: 3.
- the recombinant vector pSuper1300-ZmCPS expresses a fusion protein of 1-2565 encoding protein and GFP.
- the first 1-2565 of the sequence 3 is the nucleotide sequence of the ZmCPS gene, and the ZmCPS gene is derived from maize (B73).
- pSuper1300-HRP was introduced into Agrobacterium tumefaciens GV3101 to obtain a recombinant strain, and the recombinant strain was named GV3101-pSuper1300-HRP
- pSuper1300-ZmCPS was introduced into Agrobacterium tumefaciens GV3101 to obtain a recombinant strain, and the recombinant strain was named as GV3101-pSuper1300-ZmCPS
- pSuper1300 was introduced into Agrobacterium tumefaciens GV3101 to obtain a recombinant strain, and the recombinant strain was named GV3101-pSuper1300.
- the transgenic Arabidopsis thaliana was constructed by transforming Arabidopsis thaliana mutants ga1-1 and ga1-5 with GV3101-pSuper1300-HRP, GV3101-pSuper1300-ZmCPS and GV3101-pSuper1300 of step 1, respectively.
- the method for transgenic the Arabidopsis mutant ga1-1 to the HRP gene is as follows:
- the mutant ga1-1 seed was vernalized at 4 °C for 72 h, and seeded in gibberellin medium (gibberellin medium was added with gibberellin (GA 3 ) to MS medium.
- the obtained solid medium having a concentration of GA 3 of 10 -4 M is cultured in a culture chamber at 22 ° C, 16 h light / 8 h dark, light intensity of 60 ⁇ mol / m 2 /s, humidity 60% - 70%, and grown.
- the mutant should spray 10 -4 M GA 3 twice in two days.
- Agrobacterium liquid The Agrobacterium vaccinated with the monoclonal GV3101-pSuper1300-HRP, which was correctly detected and streaked, was inoculated into 5 ml of YEP liquid medium (containing antibiotics), and cultured at 28 ° C, 220 rpm for 30 h, according to the volume.
- the ratio of 1:10 was transferred to 50 ml of YEP (containing antibiotics), cultured at 28 ° C, 220 rpm overnight to OD 600 of 0.6-0.8; 4 ° C was centrifuged at 6000 g for 15 min, and the collected bacteria were suspended in 1/2 MS + 5% sucrose solution, 0.02%-0.05% Silwet L-77 (a surfactant, a product of AMRESCO, USA) was added before conversion.
- YEP containing antibiotics
- the anti-hygromycin plants showed that the true leaves were dark green and the roots were elongated into the medium; Hygromycin plants showed: yellow leaves of true leaves, roots did not elongate); 3 seeds of T 3 generation HRP::ga1-1 were seeded on MS medium containing hygromycin to obtain T 3 generation HRP::ga1 -1 plants were screened using hygromycin to select all T 3 generations HRP :: ga1-1 hygromycin resistant plants and transplanted to soil it, to give pure line of HRP :: ga1-1 plants.
- ga1-1 was replaced with ga1-5, and the other steps were unchanged, and the transgenic HRP gene ga1-1 and its pure plant having the name HRP::ga1-1 were respectively obtained.
- the expression level of HRP gene in the pure plant of HRP::ga1-1 and the pure plant of HRP::ga1-5 in step 2 and the pure plant of ZmCPS::ga1-1 in step 2 were identified by Real-Time PCR.
- the expression level of ZmCPS gene in the pure plant of ZmCPS::ga1-5 are 5'-ACCGAGGACTCGCAGAGTTA-3' and 5'-ACCTTTAGCATTTGGCGATG-3', and the expression level of ZmCPS gene is detected.
- the primers were 5'-TGCAGCCACTTATCGACCAG-3' and 5'-AGGCGAGGGTGTTGATCATG-3'.
- the internal reference is the AtUbI gene, and the primers of the internal reference are 5'-ATTACCCGATGGGCAAGTCA-3' and 5'-CACAAACGAGGGCTGGAACA-3'.
- the results showed that the HRP gene was found in the pure plant of HRP::ga1-1 and the pure plant of HRP::ga1-5, the pure line of ZmCPS::ga1-1 and the pure line of ZmCPS::ga1-5.
- the ZmCPS gene was expressed in all plants.
- the pure plants of HRP::ga1-1 were replaced with pure plants of HRP::ga1-5, pure plants of ZmCPS::ga1-1, and pure plants of ZmCPS::ga1-5.
- Pure plants of PsSuper1300::ga1-1 and pure plants of PsSuper1300::ga1-5, the other steps are unchanged, respectively, untreated HRP::ga1-5, 30mg/L DPC treated HRP:: Ga1-5, 500mg/L DPC treated HRP::ga1-5, untreated ZmCPS::ga1-1, 30mg/L DPC treated ZmCPS::ga1-1, 500mg/L DPC treated ZmCPS::ga1-1, untreated ZmCPS::ga1 -5, 30 mg/L DPC-treated ZmCPS::ga1-5, 500 mg/L DPC-treated ZmCPS::ga1-5, untreated PsSuper1300:
- ga1-1 When ga1-1 was cultured to the 33rd day of self-seeding (the day of sowing was recorded as the first day of sowing), 30 strains were taken and randomly divided into three groups of 10 plants each, and the following treatments were carried out in each of the three groups of plants: One group was sprayed with 0 mg/L aqueous DPC solution, and cultured for 12 days to obtain untreated ga1-1; one group was sprayed with 30 mg/L aqueous DPC solution and cultured for 12 days to obtain 30 mg/L DPC-treated ga1-1. A group of 500 mg/L aqueous DPC solution was sprayed for 12 days to obtain 500 mg/L DPC-treated ga1-1.
- ga1-1 was replaced with ga1-5, and the other steps were unchanged, and untreated ga1-5, 30 mg/L DPC-treated ga1-5, and 500 mg/L DPC-treated ga1-5 were respectively obtained.
- the decrease rate of plant height after pSuper1300::ga1-5 and ga1-5 was not significantly different by DPC treatment, while DPC had no effect on ZmCPS::ga1-5, and DPC treated HRP::ga1-
- the plant height reduction rate of 5 is much higher than the corresponding DPC concentration treated pSuper1300::ga1-5 and ga1-5:30mg/L DPC treated HRP::ga1-5 plant height reduction rate was 4.68 times ga1-5; 500mg/L DPC treated HRP::ga1
- the plant height reduction rate of -5 was 6.66 times that of ga1-5. It is indicated that HRP can increase the sensitivity of Arabidopsis to DPC.
- the plant height-related protein HRP of the present invention can increase the plant height of Arabidopsis thaliana: untreated HRP::ga1-1, 30 mg/L DPC-treated HRP::ga1-1 and 500 mg/L DPC-treated HRP
- the plant height of ::ga1-1 was 1.03, 0.68 and 0.41 times of the correspondingly treated ZmCPS::ga1-1 plant height, respectively 3.54 times, 2.71 times and 1.71 times the height of the corresponding treated ga1-1 plant.
- Untreated HRP::ga1-5, 30mg/L DPC-treated HRP::ga1-5 and 500mg/L DPC-treated HRP::ga1-5 plant heights are respectively treated ZmCPS::ga1-5
- the plant height was 0.95 times, 0.64 times and 0.38 times, which were 2.26 times, 1.65 times and 1.00 times higher than the corresponding treated ga1-5 plants, respectively.
- Treated HRP::ga1-1 a group of 500 mg/L DPC aqueous solution was sprayed for 12 days to obtain 500 mg/L DPC-treated HRP::ga1-1 sprayed with 1000 mg/L DPC aqueous solution. After 12 days of culture, 1000 mg/L DPC-treated HRP::ga1-1 was obtained.
- the pure plant of HRP::ga1-1 was replaced with the pure plant of HRP::ga1-5, and the other steps were unchanged, respectively, to obtain untreated HRP:: ga1-5, 30 mg/L DPC Treated HRP::ga1-5, 50 mg/L DPC-treated HRP::ga1-5, 100 mg/L DPC-treated HRP::ga1-5, 300 mg/L DPC-treated HRP::ga1-5, 500 mg/ L DPC treated HRP::ga1-5 and 1000 mg/L DPC treated HRP::ga1-5.
- the pBUE411-2gR CRISPR-Cas9ZmCPS vector was successfully constructed, and the maize inbred line B73 was infested with Agrobacterium (Wilson R K. The B73maize genome: complexity, diversity, and dynamics. [J]. Science, 2009, 326 (5956): 1112.) (WT) method of immature embryos.
- the vector was successfully transferred into maize embryos, and the ZmCPS gene was knocked out.
- the target sequence used was AGCTGAAGCGGATCCCAAG. After screening, the zmcps Knock out mutant was successfully obtained (ZmCPSKO, Zmcps-ko). ) plants.
- the primers are as follows:
- MT1-BsF ATATATGGTCTCTGGCGAAATTGCGAAA TGGCCA GGTT
- MT1-F0 TGAAATTGCGAAA TGGCCA GGTTTTAGAGCTAGAAATAGC
- MT2-R0 AACCTTG GGATCC GCTTCAGCTGCTTCTTGGTGCC
- MT2-BsR ATTATTGGTCTCTAAACCTTG GGATCC GCTTCAGCT
- PCR amplification 100-fold dilution of pCBC-MT1T2 (Xing H L, Dong L, Wang Z P, et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants [J]. BMC Plant Biology, 2014, 14 (1) ): 327.) Four primer PCR amplification was performed for the template. -BsF/-BsR is the normal primer concentration; -F0/-R0 is diluted 20 times.
- TaU3-RD CTCACAAATTATCAGCACGCTAGTC[rc:GACTAGCGTGCTGATAATTTGTGAG]
- TaU3-FD TTAGTCCCACCTCGCCAGTTTACAG
- TaU3-FD2 TTGACTAGCGTGCTGATAATTTGTG
- Agrobacterium tumefaciens-mediated transformation of maize immature embryos is as follows:
- Agrobacterium EHA105 (high rate of monocotyledon infection)
- the CRISPR/Cas9 system-related plasmid was provided by Mr. Chen Qijun: pBUE411; pCBC-MT1T2.
