CN104892736B - Plant stress tolerance correlative protein GmNF-YA20 and its encoding gene and application - Google Patents

Abstract

The invention discloses a plant stress tolerance-related protein GmNF-YA20, its coding gene and application. The protein provided by the present invention is the following (a) or (b): (a) a protein composed of the amino acid sequence shown in Sequence 1 in the sequence listing; (b) the amino acid sequence of Sequence 1 after one or several amino acid residues A protein derived from Sequence 1 that has substitution and/or deletion and/or addition of a group and is related to plant stress tolerance. GmNF-YA20 of the present invention is expressed under the induction of drought, high salt and low temperature, and the encoded protein is localized to the nucleus, and can specifically regulate the transcription and expression of genes containing CCAAT-box cis elements (core sequence: CCAAT). The GmNF-YA20 of the present invention can improve the drought tolerance and salt tolerance of plants, provide a basis for artificially controlling the expression of stress resistance and stress tolerance related genes, and will play a role in cultivating stress resistance and stress tolerance enhanced plant breeding. important role.

Description

Plant Stress Tolerance Related Protein GmNF-YA20 and Its Encoding Gene and Application

technical field

The invention relates to the field of biotechnology, in particular to a plant stress tolerance-related protein GmNF-YA20, its coding gene and application.

Background technique

Adversity stresses such as drought, high salinity and low temperature severely restrict the growth and development of soybean. Therefore, understanding the response and signal transduction mechanism of soybean to stress conditions and improving the stress resistance of soybean varieties have become one of the important tasks of soybean genetic research and variety improvement.

Under adversity stress, plants will produce a series of responses, accompanied by many physiological, biochemical and developmental changes. Clarifying the response mechanism of plants to stress will provide scientific evidence for the research and application of stress-resistant genetic engineering. At present, the research on plant stress resistance has gradually penetrated into the cellular and molecular levels, combined with genetics and genetic engineering research, and explored the use of biotechnology to improve plant growth characteristics, the purpose of which is to improve the adaptability of plants to adversity.

Under adverse conditions of environmental stresses such as drought, high salinity, and low temperature, plants are able to make corresponding adjustments at the molecular, cellular, and overall levels to minimize the damage caused by the environment and survive. Many genes are induced by stress, and the products of these genes can not only directly participate in the stress response of plants, but also regulate the expression of other related genes or participate in signal transduction pathways, so that plants can avoid or reduce damage and enhance resistance to stress environments. Stress-related gene products can be divided into two categories: the first category of gene-encoded products include ion channel proteins, aquaporins, osmotic regulators (sucrose, proline and betaine, etc.) Responsive gene products; the second type of gene-encoded products include protein factors involved in stress-related signal transmission and gene expression regulation, such as protein kinases, transcription factors, etc. Among them, transcription factors play an important role in the regulation of gene expression in plant stress response.

Transcription factors, also known as trans-acting factors, are DNA-binding proteins that can specifically interact with cis-acting elements in eukaryotic gene promoter regions. Through interactions between them and other related proteins, they activate or Inhibits transcription. The DNA binding region of a transcription factor determines its specificity for binding to cis-acting elements, while the transcriptional regulatory region determines whether it activates or inhibits gene expression. In addition, its own activity is also affected by nuclear localization and oligomerization.

Currently known stress-related transcription factors in plants mainly include: AP2 (APETALA2)/EREBP (ethylene responsive element binding protein, ethylene responsive element binding protein) transcription factor family with AP2 domain, basic region and leucine Zipper bZIP (basic region/leucine zipper motif transcription factors) transcription factor, WRKY transcription factor family containing conserved WRKY amino acid sequence, CBF (CCAAT binding factor) transcription factor that binds to the main nuclear transcription factor of CCAAT-box, base MYC family with sex helix-loop-helix (bHLH) and leucine zipper and MYB family with tryptophan cluster (Trp cluster). Among these transcription factor families, except the WRKY family which is not involved in the water stress response of plants, the other four families are all involved in regulating the stress response of plants to drought, high salinity and low temperature.

NF-Y is a type of transcription factor that binds to the cis-acting element CCAAT-box, and specifically recognizes and binds to cis-acting elements in the promoters or enhancers of many eukaryotic constitutive, inducible and cell cycle-dependent genes CCAAT-box, and then regulate the expression of these genes at the transcriptional level. NF-Y is a heterotrimer composed of three different subunits of NF-YA, NF-YB and NF-YC. NF-YB protein and NF-YC protein form a heterodimer interaction platform in a head-to-tail manner through each other's HFM conserved domains, attracting NF-YA protein to bind to this dimer platform to form an active Heterotrimeric nuclear transcription factor. NF-Y binds to the CCAAT box of the promoter part of the target gene through the DNA binding domain on the NF-YA subunit, and performs transcriptional activation or transcriptional repression. The conserved domains of the three subunits of NF-Y have different protein domains, among which the conserved domain of NF-YA has a DNA binding domain (DNA binding domain) and a heterodimer interaction structure with NF-YB/C Domain (subunit interaction domain). The conserved domains of NF-YB and NF-YC proteins are composed of histone-fold motifs. Among them, NF-YB is similar to the H2B histone folding motif, while NF-YC is similar to the H2A histone folding motif. The histone motif consists of three α-helices and two loops and is responsible for the formation of the H2A/H2B dimer .

So far, NF-Y genes have been cloned in plants such as Arabidopsis thaliana, soybean, corn, and rice. Functional studies showed that overexpressing AtNF-YB1 of Arabidopsis thaliana significantly improved the drought resistance of transgenic Arabidopsis, and further studies found that overexpressing the homologous gene ZmNF-YB2 of Arabidopsis AtNF-YB1, transgenic maize under water-deficient conditions Drought resistance can be significantly enhanced. Transgenic maize can maintain normal photosynthesis under drought conditions by increasing stomatal conductance, reducing leaf temperature, and preventing leaf wilting, thereby increasing maize yield. In addition, Arabidopsis AtNF-YA5 is induced by drought and ABA, and is specifically expressed in vascular bundles and guard cells. It can maintain cells under drought conditions by reducing stomatal opening, reducing water transpiration, and activating stress-responsive genes. Normal osmotic potential, which improves the drought resistance of plants by maintaining the normal physiological functions of cells. Since the stress tolerance of plants is a complex trait regulated by multiple genes, it is difficult to achieve comprehensive improvement of plant stress resistance by introducing a single functional protein gene. Therefore, using a key transcription factor to promote the expression of multiple functional genes and enhance the stress resistance of plants has become a research hotspot in plant stress resistance genetic engineering.

Contents of the invention

One object of the present invention is to provide a plant stress tolerance-related protein GmNF-YA20 and its coding gene.

The protein provided by the present invention is a nuclear transcription factor protein binding to CCAAT-box, the name is GmNF-YA20, and it is derived from soybean (Glycine max L.), which is as follows (a) or (b):

(a) A protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing;

(b) The amino acid sequence of sequence 1 is subjected to the substitution and/or deletion and/or addition of one or several amino acid residues, and the protein derived from sequence 1 is related to plant stress tolerance.

The amino acid residue sequence of Sequence 1 in the sequence listing is a protein consisting of 205 amino acid residues, wherein the amino acid residues from the 110th to 166 amino acid residues are conserved histone folding motifs.

In order to facilitate the purification of GmNF-YA20 in a), tags shown in Table 1 can be attached to the amino-terminus or carboxy-terminus of the protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing.

Table 1. Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The GmNF-YA20 in the above 1) can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed. The coding gene of GmNF-YA20 in the above 1 can be obtained by deleting the codon of one or several amino acid residues from the DNA sequence shown in the 475th-1092th base of the 5' end of the sequence 2 in the sequence listing, and/or Or carry out a missense mutation of one or several base pairs, and/or connect the coding sequence of the tag shown in Table 1 to its 5' end and/or 3' end.

The DNA molecules encoding the above proteins are also within the protection scope of the present invention.

The above-mentioned DNA molecule is any DNA molecule in the following 1)-5):

1) The coding region is the DNA molecule shown in sequence 2 in the sequence listing;

2) The coding region is the DNA molecule shown in nucleotides 475-1092 from the 5' end of Sequence 2 in the sequence listing;

3) The coding region is the DNA molecule shown in nucleotides 475-1089 from the 5' end of sequence 2 in the sequence listing;

4) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) or 3) under stringent conditions and encodes a stress tolerance-related protein;

5) A DNA molecule that has more than 90% homology with the DNA sequence defined in 1) or 2) or 3) and encodes a stress tolerance-related protein.

The cDNA sequence in Sequence 2 consists of 1604 nucleotides, and the open reading frame is the 475th to 1092nd bases from the 5' end.

The above stringent conditions can be hybridized at 65oC in a solution of 6×SSC, 0.5% SDS, and then the membrane is washed once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the above-mentioned DNA molecules are also within the protection scope of the present invention.

The above-mentioned recombinant vector is a vector in which the above-mentioned DNA molecule is inserted into an expression vector to obtain the expression of the above-mentioned protein.