- LB medium Tryptone 10g, NaCl 10g, Yeast Extract 5g. If it is a solid medium, add 15g Agar,
- YEP medium Tryptone 10g, NaCl 5g, Yeast Extract 10g. If it is a solid medium, add 15 g of Agar and make up to 1 L.
- YEB medium Tryptone 10g, Sucrose 5g, Yeast Extract 1g, MgS04 ⁇ 7H20 0.5g. If it is a solid medium, add 15 g of Agar and make up to 1 L.
- the above various vitamins are first dissolved in water, and folic acid is first dissolved in 1mL of dilute ammonia water, and then distilled water to make up to 1L.
- Amp 100 mg/mL: Weigh 5 g of Amp into 50 mL of sterile water, filter and sterilize, and store at -20 °C.
- Kan 100 mg/mL: Weigh 5 g of Kan to a volume of 50 mL of sterile water, filter and sterilize, and store at -20 °C.
- AS (200mmoL/L): Weigh 0.3924g of AS drug into 10mL DMSO, filter and sterilize, and store at -20°C in the dark.
- Cef 100 mg/mL: 25 g of Cef was weighed into 250 mL of sterile water, filtered and sterilized, and stored at -20 °C.
- Cb 100 mg/mL: Weigh 25 g of Cb into 250 mL of sterile water, filter and sterilize, and store at -20 °C.
- AgNO 3 (5 mg/mL): Weigh 50 mg of AgNO 3 into 10 mL of sterile water, filter and sterilize, and store at -20 °C in the dark. Note: Wear gloves when configuring.
- Glufosinate (10 mg/L) 100 mg of glufosinate was weighed and dissolved in 10 ml of sterile water, filtered and sterilized, and stored at -20 °C.
- 6-BA (2mg/mL): Weigh 0.2g of 6-BA and dissolve it with 1mol/L NaOH, completely dilute to 100mL, filter and sterilize, and store at 4°C.
- 2,4-D (1mg/mL): Weigh 100mg 2,4-D dissolved in a small amount of absolute ethanol, dilute to 100mL with sterile water, filter and sterilize, and store at 4 °C.
- NAA (1 mg/mL) Weigh 100 mg of NAA in a small amount of 1 mol/L NaOH. After completely dissolving, make up to 100 mL with sterile water, filter and sterilize, and store at 4 °C.
- the sterilized centrifuge tube is prepared in advance, generally a corn tube, pay attention to avoid light, cover with newspaper, AS must be used before adding Because it takes time to activate;
- the exfoliated embryo is washed with the infecting solution at least twice for 30 seconds each time;
- the preserved bacterial solution was taken out from the -80 ° C refrigerator in advance, and after sufficient thawing, 200 ul was added to 30 mL of LB medium (containing Kan, Rif) at 26 ° C, and cultured at 180 rpm overnight. On the next day, when the bacterial solution becomes cloudy, remove it from the shaker and perform secondary activation. Add 3 to 5 mL to 30 mL of LB medium, shake at 26 ° C, 180 rpm for 4 to 6 hours, and pay attention to one hour before the bacterial solution is used. Add AS (30 mL plus 3 ul) to the bacterial solution.
- LB medium containing Kan, Rif
- the shaken bacteria were separately poured into two 50 mL centrifuge tubes and centrifuged, and the centrifuge was set at 26 ° C, 4000 rpm, 10 min. After centrifugation, the supernatant is poured off, and then the inoculum is added to resuspend the cells to adjust the OD. Value to 0.6 ⁇ 0.8, pay attention to add a small part first, not enough to add, if the measured high, then add infecting solution, adjust the spare.
- (1) Restoration culture Transfer the immature embryos co-cultured for three days to the recovery medium. If the co-cultured young embryos are long-lived, carefully pick the young embryos of the long-term bacteria to 2 ml sterile centrifuge tubes or flasks. Wash the bacteria with sterile water until the water is clear, then wash it with sterile water containing 100mg/mL cephalosporin for 30min, and finally pour the young embryos into the culture dish with sterile filter paper, then dry and transfer the young embryos to the recovery culture. In the base, cultured in the dark at 28 ° C for 7 days;
- Screening culture Transfer the cultured immature embryos to the screening medium for dark culture, subculture once every two weeks, the number of screening and screening concentration depending on the situation; (currently PPT screening, 2 to 3 times, screening pressure is 5 ⁇ 10mg/L)
- the inserted mutations caused the entire protein to be misinterpreted.
- the target sequence AGCTGAAGCGGATCCCAAG was mutated to AGCTGAAGCGGATCTCCAAG.
- the other sequences of the ZmCPS gene were not encoded, and finally the active CPS protein could not be synthesized, resulting in GA synthesis.
- the gibberellin could not be synthesized downstream of the pathway, resulting in dwarfing of the plants (Fig. 4).
- the Gm3101-pSuper1300-HRP of Example 1 was transformed into the ZmCPSKO corn embryo of step 1.1 by using Agrobacterium to infect the maize immature embryos, and the positively transgenic HRP corn was obtained by screening, and the transgenic HRP corn contained the target gene HRP, and Using GV3101-pSuper1300-ZmCPS and GV3101-pSuper1300 as controls, ZmCPS control corn and empty control corn were obtained, respectively.
- WT and transgenic plants were sprayed with DPC during jointing to test their DPC sensitivity: the whole plant was sprayed with 500 ppm (5 mM) DPC when the corn was grown to 7-leaf and 12-leaf leaves, respectively, and 2 weeks after treatment. The plant height and ear height were determined afterwards.
- the experiment proves that the method for improving the sensitivity of the gibberellin inhibitor of the present invention can improve the sensitivity of the plant to the gibberellin inhibitor, and the plant height of the method for improving the sensitivity of the gibberellin inhibitor of the present invention
- Related proteins HRP and DPC can regulate the plant height of Arabidopsis thaliana and the plant height of transgenic Arabidopsis Decreased as the concentration of DPC increased.
- the plant height reduction rate of HRP::ga1-1 treated by DPC was much higher than that of the corresponding DPC concentration treated ZmCPS::ga1-1, pSuper1300::ga1-1 and ga1-1:30mg/L DPC treated HRP::
- the plant height reduction rate of ga1-1 was 2.49 times that of ga1-1; the plant height reduction rate of 500 mg/L DPC-treated HRP::ga1-1 was 3.47 times that of ga1-1.
- the plant height reduction rate of HRP::ga1-5 treated by DPC was much higher than that of ZmCPS::ga1-1, pSuper1300::ga1-5 and ga1-5:30mg/L DPC treated by corresponding DPC concentration::
- the plant height reduction rate of ga1-5 was 4.68 times of ga1-5; the plant height reduction rate of 500 mg/L DPC-treated HRP::ga1-5 was 6.66 times of ga1-5.
- the plant height-related protein HRP of the present invention can increase the plant height of Arabidopsis thaliana: untreated HRP::ga1-1, 30 mg/L DPC-treated HRP::ga1-1 and 500 mg/L DPC-treated HRP
- the plant height of ::ga1-1 was 3.54 times, 2.71 times and 1.71 times higher than the corresponding treated ga1-1 plant height; untreated HRP::ga1-5, 30mg/L DPC treated HRP::ga1-5
- the plant height of HRP::ga1-5 treated with 500 mg/L DPC was 2.26 times, 1.65 times and 1.00 times higher than the height of the corresponding treated ga1-5 strain, respectively.