An existing plant expression vector can be used to construct a recombinant expression vector containing the gene. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. The plant expression vector can also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyA signal can direct polyA to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopain synthase Nos gene), plant gene (such as soybean storage The untranslated region transcribed at the 3' end of protein gene) has similar functions. When using the gene to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription start nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, maize Ubiquitin promoters (Ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used, These enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) Genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

In the embodiment of the present invention, the expression vector is YEP-GAP, and the corresponding recombinant vector is YEP-GAP-GmNF-YA20, and YEP-GAP-GmNF-YA20 is obtained by inserting the above DNA molecule into the multiple cloning site of YEP-GAP The recombinant plasmid is preferably a recombinant vector obtained by inserting the DNA fragment shown in the 475th to 1089th nucleotides from the 5' end of sequence 2 in the sequence listing between the BamHI and XhoI restriction recognition sites of YEP-GAP;

The expression vector is pBI121, and the corresponding recombinant vector is pBI121-GmNF-YA20, pBI121-GmNF-YA20 is a recombinant plasmid obtained by inserting the above DNA molecule into the multiple cloning site of pBI121, preferably by inserting sequence 2 of the sequence table from the 5' end The recombinant vector obtained by inserting the DNA fragment indicated by nucleotides 475 to 1092 between the SmaI and SacI restriction sites of pBI121.

A primer pair for amplifying the aforementioned DNA molecule or any fragment thereof.

The primer pair is GmNF-YA20-BHI and GmNF-YA20-XI or GmNF-YA20-121F and GmNF-YA20-121R:

GmNF-YA20–BHI: 5’-CGGGATCCATGACTACATCTACTCGTGACAT-3’;

GmNF-YA20-XI: 5'-CCGCTCGAGGGATGTTCTATCTGATGGT-3'.

GmNF-YA20-121F: 5'-TCCCCCGGGATGACTACATCTACTCGTGACAT-3';

GmNF-YA20-121R: 5'-CGAGCTCTTAGGATGTTCTATCTGATGGT-3'.

The application of the above-mentioned protein, above-mentioned DNA molecule or above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacteria in regulating plant stress tolerance is also within the protection scope of the present invention.

In the above application, the stress tolerance is drought tolerance and/or salt tolerance;

The aforementioned adjustment of plant stress tolerance is to improve plant salt tolerance or increase plant drought tolerance.

The plant is a dicotyledonous plant or a monocotyledonous plant; the specific cotyledonous plant is Arabidopsis thaliana.

Another object of the present invention is to provide a method for breeding transgenic plants.

The method provided by the invention is to introduce the above-mentioned DNA molecule into the target plant to obtain a transgenic plant with higher stress tolerance than the target plant.

In the above method, the above-mentioned DNA molecule is introduced into the target plant through the above-mentioned recombinant vector;

The stress tolerance is drought tolerance and/or salt tolerance;

The drought tolerance is reflected in the stress of 4% PEG, and the length of the main root of the transgenic plant is greater than that of the target plant;

The salt tolerance is reflected in the stress of 150mM NaCl, and the length of the main root of the transgenic plant is longer than that of the target plant.

The target plant is a dicotyledonous plant or a monocotyledonous plant; the specific dicotyledonous plant is Arabidopsis thaliana.

The application of the above proteins as transcription factors is also within the protection scope of the present invention.

Experiments of the present invention prove that the gene GmNF-YA20 is cloned from soybean, expressed under the induction of drought, high salt and ethylene, and the encoded protein is localized in the nucleus, and can specifically regulate the cis element containing CCAAT-box (core sequence: CCAAT) gene transcription and expression, and the GmNF-YA20 of the present invention can improve the drought tolerance of plants, which provides a basis for artificially controlling the expression of stress resistance and stress tolerance related genes, and will be used in the cultivation of stress resistance and stress tolerance play an important role in plant breeding for enhanced stress tolerance.