- the method of the present invention for increasing the sensitivity of phytoerythromycin inhibitors can be used to increase the sensitivity of plants to gibberellin inhibitors, and the plant height of plants can be regulated by the plant height-related protein HRP of the present invention.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Endocrinology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明公开了提高植物对赤霉素抑制剂敏感性的方法及其应用。本发明所提供的提高植物对赤霉素抑制剂敏感性的方法,包括向受体植物中导入蛋白质的编码基因得到转基因植物的步骤;所述转基因植物与所述受体植物相比所述赤霉素抑制剂敏感性增加;所述蛋白质为如下a)或b)或c)的蛋白质:a)氨基酸序列是序列1的第1-821位氨基酸的蛋白质;b)氨基酸序列是序列1的蛋白质;c)在a)或b)的N端或/和C端连接标签得到的融合蛋白质。实验证明,本发明的提高植物赤霉素抑制剂敏感性的方法可以用来提高植物对赤霉素抑制剂的敏感性,并可用本发明的株高相关蛋白HRP调控植物的株高。
Description
本发明涉及生物技术领域中提高植物对赤霉素抑制剂敏感性的方法及其应用。
缩节安(N,N-dimethylpiperidinium chloride,DPC),其化学名称为1,1-二甲基哌啶翁氯化物,是一种抑制型的植物生长调节剂,对植物营养生长有延缓作用,可抑制细胞伸长,减弱植株顶芽长势,控制其纵横生长。棉花应用DPC后,叶片加厚,叶色变深,叶绿素含量加大,叶片光合速率提高(何钟佩等,1991;李丕明等,1991;田晓莉等,2004),因而有利于田间透光,增强棉株下部的光照,并可抑制棉株主茎的生长,使棉株节间缩短,株型紧凑,从而防止了棉株旺长,推迟其封行期。DPC还可提高棉株根系活力(田晓莉等,2006),同时DPC还能提高细胞膜的稳定性,增加棉株抗逆性(邵莉相等,2004)。
DPC在全国棉花上的应用占种植面积的80%以上,可在田间生长环境条件下对棉花进行调控,可通过缩短节间和减少节间数目,抑制棉花主茎的生长,降低株高,使其株型紧凑,有效控制棉花的旺长,塑造理想的株型从而优化其经济性状(何钟佩等,1991;Reddy et al.,1992)。在番茄栽培过程中,使用DPC能够抑制番茄植株徒长,增加幼苗叶绿素和可溶性糖的含量,降低相对电导率,显著提高大果番茄的产量(毛秀杰等,1999;王梅等,2012)。但DPC对禾本科作物如玉米调控效应不显著,应用1000mg/L DPC处理玉米郑单958,株高和茎粗与对照都没有显著的差异(陈吟,2012)。部分植物对DPC不敏感,局限了DPC在作物生产中的应用。
发明公开
本发明所要解决的技术问题是如何提高植物的赤霉素抑制剂敏感性。
为解决上述技术问题,本发明首先提供了提高植物对赤霉素抑制剂敏感性的方法。
本发明所提供的提高植物对赤霉素抑制剂敏感性的方法,包括A1)或A2):
A1)增加受体植物中蛋白质的含量或增强所述受体植物中所述蛋白质的活性得到转基因植物;
A2)向所述受体植物中导入所述蛋白质的编码基因得到转基因植物;
所述转基因植物与所述受体植物相比所述赤霉素抑制剂敏感性增加;
所述蛋白质的名称为株高相关蛋白(HRP),为如下a)或b)或c)的蛋白质:
a)氨基酸序列是序列1的第1-821位氨基酸的蛋白质;
b)氨基酸序列是序列1的蛋白质;
c)在a)或b)的N端或/和C端连接标签得到的融合蛋白质。
其中,序列1的第1-821位氨基酸为株高相关蛋白HRP的氨基酸序列,由
序列2的第1-2463位核苷酸所示的株高相关蛋白HRP基因编码;序列1的第824-996位氨基酸为GFP的氨基酸序列,由序列2的第2470-2720位核苷酸所示的DNA分子编码。
为了使a)中的蛋白质便于纯化,可在序列表中序列1所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1、标签的序列
标签 | 残基 | 序列 |
Poly-Arg | 5-6(通常为5个) | RRRRR |
Poly-His | 2-10(通常为6个) | HHHHHH |
FLAG | 8 | DYKDDDDK |
Strep-tag II | 8 | WSHPQFEK |
c-myc | 10 | EQKLISEEDL |
上述b)中的HRP可人工合成,也可先合成其编码基因,再进行生物表达得到。上述b)中的HRP的编码基因可通过将序列表中序列2的第1-2463位所示的DNA序列中缺失一个或几个氨基酸残基的密码子,和/或进行一个或几个碱基对的错义突变,和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
上述d)中的HRP可人工合成,也可先合成其编码基因,再进行生物表达得到。上述d)中的HRP的编码基因可通过将序列表中序列2所示的DNA序列中缺失一个或几个氨基酸残基的密码子,和/或进行一个或几个碱基对的错义突变,和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
所述方法还包括将所述受体植物中的CPS基因敲除。所述CPS基因可为序列表中序列4所示的DNA分子。所述敲除具体可为将所述CPS基因的AGCTGAAGCGGATCCCAAG突变为AGCTGAAGCGGATCTCCAAG。
上述方法中,所述HRP的编码基因为如下1)或2)或3)或4)或5)或6)所示的基因:
1)核苷酸序列是序列表中序列2的第1-2463位核苷酸的cDNA分子或DNA分子;
2)与1)限定的核苷酸序列具有75%或75%以上同一性,且编码所述HRP的cDNA分子或基因组DNA分子;
3)在严格条件下与1)限定的核苷酸序列杂交,且编码所述HRP的cDNA分子或基因组DNA分子;
4)核苷酸序列是序列表中序列2的cDNA分子或DNA分子;
5)与4)限定的核苷酸序列具有75%或75%以上同一性,且编码所述HRP的cDNA分子或基因组DNA分子;
6)在严格条件下与4)限定的核苷酸序列杂交,且编码所述HRP的cDNA分
子或基因组DNA分子。
其中,所述核酸分子可以是DNA,如cDNA、基因组DNA或重组DNA;所述核酸分子也可以是RNA,如mRNA或hnRNA等。
其中,序列2的核苷酸序列编码序列1所示的氨基酸序列。
本领域普通技术人员可以很容易地采用已知的方法,例如定向进化和点突变的方法,对本发明的编码HRP的核苷酸序列进行突变。那些经过人工修饰的,具有与本发明分离得到的HRP的核苷酸序列75%或者更高同一性的核苷酸,只要编码HRP且具有HRP功能,均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码序列1或序列1的第1-821位氨基酸所示的蛋白质的核苷酸序列具有75%或更高,或85%或更高,或90%或更高,或95%或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间的同一性可以用百分比(%)表示,其可以用来评价相关序列之间的同一性。
上述方法中,所述严格条件是在2×SSC,0.1%SDS的溶液中,在68℃下杂交并洗膜2次,每次5min,又于0.5×SSC,0.1%SDS的溶液中,在68℃下杂交并洗膜2次,每次15min;或,0.1×SSPE(或0.1×SSC)、0.1%SDS的溶液中,65℃条件下杂交并洗膜。
上述75%或75%以上同一性,可为80%、85%、90%或95%以上的同一性。
在本发明的实施例中,所述HRP的编码基因(即序列2的第1-821位核苷酸所示的DNA分子)通过含有HRP基因表达盒的HRP基因重组表达载体导入目的植物中。
上述方法中,其中所述HRP基因可先进行如下修饰,再导入受体种子植物中,以达到更好的表达效果:
1)根据实际需要进行修饰和优化,以使基因高效表达;例如,可根据受体植物所偏爱的密码子,在保持本发明所述HRP基因的氨基酸序列的同时改变其密码子以符合植物偏爱性;优化过程中,最好能使优化后的编码序列中保持一定的GC含量,以最好地实现植物中导入基因的高水平表达,其中GC含量可为35%、多于45%、多于50%或多于约60%;
2)修饰邻近起始甲硫氨酸的基因序列,以使翻译有效起始;例如,利用在植物中已知的有效的序列进行修饰;
3)与各种植物表达的启动子连接,以利于其在植物中的表达;所述启动子可包括组成型、诱导型、时序调节、发育调节、化学调节、组织优选和组织特异性启动子;启动子的选择将随着表达时间和空间需要而变化,而且也取决于靶物种;例如组织或器官的特异性表达启动子,根据需要受体在发育的什么时期而定;尽管证明了来源于双子叶植物的许多启动子在单子叶植物中是可起作
用的,反之亦然,但是理想地,选择双子叶植物启动子用于双子叶植物中的表达,单子叶植物的启动子用于单子叶植物中的表达;
4)与适合的转录终止子连接,也可以提高本发明基因的表达效率;例如来源于CaMV的tml,来源于rbcS的E9;任何已知在植物中起作用的可得到的终止子都可以与本发明基因进行连接;
5)引入增强子序列,如内含子序列(例如来源于Adhl和bronzel)和病毒前导序列(例如来源于TMV,MCMV和AMV)。
所述HRP基因重组表达载体可通过使用Ti质粒,植物病毒栽体,直接DNA转化,微注射,电穿孔等常规生物技术方法导入植物细胞(Weissbach,1998,Method for Plant Molecular Biology VIII,Academy Press,New York,pp.411-463;Geiserson and Corey,1998,Plant Molecular Biology(2nd Edition).)。
上述方法中,所述转基因植物理解为不仅包含将所述HRP基因转化目的植物得到的第一代转基因植物,也包括其子代。对于转基因植物,可以在该物种中繁殖该基因,也可用常规育种技术将该基因转移进入相同物种的其它品种,特别包括商业品种中。所述转基因植物包括种子、愈伤组织、完整植株和细胞。