Description of drawings

Figure 1 is the homology comparison result of the amino acid sequence of GmNF-YA20 and Arabidopsis amino acid AtNF-YA7

Figure 2 is the real-time PCR map of stress-induced expression of GmNF-YA20

Figure 3 is a schematic diagram of the principle of yeast one-hybrid system to prove the binding specificity and activation characteristics of transcription factors in vivo

Figure 4 shows the target gene of molecular detection in transgenic Arabidopsis

Figure 5 is a comparison of drought tolerance between wild type and transgenic Arabidopsis

Figure 6 is a comparison of salt tolerance between wild type and transgenic Arabidopsis

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

% in the following examples, unless otherwise specified, are mass percentages. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Embodiment 1, the acquisition of gene GmNF-YA20

1. Acquisition of the gene GmNF-YA15

Soybean Tiefeng No. 8 grown in hydroponics for about 10 days (National Germplasm Bank, No. ZM242; recorded in the following documents: Wang Caijie, Sun Shi, Wu Baomei, Changruzhen, Han Tianfu. Since the 1940s in China Pedigree Analysis of Soybean Varieties Planted by Area. Chinese Journal of Oil Crops. 2013, 35(3):246-252, available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences) Seedlings at the four-leaf stage were drought-treated for 2 hours, quick-frozen with liquid nitrogen, Store at -80°C for later use. mRNA was isolated using QuikprepMicro mRNA Purification Kit (Pharmacia). First-strand cDNA synthesis was performed with Reverse Transcriptase XL (AMV). The ds cDNA was synthesized by SMART method, and the PCR products were detected by 1.0% agarose gel electrophoresis.

Sequence 2 in the full-length sequence list of soybean CCAAT-box nuclear transcription factor C family gene was obtained by 5'RACE and 3'RACE methods.

The gene shown in sequence 2 in the sequence listing is named GmNF-YA20, and its open reading frame is from nucleotides 475 to 1092 at the 5' end of sequence 2 in the sequence listing, and the protein encoded by the gene is named GmNF-YA20 , the amino acid sequence of the protein is sequence 1 in the sequence table, sequence 1 consists of 205 amino acid residues, and has a conserved histone folding motif.

The above sequence 2 can also be artificially synthesized.

The sequence of GmNF-YA20 was compared on Genabnk, and it has high homology with the protein AtNF-YC7 in Arabidopsis (Figure 1), but no homologous protein was found in soybean, proving that the GmNF-YA20 protein is a new protein.

2. Real-time fluorescent quantitative PCR analysis of the expression characteristics of GmNF-YA20

1. Coercion treatment

Seedling age is Tiefeng No. 8 seedling of 10 days, carries out following processing:

(1) Drought treatment (Figure 2A): Take out the potted soybean seedlings to absorb the moisture from the roots, place them on dry filter paper, and cultivate them in a drought for 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, and 24 hours, then take them out Materials were quick-frozen in liquid nitrogen and stored at -80°C for later use.

(2) Salt treatment (Fig. 2B): Put the soybean seedlings in 200mM NaCl solution, and cultivate them under light for 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, and 24 hours. Quick-frozen in liquid nitrogen and stored at -80°C for later use.

(3) Low temperature treatment (Fig. 2C): Put the soybean seedlings in a 4°C incubator, incubate under light for 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, and 24 hours, take them out and freeze them with liquid nitrogen, and store them at -80°C spare.

(4) High temperature treatment (Fig. 2D): Put the soybean seedlings at 42°C, cultivate them under light for 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, and 24 hours, then take them out and freeze them with liquid nitrogen, and store them at -80°C spare.

(5) Oxidation treatment (Fig. 2E): Put the soybean seedlings in 30mM hydrogen peroxide (H2O2) solution, cultivate them under light for 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, and 24 hours, then take them out and freeze them with liquid nitrogen , stored at -80°C for later use.

(6) Ethylene treatment (Figure 2F): Soybean seedlings were placed in a solution of 100 μM ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and incubated under light for 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours. After 1 hour and 24 hours, they were taken out and quick-frozen in liquid nitrogen, and stored at -80°C for later use.

(7) Control treatment: soybean seedlings without any treatment were directly frozen at -80°C as a control (0 hour).

2. Isolation of mRNA

The soybean seedlings treated with the above treatments were isolated from mRNA using Quikprep Micro mRNA Purification Kit (Pharmacia).

3. Reverse transcription into cDNA

The purified mRNA was reverse transcribed into cDNA.

4. Real-time fluorescent quantitative PCR

The cDNA was diluted 50 times and used as a template for Q-RT-PCR. Use the specific primer pair of the 3′ non-coding region of the gene (F: 5′-AGAGTGACTGCAATGGGATG-3′, R: 5′-GAATAGCAAGGCTCGGAAA-3′) to amplify the sample by Q-RT-PCR, and analyze the effect of the gene on various treatments For the response, actin (F:5'-CGGTGGTTCTATCTTGGCATC-3', R:5'-GTCTTTCGCTTCAATAACCCTA-3') was used as internal reference. Q-RT-PCR at ABI It was carried out on a 7000 real-time fluorescent quantitative PCR instrument, and a parallel experiment was set for 3 repetitions. The method reported by Livak KJ and Schmittgen TD (2001), ie, 2 ^-ΔΔCT, was used to calculate the relative expression level.