为解决上述技术问题,本发明还提供了下述M1或M2的产品:
M1、所述蛋白质;
M2、与所述蛋白质相关的生物材料,为下述B1)至B20)中的任一种:
B1)编码所述蛋白质的核酸分子;
B2)含有B1)所述核酸分子的表达盒;
B3)含有B1)所述核酸分子的重组载体;
B4)含有B2)所述表达盒的重组载体;
B5)含有B1)所述核酸分子的重组微生物;
B6)含有B2)所述表达盒的重组微生物;
B7)含有B3)所述重组载体的重组微生物;
B8)含有B4)所述重组载体的重组微生物;
B9)含有B1)所述核酸分子的转基因植物细胞系;
B10)含有B2)所述表达盒的转基因植物细胞系;
B11)含有B3)所述重组载体的转基因植物细胞系;
B12)含有B4)所述重组载体的转基因植物细胞系;
B13)含有B1)所述核酸分子的转基因植物组织;
B14)含有B2)所述表达盒的转基因植物组织;
B15)含有B3)所述重组载体的转基因植物组织;
B16)含有B4)所述重组载体的转基因植物组织;
B17)含有B1)所述核酸分子的转基因植物器官;
B18)含有B2)所述表达盒的转基因植物器官;
B19)含有B3)所述重组载体的转基因植物器官;
B20)含有B4)所述重组载体的转基因植物器官。
上述产品中,B2)所述的含有编码HRP的核酸分子的表达盒(HRP基因表达盒),是指能够在宿主细胞中表达HRP的DNA,该DNA不但可包括启动HRP基因转录的启动子,还可包括终止HRP基因转录的终止子。进一步,所述表达盒还可包括增强子序列。可用于本发明的启动子包括但不限于:组成型启动子,组织、器官和发育特异的启动子,和诱导型启动子。启动子的例子包括但不限于:花椰菜花叶病毒的组成型启动子35S:来自西红柿的创伤诱导型启动子,亮氨酸氨基肽酶("LAP",Chao等人(1999)Plant Physiol 120:979-992);来自烟草的化学诱导型启动子,发病机理相关1(PR1)(由水杨酸和BTH(苯并噻二唑-7-硫代羟酸S-甲酯)诱导);西红柿蛋白酶抑制剂II启动子(PIN2)或LAP启动子(均可用茉莉酮酸甲酯诱导);热休克启动子(美国专利5,187,267);四环素诱导型启动子(美国专利5,057,422);种子特异性启动子,如谷子种子特异性启动子pF128(CN101063139B(中国专利200710099169.7)),种子贮存蛋白质特异的启动子(例如,菜豆球蛋白、napin,oleosin和大豆beta conglycin的启动子(Beachy等人(1985)EMBO J.4:3047-3053))。它们可单独使用或与其它的植物启动子结合使用。此处引用的所有参考文献均全文引用。合适的转录终止子包括但不限于:农杆菌胭脂碱合成酶终止子(NOS终止子)、花椰菜花叶病毒CaMV 35S终止子、tml终止子、豌豆rbcS E9终止子和胭脂氨酸和章鱼氨酸合酶终止子(参见,例如:Odell等人(I985)Nature313:810;Rosenberg等人(1987)Gene,56:125;Guerineau等人(1991)Mol.Gen.Genet,262:141;Proudfoot(1991)Cell,64:671;Sanfacon等人Genes Dev.,5:141;Mogen等人(1990)Plant Cell,2:1261;Munroe等人(1990)Gene,91:151;Ballad等人(1989)Nucleic Acids Res.17:7891;Joshi等人(1987)Nucleic Acid Res.,15:9627)。
可用现有的表达载体构建含有所述HRP基因表达盒的重组载体。所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等。如pAHC25、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa或pCAMBIA1391-Xb(CAMBIA公司)等。所述植物表达载体还可包含外源基因的3′端非翻译区域,即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3′端,如农杆菌冠瘿瘤诱导(Ti)质粒基因(如胭脂碱合成酶基因Nos)、植物基因(如大豆贮存蛋白基因)3′端转录的非翻译区均具有类似功能。使用本发明的基因构建植物表达载体时,还可使用增强子,包括翻译增强子或转录增强子,这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等,但必需与编码序列的阅读框相同,以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的,可以是天然的,也可以是合成的。翻译起始
区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选,可对所用植物表达载体进行加工,如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因(GUS基因、萤光素酶基因等)、抗生素的标记基因(如赋予对卡那霉素和相关抗生素抗性的nptII基因,赋予对除草剂膦丝菌素抗性的bar基因,赋予对抗生素潮霉素抗性的hph基因,和赋予对氨甲喋呤抗性的dhfr基因,赋予对草甘磷抗性的EPSPS基因)或是抗化学试剂标记基因等(如抗除莠剂基因)、提供代谢甘露糖能力的甘露糖-6-磷酸异构酶基因。从转基因植物的安全性考虑,可不加任何选择性标记基因,直接以逆境筛选转化植株。
上述产品中,所述载体可为质粒、黏粒、噬菌体或病毒载体。
上述产品中,所述微生物可为酵母、细菌、藻或真菌,如农杆菌。
上述产品中,所述转基因植物细胞系、转基因植物组织和转基因植物器官均不包括繁殖材料。
在本发明的一个实施方式中,HRP的编码基因(即序列2的第1-2463位所示的DNA分子)通过含有HRP的编码基因的表达盒的重组载体导入根癌农杆菌GV3101中。所述重组载体为用序列2的第1-2463位所示的DNA分子替换载体pSuper1300的Xba I和Kpn I识别序列间的DNA片段得到的重组载体pSuper1300-HRP,所述pSuper1300-HRP与所述pSuper1300的差别仅在于将所述pSuper1300-HRP的Xba I和Kpn I识别序列间的DNA片段替换为序列2的第1-2463位核苷酸所示的DNA分子。所述重组载体pSuper1300-HRP表达序列1所示的蛋白质。
上述产品中,B1)所述核酸分子可为所述1)或所述2)或所述3)或所述4)或所述5)或所述6)所示的基因。
为解决上述技术问题,本发明还提供了下述N1-N4中任一应用:
N1、所述方法在调控植物株高中的应用;
N2、所述产品在调控植物株高中的应用;
N3、所述产品在培育对赤霉素抑制剂敏感性增加植物中的应用;
N4、所述产品在培育株高增加植物中的应用。
上述应用中,所述植物为转基因植物。
上述应用中,N4所述应用包括向受体植物中导入所述HRP的编码基因得到转基因植物的步骤;所述转基因植物与所述受体植物相比株高增加。
上述应用中,N1或N2所述应用包括S1)和S2):
S1)向受体植物中导入所HRP的编码基因得到转基因植物;
S2)对所述转基因植物施用所述赤霉素抑制剂得到与所述转基因植物相比株高降低的植物。
上述应用中,所述HRP的编码基因的编码序列是序列表中序列2的DNA分子。
在本发明的实施例中,所述HRP的编码基因(即序列2的第1-821位核苷酸所示的DNA分子)通过含有HRP基因表达盒的HRP基因重组表达载体导入目的植物中。
上述应用中,其中所述HRP基因可先进行如下修饰,再导入受体种子植物中,以达到更好的表达效果:
1)根据实际需要进行修饰和优化,以使基因高效表达;例如,可根据受体植物所偏爱的密码子,在保持本发明所述HRP基因的氨基酸序列的同时改变其密码子以符合植物偏爱性;优化过程中,最好能使优化后的编码序列中保持一定的GC含量,以最好地实现植物中导入基因的高水平表达,其中GC含量可为35%、多于45%、多于50%或多于约60%;
2)修饰邻近起始甲硫氨酸的基因序列,以使翻译有效起始;例如,利用在植物中已知的有效的序列进行修饰;
3)与各种植物表达的启动子连接,以利于其在植物中的表达;所述启动子可包括组成型、诱导型、时序调节、发育调节、化学调节、组织优选和组织特异性启动子;启动子的选择将随着表达时间和空间需要而变化,而且也取决于靶物种;例如组织或器官的特异性表达启动子,根据需要受体在发育的什么时期而定;尽管证明了来源于双子叶植物的许多启动子在单子叶植物中是可起作用的,反之亦然,但是理想地,选择双子叶植物启动子用于双子叶植物中的表达,单子叶植物的启动子用于单子叶植物中的表达;
4)与适合的转录终止子连接,也可以提高本发明基因的表达效率;例如来源于CaMV的tml,来源于rbcS的E9;任何已知在植物中起作用的可得到的终止子都可以与本发明基因进行连接;
5)引入增强子序列,如内含子序列(例如来源于Adhl和bronzel)和病毒前导序列(例如来源于TMV,MCMV和AMV)。
所述HRP基因重组表达载体可通过使用Ti质粒,植物病毒栽体,直接DNA转化,微注射,电穿孔等常规生物技术方法导入植物细胞(Weissbach,1998,Method for Plant Molecular Biology VIII,Academy Press,New York,pp.411-463;Geiserson and Corey,1998,Plant Molecular Biology(2nd Edition).)。
上述应用中,所述转基因植物理解为不仅包含将所述HRP基因转化目的植物得到的第一代转基因植物,也包括其子代。对于转基因植物,可以在该物种中繁殖该基因,也可用常规育种技术将该基因转移进入相同物种的其它品种,特别包括商业品种中。所述转基因植物包括种子、愈伤组织、完整植株和细胞。
本发明中,所述赤霉素抑制剂可为赤霉素合成抑制剂,如赤霉素生物合成抑制剂。所述赤霉素抑制剂具体可为缩节安DPC。所述缩节安具体可为江苏润泽农化有限公司公司产品,货号为HG/T2856-1997。
本发明中,所述植物可为双子叶植物或单子叶植物。所述双子叶植物可为
十字花科植物,如拟南芥(Arabidopsis thaliana)。所述单子叶植物具体可为玉米。
本发明中,所述赤霉素抑制剂敏感性增加体现在在相同的赤霉素抑制剂浓度增加量下,转基因植物的株高降低率高于受体植物。如,30mg/L缩节胺处理的转HRP基因植物的株高相对于0mg/L缩节胺处理的转HRP基因植物的株高的株高降低率为33.92%,30mg/L缩节胺处理的受体植物的株高相对于0mg/L缩节胺处理的受体植物的株高的株高降低率为13.60%。
图1为DPC处理前的转HRP和ZmCPS基因拟南芥的株高。其中,A为ZmCPS::ga1-1的株高,B为HRP::ga1-1的株高,C为ZmCPS::ga1-5的株高,D为HRP::ga1-5的株高。
图2为不同处理的拟南芥的株高。其中,A为不同浓度的DPC处理的ZmCPS::ga1-1的株高,B为不同浓度的DPC处理的HRP::ga1-1的株高,C为不同浓度的DPC处理的ZmCPS::ga1-5的株高,D为不同浓度的DPC处理的HRP::ga1-5的株高。
图3为不同浓度DPC处理的HRP::ga1-1和HRP::ga1-5的株高。其中,A为不同浓度DPC处理的HRP::ga1-1的株高;B为不同浓度DPC处理的HRP::ga1-5的株高。
图4为ZmCPSKO与WT的植株表型。
图5为转HRP的zmcps-ko植株对DPC敏感性分析。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中的载体pSuper1300为文献(Lipid transfer protein 3as a target of MYB96mediates freezing and drought stress in Arabidopsis,Journal of Experimental Botany,Guo et al.,Vol.64,No.6,pp.1755–1767,2013)中的pSuper1300::GFP,公众可从申请人处获得该生物材料,该生物材料只为重复本发明的相关实验所用,不可作为其它用途使用。
下述实施例中的根癌农杆菌GV3101为文献(A Plasma Membrane Receptor Kinase,GHR1,Mediates Abscisic Acid-and Hydrogen Peroxide-Regulated Stomatal Movement in Arabidopsis,Hua et al.,The Plant Cell,Vol.24:2546–2561,June 2012)中的Agrobacterium strain GV3101,公众可从申请人处获得该生物材料,该生物材料只为重复本发明的相关实验所用,不可作为其它用途使用。
下述实施例中的拟南芥突变体ga1-1和ga1-5为俄亥俄州立大学拟南芥生
物资源中心产品。
下述实施例中的0mg/L的DPC水溶液为超纯水,下述实施例中的30mg/L的DPC水溶液为向1L超纯水中加入30mg DPC得到的溶液,下述实施例中的500mg/L的DPC水溶液为向1L超纯水中加入500mg DPC得到的溶液。
下述实施例中拟南芥的生长条件均为:光周期为16h光/8h暗,光强为60μmol/m2/s,湿度60%-70%,温度为22℃的培养室中培养。
下述实施例中的缩节胺为江苏润泽农化有限公司公司产品,货号为HG/T2856-1997,缩节安为赤霉素生物合成抑制剂。
实施例1、利用调控植物株高的成套试剂调控拟南芥的株高
调控植物株高的成套试剂,由株高相关蛋白和缩节安(N,N-dimethylpiperidinium chloride,,DPC,也被称为1,1-二甲基哌啶翁氯化物)组成;所述株高相关蛋白,名称为HRP,其氨基酸序列为序列表中序列1的第1-821位。株高相关蛋白(HRP)基因为序列2的第1-2463位所示的DNA分子。
HRP基因来源于亚洲棉(G.arboreum)石系亚1号(SXY1)(Li et al.,Genome sequence of the cultivated cotton Gossypium arboreum,Nature GenNetics VOLUME 46NUMBER 6JUNE 2014)。株高相关蛋白HRP基因为序列表中序列2所示的DNA分子。
1、重组载体和重组农杆菌的构建
将载体pSuper1300的Xba I和Kpn I识别序列间的片段替换为序列2的第1-2463位核苷酸所示的DNA分子(即HRP基因)得到重组载体pSuper1300-HRP,pSuper1300-HRP与pSuper1300的差别仅在于将pSuper1300-HRP的Xba I和Kpn I识别序列间的DNA片段替换为序列2的第1-2463位核苷酸所示的DNA分子。重组载体pSuper1300-HRP表达序列1所示的株高相关蛋白HRP与GFP形成的融合蛋白。
其中,序列1的第1-821位氨基酸为株高相关蛋白HRP的氨基酸序列,由序列2的第1-2463位核苷酸所示的株高相关蛋白HRP基因编码;序列1的第824-996位氨基酸为GFP的氨基酸序列,由序列2的第2470-2720位核苷酸所示的DNA分子编码。
将载体pSuper1300的Spe I和Kpn I识别序列间的片段替换为序列3的第1-2565位所示的DNA分子得到重组载体pSuper1300-ZmCPS,pSuper1300-ZmCPS与pSuper1300的差别仅在于将pSuper1300-ZmCPS的Spe I和Kpn I识别序列间的DNA片段替换为序列3的所示的DNA分子。重组载体pSuper1300-ZmCPS表达序列3第1-2565位编码蛋白与GFP形成的融合蛋白。
其中,序列3的第1-2565位为ZmCPS基因的核苷酸序列,ZmCPS基因来源于玉米(B73)。
将pSuper1300-HRP导入根癌农杆菌GV3101中,得到重组菌,将该重组菌命名为GV3101-pSuper1300-HRP;将pSuper1300-ZmCPS导入根癌农杆菌GV3101中,得到重组菌,将该重组菌命名为GV3101-pSuper1300-ZmCPS;将pSuper1300导入根癌农杆菌GV3101中,得到重组菌,将该重组菌命名为GV3101-pSuper1300。
2、转基因拟南芥的构建
分别利用步骤1的GV3101-pSuper1300-HRP、GV3101-pSuper1300-ZmCPS和GV3101-pSuper1300分别转化拟南芥突变体ga1-1和ga1-5,构建转基因拟南芥。向拟南芥突变体ga1-1转HRP基因的方法如下:
2.1拟南芥的培养:将突变体ga1-1种子在4℃条件下春化72h,点种于赤霉素培养基(赤霉素培养基为向MS培养基中加入赤霉素(GA3)得到的GA3浓度为10-4M的固体培养基)中于22℃、16h光/8h暗、光强为60μmol/m2/s、湿度60%-70%的培养室中培养,生长到4片真叶时移栽到营养土与蛭石等比例混合的种植钵中,其中突变体要两天喷一次10-4M GA3。
2.2农杆菌菌液的制备:取检测正确、划线培养的单克隆GV3101-pSuper1300-HRP接种的农杆菌接种于5ml YEP液体培养基(含抗生素)中,28℃、220rpm下培养30h,按体积比为1:100将得到的菌液转接到50ml YEP(含抗生素)中,28℃,220rpm培养过夜,至OD600为0.6-0.8;4℃于6000g离心15min,所收集的菌体重悬于1/2MS+5%蔗糖溶液,转化前加入0.02%-0.05%Silwet L-77(一种表面活性剂,为美国AMRESCO公司产品)。
2.3转化:待拟南芥植株开花后,剪去主枝顶端,促进侧枝发展。在剪枝后的6天内,将准备好的农杆菌蘸湿拟南芥未露白的花序上。将转化后的拟南芥用充满气的黑色塑料袋包住,暗培养24h后去掉黑色塑料袋。恢复光照按正常方法培养植株至结实,收获成熟的转HRP基因ga1-1(HRP::ga1-1)T1代种子。
按照如下方法获得HRP::ga1-1的纯系植株:①将T1代HRP::ga1-1种子播种在含有潮霉素的MS固体培养基上进行筛选,将真叶健康呈深绿色、根伸长至培养基中的抗潮霉素的阳性植株移入土壤中,收获T2代HRP::ga1-1种子;②将T2代HRP::ga1-1种子播种在含有潮霉素的MS固体培养基上得到T2代HRP::ga1-1植株,利用潮霉素进行筛选,选择抗潮霉素:不抗潮霉素=3:1的T2代HRP::ga1-1植株并将其中的抗潮霉素植株移入土壤中,收获T3代HRP::ga1-1种子(抗潮霉素植株表现为:真叶健康呈深绿色,根伸长至培养基中;不抗潮霉素植株表现为:真叶发黄,根不伸长);③将T3代HRP::ga1-1种子播种在含有潮霉素的MS培养基上,得到T3代HRP::ga1-1植株,利用潮霉素进行筛选,选择全部为抗潮霉素的T3代HRP::ga1-1植株并将其移栽到土壤中,得到HRP::ga1-1的纯系植株。
按照上述方法,将ga1-1替换为ga1-5,其他步骤均不变,分别得到名称为HRP::ga1-1的转HRP基因ga1-1及其纯系植株。
按照上述方法,将GV3101-pSuper1300-HRP分别替换为GV3101-pSuper1300-ZmCPS和GV3101-pSuper1300,其他步骤均不变,分别得到
名称为ZmCPS::ga1-1的转ZmCPS基因ga1-1及其纯系植株和名称为pSuper1300::ga1-1的转空载体ga1-1及其纯系植株。
按照上述方法,将ga1-1替换为ga1-5,并将GV3101-pSuper1300-HRP分别替换为GV3101-pSuper1300-ZmCPS和GV3101-pSuper1300,其他步骤均不变,分别得到名称为ZmCPS::ga1-5的转ZmCPS基因ga1-5及其纯系植株和名称为pSuper1300::ga1-5的转空载体ga1-5及其纯系植株。
3、转基因植株的鉴定
3.1转HRP基因的纯系拟南芥中HRP基因表达量的检测
利用Real-Time PCR鉴定步骤2的HRP::ga1-1的纯系植株和HRP::ga1-5的纯系植株中HRP基因的表达水平及步骤2的ZmCPS::ga1-1的纯系植株和ZmCPS::ga1-5的纯系植株中ZmCPS基因的表达水平,检测HRP基因的表达水平的引物为5’-ACCGAGGACTCGCAGAGTTA-3’和5’-ACCTTTAGCATTTGGCGATG-3’,检测ZmCPS基因的表达水平的引物为5’-TGCAGCCACTTATCGACCAG-3’和5’-AGGCGAGGGTGTTGATCATG-3’。内参为AtUbI基因,内参的引物为5’-ATTACCCGATGGGCAAGTCA-3’和5’-CACAAACGAGGGCTGGAACA-3’。结果显示,HRP::ga1-1的纯系植株和HRP::ga1-5的纯系植株中均表达HRP基因,ZmCPS::ga1-1的纯系植株和ZmCPS::ga1-5的纯系植株中均表达ZmCPS基因。
3.2转HRP基因的纯系拟南芥中HRP的检测
利用Westernblot鉴定步骤2的HRP::ga1-1的纯系植株、HRP::ga1-5的纯系植株中的HRP蛋白以及步骤2的ZmCPS::ga1-1的纯系植株和ZmCPS::ga1-5的纯系植株中的ZmCPS蛋白,一抗为Anti-GFP Tag Rabbit(Roche产品,货号为14717400)。结果显示,HRP::ga1-1的纯系植株和HRP::ga1-5的纯系植株中均有HRP蛋白表达,ZmCPS::ga1-1的纯系植株和ZmCPS::ga1-5的纯系植株中均有ZmCPS蛋白表达。
4、DPC对转HRP基因拟南芥株高的影响
实验重复三次,每次重复试验的具体步骤如下:
4.1DPC可以降低转HRP基因拟南芥株高
随机选取步骤2的HRP::ga1-1的纯系植株30株,将其随机分为三组,每组10株,在这三组植株自播种(播种当天记为播种第1天)的第33天时分别进行以下处理:一组喷施水(即0mg/L的DPC水溶液)后,培养12天,得到未处理的HRP::ga1-1;一组喷施30mg/L的DPC水溶液后,培养12天,得到30mg/L DPC处理的HRP::ga1-1;一组喷施500mg/L的DPC水溶液后,培养12天,得到500mg/L DPC处理的HRP::ga1-1。
按照上述方法,将HRP::ga1-1的纯系植株分别替换为HRP::ga1-5的纯系植株、ZmCPS::ga1-1的纯系植株、ZmCPS::ga1-5的纯系植株、PsSuper1300::ga1-1的纯系植株和PsSuper1300::ga1-5的纯系植株,其他步骤均不变,分别得到未处理的HRP::ga1-5、30mg/L DPC处理的HRP::ga1-5、500mg/L DPC处理的
HRP::ga1-5、未处理的ZmCPS::ga1-1、30mg/L DPC处理的ZmCPS::ga1-1、500mg/L DPC处理的ZmCPS::ga1-1、未处理的ZmCPS::ga1-5、30mg/L DPC处理的ZmCPS::ga1-5、500mg/L DPC处理的ZmCPS::ga1-5、未处理的PsSuper1300::ga1-1、30mg/L DPC处理的PsSuper1300::ga1-1、500mg/L DPC处理的PsSuper1300::ga1-1、未处理的PsSuper1300::ga1-5、30mg/L DPC处理的PsSuper1300::ga1-5和500mg/L DPC处理的PsSuper1300::ga1-5。
将ga1-1培养至自播种(播种当天记为播种第1天)的第33天时,取30株,将其随机分为三组,每组10株,在这三组植株分别进行以下处理:一组喷施0mg/L的DPC水溶液后,培养12天,得到未处理的ga1-1;一组喷施30mg/L的DPC水溶液后,培养12天,得到30mg/L DPC处理的ga1-1;一组喷施500mg/L的DPC水溶液后,培养12天,得到500mg/L DPC处理的ga1-1。
按照上述方法,将ga1-1替换为ga1-5,其他步骤均不变,分别得到未处理的ga1-5、30mg/L DPC处理的ga1-5和500mg/L DPC处理的ga1-5。
分别测量上述各拟南芥在处理前的的株高(图1)及不同处理后的株高(图2和表2)。
表2、不同处理的拟南芥的株高及株高降低率
结果显示,在相同的DPC浓度下,pSuper1300::ga1-1和ga1-1经DPC处理后株高降低率差异不大,而DPC对ZmCPS::ga1-1基本无影响,DPC处理的HRP::ga1-1的株高降低率均远高于相应DPC浓度处理的pSuper1300::ga1-1和ga1-1:30mg/L DPC处理的HRP::ga1-1的株高降低率为ga1-1的2.49倍;500mg/L DPC处理的HRP::ga1-1的株高降低率为ga1-1的3.47倍。在相同的DPC浓度下,pSuper1300::ga1-5和ga1-5经DPC处理后株高降低率差异不大,而DPC对ZmCPS::ga1-5基本无影响,DPC处理的HRP::ga1-5的株高降低率均远高于相应
DPC浓度处理的pSuper1300::ga1-5和ga1-5:30mg/L DPC处理的HRP::ga1-5的株高降低率为ga1-5的4.68倍;500mg/L DPC处理的HRP::ga1-5的株高降低率为ga1-5的6.66倍。表明,HRP可以提高拟南芥对DPC的敏感性。
结果显示,本发明的株高相关蛋白HRP可以提高拟南芥的株高:未处理的HRP::ga1-1、30mg/L DPC处理的HRP::ga1-1和500mg/L DPC处理的HRP::ga1-1的株高分别为相应处理的ZmCPS::ga1-1株高的1.03倍、0.68倍和0.41倍,分别为相应处理的ga1-1株高的3.54倍、2.71倍和1.71倍;未处理的HRP::ga1-5、30mg/L DPC处理的HRP::ga1-5和500mg/L DPC处理的HRP::ga1-5的株高分别为相应处理的ZmCPS::ga1-5株高的0.95倍、0.64倍和0.38倍,分别为相应处理的ga1-5株高的2.26倍、1.65倍和1.00倍。
4.2不同浓度的DPC对转HRP基因拟南芥株高的影响
随机选取步骤2的HRP::ga1-1的纯系植株70株,将其随机分为七组,每组10株,在这七组植株自播种(播种当天记为播种第1天)的第33天时时分别进行以下处理:一组喷施水(即0mg/L的DPC水溶液)后,培养12天,得到未处理的HRP::ga1-1;一组喷施30mg/L的DPC水溶液后,培养12天,得到30mg/L DPC处理的HRP::ga1-1;一组喷施50mg/L的DPC水溶液后,培养12天,得到50mg/L DPC处理的HRP::ga1-1;一组喷施100mg/L的DPC水溶液后,培养12天,得到100mg/L DPC处理的HRP::ga1-1;一组喷施300mg/L的DPC水溶液后,培养12天,得到300mg/L DPC处理的HRP::ga1-1;一组喷施500mg/L的DPC水溶液后,培养12天,得到500mg/L DPC处理的HRP::ga1-1一组喷施1000mg/L的DPC水溶液后,培养12天,得到1000mg/L DPC处理的HRP::ga1-1。
按照上述方法,将HRP::ga1-1的纯系植株替换为HRP::ga1-5的纯系植株,其他步骤均不变,分别得到未处理的HRP::ga1-5、30mg/L DPC处理的HRP::ga1-5、50mg/L DPC处理的HRP::ga1-5、100mg/L DPC处理的HRP::ga1-5、300mg/L DPC处理的HRP::ga1-5、500mg/L DPC处理的HRP::ga1-5和1000mg/L DPC处理的HRP::ga1-5。
分别测量上述不同处理拟南芥的株高(图3),平均株高见表3。
结果显示,转HRP基因拟南芥的株高受DPC的浓度的影响,转HRP基因拟南芥的株高随DPC浓度的增加而降低。
表3、不同浓度DPC处理的转HRP基因拟南芥的株高(cm)
实施例2、DPC对转HRP到Zmcps-ko植株敏感性分析
一、利用CRISPR的技术获得了矮化的zmcps ko突变体植株
成功构建了pBUE411-2gR CRISPR-Cas9ZmCPS载体,利用农杆菌侵染玉米自交系B73(Schnable P S,Wilson R K.The B73maize genome:complexity,diversity,and dynamics.[J].Science,2009,326(5956):1112.)(WT)幼胚的方法成功把载体转入玉米胚中,敲除ZmCPS基因,所用靶序列为AGCTGAAGCGGATCCCAAG,经过筛选最后成功获得zmcps Knock out突变体(ZmCPSKO,Zmcps-ko)植株。
CRISPR实验方案—pBUE411-2gR载体构建、PCR鉴定及测序确认,参考中国农业大学陈其军教授方法(Xing H L,Dong L,Wang Z P,et al.A CRISPR/Cas9toolkit for multiplex genome editing in plants[J].BMC Plant Biology,2014,14(1):327.)
ZmCPS基因序列:
引物如下:
MT1-BsF:ATATATGGTCTCTGGCGAAATTGCGAAATGGCCAGGTT
MT1-F0:TGAAATTGCGAAATGGCCAGGTTTTAGAGCTAGAAATAGC
MT2-R0:AACCTTGGGATCCGCTTCAGCTGCTTCTTGGTGCC
MT2-BsR:ATTATTGGTCTCTAAACCTTGGGATCCGCTTCAGCT
下划线的是Msc1和BamH1的识别序列。
PCR扩增:以稀释100倍的pCBC-MT1T2(Xing H L,Dong L,Wang Z P,et al.A CRISPR/Cas9toolkit for multiplex genome editing in plants[J].BMC Plant Biology,2014,14(1):327.)为模板进行四引物PCR扩增。-BsF/-BsR为正常引物浓度;-F0/-R0稀释20倍。
纯化回收PCR产物,建立如下酶切-连接体系(restriction-ligation):
取5ul转化大肠杆菌感受态。Kan板筛选。OsU3-FD3+TaU3-RD=831bp菌落PCR鉴定,OsU3-FD3和TaU3-FD2测序确认。
注1:菌落PCR及测序引物:
OsU3-FD3GACAGGCGTCTTCTACTGGTGCTAC
TaU3-RD:CTCACAAATTATCAGCACGCTAGTC[rc:GACTAGCGTGCTGATAATTTGTGAG]
TaU3-FD:TTAGTCCCACCTCGCCAGTTTACAG
TaU3-FD2:TTGACTAGCGTGCTGATAATTTGTG
农杆菌介导玉米幼胚转化流程如下:
1.材料与方法
1.1实验材料
1.1.1植物材料
玉米(Zea mays L.)自交系B73(Schnable P S,Wilson R K.The B73maize genome:complexity,diversity,and dynamics.[J].Science,2009,326(5956):1112.)
1.1.2实验菌株
农杆菌EHA105(单子叶植物侵染率高)
1.1.3质粒载体
由陈其军老师提供CRISPR/Cas9体系相关质粒:pBUE411;pCBC-MT1T2。
1.2常用的培养基和溶液的配置
1.2.1常用培养基的配置
LB培养基:Tryptone 10g,NaCl 10g,Yeast Extract 5g。若为固体培养基,则再加15g Agar,
定容至1L。
YEP培养基:Tryptone 10g,NaCl 5g,Yeast Extract 10g。若为固体培养基,则再加15g Agar,定容至1L。
YEB培养基:Tryptone 10g,Sucrose 5g,Yeast Extract 1g,MgS04}7H20 0.5g。若为固体培养基,则再加15g Agar,定容至1L。
1.2.2母液的配置
(1)N6大量元素(母液浓度20×):2L
配2L母液的方法:先称取6.64g CaCl2·2H20溶于700mL蒸馏水中,再称取其余4种成分,溶于700mL蒸馏水中,待两种溶液均完全溶解后,然后混在一起后,定容至2L。
(2)B 5微量(母液浓度100×):1L
(3)Fe盐(母液浓度100×):1L
方法:先用热水将Na2EDTA溶解,然后将FeS04·7H20逐渐溶于Na2EDTA溶液中,定容至
1L。
(4)RT V(母液浓度200×):1L
以上各种维生素均先溶于水,而叶酸先用1mL稀氨水溶解后,再加蒸馏水定容至1L。
(5)MS大量元素(母液浓度20×):1L
(6)MS微量元素(母液浓度200×):500mL
(7)MS有机物(母液浓度200×):500mL
(8)Fe盐(母液浓度100×):1L
方法:先用热水将Na2EDTA溶解,然后将FeS04·7H20逐渐溶于Na2EDTA溶液中,定容至
1L。
1.2.3转化玉米所用的培养基的配置
1.2.4常用溶液的配置
Amp(100mg/mL):称取5g Amp定容于50mL无菌水中,过滤灭菌分装,-20℃保存。
Kan(100mg/mL):称取5g Kan定容于50mL无菌水中,过滤灭菌分装,-20℃保存。
Rif(50mg/mL):称取0.5gRif定容于10mLDMSO中,过滤灭菌分装,-20℃保存。
AS(200mmoL/L):称取0.3924gAS药品定容于10mLDMSO中,过滤灭菌分装,-20℃避光保存。
Cef(100mg/mL):称取25g Cef定容于250mL无菌水中,过滤灭菌分装,-20℃保存。
Cb(100mg/mL):称取25g Cb定容于250mL无菌水中,过滤灭菌分装,-20℃保存。
AgNO3(5mg/mL):称取50mg AgNO3定容于10mL无菌水中,过滤灭菌分装,-20℃避光保存。注意:配置的时候要戴手套。
草丁膦(10mg/L):称取100mg草丁膦溶于10ml无菌水中,过滤灭菌后分装,-20℃保存。
6-BA(2mg/mL):称取0.2g6-BA先用1mol/L NaOH溶解完全加水定容至100mL,过滤灭菌分装,4℃保存。
2,4-D(1mg/mL):称取100mg 2,4-D溶于少量的无水乙醇中,用无菌水定容至100mL,过滤灭菌分装,4℃保存。
NAA(1mg/mL):称取100mg NAA溶于少量的1mol/L NaOH中,待完全溶解后用无菌水定容至100mL,过滤灭菌分装,4℃保存。
1.3农杆菌转化玉米幼胚
1.3.1玉米的授粉
(1)待雌穗出现时及时套袋,花丝抽出后授粉,并保持套袋至收获;
(2)授粉后挂上小牌,写上授粉的父母本或授粉方式和日期,10-12天后收获。
1.3.2幼胚的剥离
(1)取授粉后10-12天,幼胚大小为1.5-2.0mm的B73雌穗;
(2)将新收获的玉米雌穗去掉苞叶、花丝以及雌穗的头尾部分,将一只枪式镊子从顶部插入雌穗,玉米喷完酒精拿到超净台中,放到装有70%酒精的广口瓶中浸泡3分钟;
(3)取出雌穗,置于空培养皿中备用;
(4)用左手持住镊子,右手用装有22号刀片的手术刀切去籽粒的上半部分;
(5)用10号刀片,将刀尖插入籽粒下部的果皮与胚乳之间,将胚乳挑出,轻轻的将幼胚剥离,置于装有侵染液(含AS和AgN03)的2mL离心管中;
剥胚过程中的注意事项:
(1)提前把侵染液配好,用之前加AS(1L侵染液加500uLAS,AS现用现加,侵染液使用之前分装到小瓶中,避免交叉污染,侵染液不能放太长时间,出现沉淀,要重新配置;
(2)加完AS的侵染液要分装到2mL的离心管中,灭菌的离心管提前准备好,一般一个玉米一个离心管,注意避光,用报纸盖住,AS一定用之前加,因为其活化需要时间;
(3)剥胚注意不要污染,一般剥完胚用侵染液清洗至少两次,每次30秒;
1.3.3农杆菌菌液的制备
提前从-80℃冰箱里取出保存的菌液,充分解冻之后,取200ul加到30mL LB培养基中(含Kan,Rif)26℃,180rpm过夜培养。第二天当菌液变浑浊时,从摇床取出,进行二次活化,取3~5mL加到30mL LB培养基中,26℃,180rpm摇4~6小时,注意在菌液使用前一个小时,向菌液中加入AS(30mL加3ul)。把摇好的菌液分别倒在两个50mL离心管中进行离心,离心机设置为26℃,4000rpm,10min。离心后将上清液倒掉,然后加入侵染液重悬菌体,调OD
值至0.6~0.8,注意添加时先加少部分,不够再加,若测出来高就再加侵染液,调好备用。
1.3.4幼胚的农杆菌侵染
(1)吸出保存幼胚的离心管中侵染液,加入1.5ml准备好的菌液,垂直静放15分钟;
(2)准备灭过菌的空皿,往里面铺2~3层滤纸,再往玉米共培养基里铺一层滤纸;
(3)15分钟后,将幼胚连同菌液倒在铺有无菌滤纸的培养皿中,超净台中吹干,但不要使幼胚失水;
(4)将吹干的幼胚转移至共培养培养基中,盾片朝上,24℃黑暗共培养三天;
1.3.5玉米转化材料的恢复、筛选、分化和生根培养
(1)恢复培养:将共培养三天的幼胚转移到恢复培养基中,如果共培养的幼胚长菌,可将长菌的幼胚小心地挑到2ml灭菌的离心管或三角瓶中,用无菌水洗菌直至水清,然后用含有100mg/mL头孢的无菌水洗30min,最后将幼胚倒在铺有无菌滤纸的培养皿中吹干,再将幼胚转移到恢复培养基中,28℃黑暗培养7天;
(2)筛选培养:将恢复培养的幼胚转移到筛选培养基中暗培养,每两周继代一次,筛选次数和筛选浓度视情况而定;(目前为PPT筛选,2~3次,筛选压力为5~10mg/L)
(3)诱导分化培养:将筛选后的愈伤转移到诱导培养基(高糖培养基)中,暗培养两周;
(4)分化培养:将恢复培养两周的抗性愈伤转移到分化培养基中,见光培养,光照周期为光照/黑暗:16h/8h,每两周继代一次;
(5)生根培养:待分化出的幼苗长至1至2个叶片以上时,将幼苗从抗性愈伤上切下,移入广口瓶中进行生根培养;当再生苗长至瓶口,根上长出足够侧根后可进行炼苗;
(6)再生苗的炼苗移栽:将小苗移到温室中放置2-3天后,将封口膜打开,加入一层无菌水,再放置2-3天,然后将小苗移栽入小盆中,移栽之前用自来水将小苗根上的培养基冲洗干净,待小苗比较壮实后移入温室大盆中继续培养。
1.4阳性植株检测
待幼苗长至4展叶后取幼叶进行检测鉴定。
针对目的片段进行PCR扩增和测序鉴定,
突变体转基因材料PCR和测序检测引物
转基因植株目的片段检测引物
棉花HRP-CDS-F CTAGTCTAGAATGTTTTCCCATTCCTTCCT
棉花HRP-CDS-R CGGGGTACCGCGTACTTTCTCAAAG
经过检测测序突变的植株与WT相比,插入了一个碱基导致整个蛋白翻译错乱,靶序列AGCTGAAGCGGATCCCAAG突变为AGCTGAAGCGGATCTCCAAG,ZmCPS基因的其他序列未发生编码,最后不能合成有活性的CPS蛋白,导致GA合成途径下游无法合成赤霉素,导致植株矮化(图4)。
二、DPC对转HRP的Zmcps-ko株系敏感性分析
利用农杆菌侵染玉米幼胚的方法,将实施例1的GV3101-pSuper1300-HRP转化步骤1.1的ZmCPSKO玉米胚,经过筛选获得阳性转HRP的玉米,该转HRP的玉米中含有目的基因HRP,并利用GV3101-pSuper1300-ZmCPS和GV3101-pSuper1300作为对照,分别得到ZmCPS对照玉米和空载对照玉米。
将WT以及转基因植株在拔节期间喷施DPC,检测其DPC敏感性:玉米长至7展叶和12展叶时利用500ppm(5mM)DPC对整株进行喷施,分别于处理后2周和抽雄后测定株高、穗位高。
结果说明,WT对DPC是不敏感的,而互补的植株对DPC变得敏感(图5,株系号为443的结果)。从表4分析,转HRP的Zmcps-ko株系与野生型(B73)在正常条件下株高没有差异,但DPC处理显著降低了转基因株系株高和穗位,但野生型对DPC不敏感。
表4、DPC对转HRP的zmcps-ko株系株型的调控
工业应用
实验证明,本发明的提高植物赤霉素抑制剂敏感性的方法可以提高植物对赤霉素抑制剂的敏感性的方法,本发明的提高植物赤霉素抑制剂敏感性的方法中的株高相关蛋白HRP和DPC可以调控拟南芥的株高,并且转基因拟南芥的株高
随DPC浓度的增加而降低。DPC处理的HRP::ga1-1的株高降低率均远高于相应DPC浓度处理的ZmCPS::ga1-1、pSuper1300::ga1-1和ga1-1:30mg/L DPC处理的HRP::ga1-1的株高降低率为ga1-1的2.49倍;500mg/L DPC处理的HRP::ga1-1的株高降低率为ga1-1的3.47倍。DPC处理的HRP::ga1-5的株高降低率均远高于相应DPC浓度处理的ZmCPS::ga1-1、pSuper1300::ga1-5和ga1-5:30mg/L DPC处理的HRP::ga1-5的株高降低率为ga1-5的4.68倍;500mg/L DPC处理的HRP::ga1-5的株高降低率为ga1-5的6.66倍。
实验证明,本发明的株高相关蛋白HRP可以提高拟南芥的株高:未处理的HRP::ga1-1、30mg/L DPC处理的HRP::ga1-1和500mg/L DPC处理的HRP::ga1-1的株高分别为相应处理的ga1-1株高的3.54倍、2.71倍和1.71倍;未处理的HRP::ga1-5、30mg/L DPC处理的HRP::ga1-5和500mg/L DPC处理的HRP::ga1-5的株高分别为相应处理的ga1-5株高的2.26倍、1.65倍和1.00倍。
实验证明,本发明的提高植物赤霉素抑制剂敏感性的方法可以用来提高植物对赤霉素抑制剂的敏感性,并可用本发明的株高相关蛋白HRP调控植物的株高。
Claims (11)
- 提高植物对赤霉素抑制剂敏感性的方法,包括A1)或A2):A1)增加受体植物中蛋白质的含量或增强所述受体植物中所述蛋白质的活性得到转基因植物;A2)向所述受体植物中导入所述蛋白质的编码基因得到转基因植物;所述转基因植物与所述受体植物相比所述赤霉素抑制剂敏感性增加;所述蛋白质为如下a)或b)或c)的蛋白质:a)氨基酸序列是序列1的第1-821位氨基酸的蛋白质;b)氨基酸序列是序列1的蛋白质;c)在a)或b)的N端或/和C端连接标签得到的融合蛋白质。
- 根据权利要求1所述的方法,其特征在于:所述方法还包括将所述受体植物中序列4所示的基因敲除。
- 根据权利要求1或2所述的方法,其特征在于:所述蛋白质的编码基因为如下1)或2)或3)或4)或5)或6)所示的基因:1)核苷酸序列是序列表中序列2的第1-2463位核苷酸的cDNA分子或DNA分子;2)与1)限定的核苷酸序列具有75%或75%以上同一性,且编码权利要求1所述蛋白质的cDNA分子或基因组DNA分子;3)在严格条件下与1)限定的核苷酸序列杂交,且编码权利要求1所述蛋白质的cDNA分子或基因组DNA分子;4)核苷酸序列是序列表中序列2的cDNA分子或DNA分子;5)与4)限定的核苷酸序列具有75%或75%以上同一性,且编码权利要求1所述蛋白质的cDNA分子或基因组DNA分子;6)在严格条件下与4)限定的核苷酸序列杂交,且编码权利要求1所述蛋白质的cDNA分子或基因组DNA分子。
- 根据权利要求1-3中任一所述的方法,其特征在于:所述赤霉素抑制剂为赤霉素合成抑制剂。
- 根据权利要求1-4中任一所述的方法,其特征在于:所述植物为A1)或A2):A1)双子叶植物或单子叶植物;A2)玉米或拟南芥。
- 权利要求1所述蛋白质。
- 与权利要求1所述蛋白质相关的生物材料,为下述B1)至B20)中的任一种:B1)编码权利要求1所述蛋白质的核酸分子;B2)含有B1)所述核酸分子的表达盒;B3)含有B1)所述核酸分子的重组载体;B4)含有B2)所述表达盒的重组载体;B5)含有B1)所述核酸分子的重组微生物;B6)含有B2)所述表达盒的重组微生物;B7)含有B3)所述重组载体的重组微生物;B8)含有B4)所述重组载体的重组微生物;B9)含有B1)所述核酸分子的转基因植物细胞系;B10)含有B2)所述表达盒的转基因植物细胞系;B11)含有B3)所述重组载体的转基因植物细胞系;B12)含有B4)所述重组载体的转基因植物细胞系;B13)含有B1)所述核酸分子的转基因植物组织;B14)含有B2)所述表达盒的转基因植物组织;B15)含有B3)所述重组载体的转基因植物组织;B16)含有B4)所述重组载体的转基因植物组织;B17)含有B1)所述核酸分子的转基因植物器官;B18)含有B2)所述表达盒的转基因植物器官;B19)含有B3)所述重组载体的转基因植物器官;B20)含有B4)所述重组载体的转基因植物器官。
- 下述N1或N2的应用:N1、权利要求1-5中任一所述的方法在调控植物株高中的应用;N2、权利要求1所述蛋白质或权利要求7所述生物材料在调控植物株高中的应用;N3、权利要求1所述蛋白质或权利要求7所述生物材料在培育对赤霉素抑制剂敏感性增加植物中的应用;N4、权利要求1所述蛋白质或权利要求7所述生物材料在培育株高增加植物中的应用。
- 根据权利要求8所述应用,其特征在于:N4所述应用包括向受体植物中导入权利要求1所述蛋白质的编码基因得到转基因植物的步骤;所述转基因植物与所述受体植物相比株高增加。
- 根据权利要求8所述应用,其特征在于:N1或N2所述应用包括S1)和S2):S1)向受体植物中导入权利要求1所述蛋白质的编码基因得到转基因植物;S2)对所述转基因植物施用权利要求1-5中任一所述赤霉素抑制剂得到与所述转基因植物相比株高降低的植物。
- 根据权利要求9或10所述的应用,其特征在于:权利要求1所述蛋白质的编码基因的编码序列是序列表中序列2的DNA分子。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/480,794 US11242537B2 (en) | 2017-01-25 | 2017-01-25 | Method for improving sensitivity of plant to gibberellin inhibitor and use thereof |
PCT/CN2017/072607 WO2018137173A1 (zh) | 2017-01-25 | 2017-01-25 | 提高植物对赤霉素抑制剂敏感性的方法及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/CN2017/072607 WO2018137173A1 (zh) | 2017-01-25 | 2017-01-25 | 提高植物对赤霉素抑制剂敏感性的方法及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2018137173A1 true WO2018137173A1 (zh) | 2018-08-02 |
Family
ID=62977847
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2017/072607 WO2018137173A1 (zh) | 2017-01-25 | 2017-01-25 | 提高植物对赤霉素抑制剂敏感性的方法及其应用 |
Country Status (2)
Country | Link |
---|---|
US (1) | US11242537B2 (zh) |
WO (1) | WO2018137173A1 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109206494A (zh) * | 2018-10-29 | 2019-01-15 | 中国农业大学 | ZmRPH1基因在调控植物株高及抗倒伏能力中的应用 |
CN116064650A (zh) * | 2023-03-20 | 2023-05-05 | 山东农业大学 | Mos3基因在调控植物抗盐性中的应用 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117821474A (zh) * | 2024-02-19 | 2024-04-05 | 甘肃农业大学 | 一种调控陆地棉早熟性状的基因及其应用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1228784A (zh) * | 1996-02-12 | 1999-09-15 | 植物生物科学有限公司 | 编码拟南芥的gai基因的核酸 |
WO2003100032A2 (en) * | 2002-05-23 | 2003-12-04 | Wisconsin Alumni Research Foundation | Dwarfism genes and dwarf plants |
US20090313725A1 (en) * | 2008-06-16 | 2009-12-17 | Academia Sinica | Gibberellin 2-Oxidase Genes And Uses Thereof |
WO2011050281A2 (en) * | 2009-10-23 | 2011-04-28 | Academia Sinica | A method of controlling plant growth and architecture by controlling expression of gibberellin 2-oxidase |
CN106367433A (zh) * | 2015-07-22 | 2017-02-01 | 中国农业大学 | 提高植物对赤霉素抑制剂敏感性的方法及其应用 |
-
2017
- 2017-01-25 US US16/480,794 patent/US11242537B2/en active Active
- 2017-01-25 WO PCT/CN2017/072607 patent/WO2018137173A1/zh active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1228784A (zh) * | 1996-02-12 | 1999-09-15 | 植物生物科学有限公司 | 编码拟南芥的gai基因的核酸 |
WO2003100032A2 (en) * | 2002-05-23 | 2003-12-04 | Wisconsin Alumni Research Foundation | Dwarfism genes and dwarf plants |
US20090313725A1 (en) * | 2008-06-16 | 2009-12-17 | Academia Sinica | Gibberellin 2-Oxidase Genes And Uses Thereof |
WO2011050281A2 (en) * | 2009-10-23 | 2011-04-28 | Academia Sinica | A method of controlling plant growth and architecture by controlling expression of gibberellin 2-oxidase |
CN106367433A (zh) * | 2015-07-22 | 2017-02-01 | 中国农业大学 | 提高植物对赤霉素抑制剂敏感性的方法及其应用 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109206494A (zh) * | 2018-10-29 | 2019-01-15 | 中国农业大学 | ZmRPH1基因在调控植物株高及抗倒伏能力中的应用 |
CN116064650A (zh) * | 2023-03-20 | 2023-05-05 | 山东农业大学 | Mos3基因在调控植物抗盐性中的应用 |
Also Published As
Publication number | Publication date |
---|---|
US11242537B2 (en) | 2022-02-08 |
US20210032648A1 (en) | 2021-02-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11254715B2 (en) | Protein associated with disease resistance and encoding gene thereof, and use thereof in regulation of plant disease resistance | |
CN111218470B (zh) | 一种调控植物抗逆性的方法 | |
CN114805522B (zh) | 水稻OsbHLH38蛋白及其编码基因在提高植物抗非生物胁迫中的用途 | |
CN107459565B (zh) | 大豆抗旱相关蛋白在调控大豆抗旱性中的应用 | |
CN104059937A (zh) | 一个来源于苜蓿的蛋白质及其编码基因的新用途 | |
WO2018137173A1 (zh) | 提高植物对赤霉素抑制剂敏感性的方法及其应用 | |
CN104745600B (zh) | 水稻基因OsVHA1在延缓植物叶片衰老和提高植物耐盐性中的应用 | |
CN106367433B (zh) | 提高植物对赤霉素抑制剂敏感性的方法及其应用 | |
CN106591324B (zh) | 谷子SiASR4基因及应用 | |
CN110684088A (zh) | 蛋白ZmbZIPa3及其编码基因在调控植物生长发育与耐逆性中的应用 | |
CN104744579A (zh) | 抗逆相关蛋白GmL16在调控植物抗逆性中的应用 | |
CN114752573B (zh) | 水稻OsGA20ox2蛋白及其编码基因在提高植物抗非生物胁迫中的用途 | |
CN108359688B (zh) | 提高植物对赤霉素抑制剂敏感性的方法及其应用 | |
CN106279386A (zh) | 一种水稻穗顶部生长发育相关蛋白及其编码基因与应用 | |
CN110627887B (zh) | SlTLFP8蛋白及其相关生物材料在调控番茄抗旱性中的应用 | |
CN108611365B (zh) | 种子相关蛋白在调控植物种子产量中的应用 | |
CN105713078A (zh) | 抗旱相关蛋白在调控植物抗旱性中的应用 | |
CN106565833B (zh) | 抗旱相关蛋白与其编码基因以及二者在调控植物抗旱性中的应用 | |
CN116410985B (zh) | 小麦TaNF-YB3D基因、其可变剪接形式及应用 | |
CN110684114A (zh) | 植物耐逆性相关蛋白TaBAKL在调控植物耐逆性中的应用 | |
CN108892714A (zh) | 植物耐盐相关蛋白GmLURP17及其编码基因的应用 | |
CN102971427A (zh) | 具有增强的产量相关性状的植物和用于产生该植物的方法 | |
CN108795975B (zh) | 野生大豆相关蛋白在提高植物抗虫性中的应用 | |
CN100424177C (zh) | 融合杀虫基因cryci及其应用 | |
Bhatti et al. | Agrobacterium mediated tobacco transformation with rice FAE gene and segregation analysis of T1 generation |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17893830 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 17893830 Country of ref document: EP Kind code of ref document: A1 |