ΔΔC _T = (C _T.Target - C _T.Actin ) _Timex - (C _T.Target - C _T.Actin ) _Time0

Time x indicates any time point, and Time ₀ indicates the expression of the target gene after correction by actin.

The results are shown in Figure 2. It can be seen that GmNF-YA20 has different responses to drought, salinity, low temperature, high temperature and oxidative stress and ethylene hormone.

Example 2, Application of GmNF-YA20 as a transcription factor

The main principle of using the yeast one-hybrid system to prove the activation properties of transcription factors is shown in Figure 3. The CCAAT cis-acting element and the mutant CCAAT cis-acting element were respectively constructed into the basic promoter Pmin of the pHISi-1 vector and the pLacZi vector ( The upstream of the minimal promoter) and the downstream of the Pmin promoter are connected to the reporter genes (His3, LacZ and URA3). When the expression vector YEP-GAP (without activation function) connected with the target gene encoding the transcription factor is transformed into the yeast cells connected with the CCAAT cis-acting element and the mutant CCAAT cis-acting element, if the mutant CCAAT The reporter gene in yeast cells with cis-acting elements cannot be expressed, but the reporter gene in yeast cells with specific CCAAT cis-acting elements can be expressed, indicating that the transcription factor can bind to CCAAT cis-acting elements and has the ability to activate function, activate the Pmin promoter, and promote the expression of the reporter gene. Thus demonstrating the in vivo binding specificity and activation function of the transcription factor of interest.

Expression vector YEP-GAP: Institute of Crop Science, Chinese Academy of Agricultural Sciences guaranteed to be available to the public; References Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Twotranscription factors, DREB1 and DREB2, with an EREBP/AP2DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis, Plant Cell 1998 Aug;10(8):1391-1406.

YPD liquid medium: 10g/L of yeast extract (Bacto-Yeast Extract) for bacterial culture, 20g/L of tryptone (Bacto-Peptone) for bacterial culture, adjust the pH to 5.8, sterilize at 121°C/15min, drop After reaching 60°C, add 40% Glucose to make the final concentration 20g/L.

SD/His ^- /Ura ^- / ^Trp -selective medium: Yeast nitrogenbase without amino acids (Yeast nitrogenbase) 6.7g/L, auxotrophic mixture (drop-out media without His/Ura/Trp) 100ml, agar Powder (Bacteriological agar) 20g/L, adjust the pH to 5.8, sterilize at 121°C/15min, add 40% Glucose after cooling down to 60°C to make the final concentration 20g/L.

Auxotrophic mixture (Drop-out mix): (10×): L-Isoleucine (isoleucine) 300mg/L, L-Valine (valine) 1500mg/L, L-Adenine (adenine) 200mg/L L, L-Arginine (arginine) 200mg/L, L-Histidine Hcl monohydrate (histidine) 200mg/L, L-Leucine (leucine) 1000mg/L, L-LysineHcl (lysine) 300mg/L L, L-Methionine (methionine) 200mg/L, L-Phenylalanine (phenylalanine) 500mg/L, L-Threonine (threonine) 2000mg/L, L-Tyrosine (tyrosine) 300mg/L L.

1×PEG/LiAc: 50% PEG33508ml, 10×TE buffer 1ml, 10×LiAc 1ml.

10×TE Buffer: 100mM Tris-Hcl, 10mM EDTA, pH=7.5, autoclaved at 121℃, stored at room temperature.

1×TE/LiAc: 10×TE buffer 1ml, 10×LiAc 1ml, ddH ₂ O 8ml.

Z Buffer: Na ₂ HPO ₄ 7H ₂ O 16.1g/L, NaH ₂ PO ₄ H ₂ O 5.5g/L, KCl 0.75g/L, MgSO ₄ 7H ₂ O 0.246g/L, adjust the pH to 7.0 , sterilized at 121°C/15min, and stored at 4°C.

X-gal stock solution (X-gal Stock Solution): Dissolve X-gal with N,N-dimethyl-formamide (DMF) to make the final concentration 20mg/ml, store at -20°C.

100ml of Z buffer containing X-gal (Z buffer with X-gal), ready-to-use: Zbuffer98ml, β-mercaptoethanol (β-mercaptoethanol) 0.27ml, X-gal stock solution (X-gal stocksolution) 1.67 ml.

10×LiAc: 100Mm Tris-Hcl, 100mM EDTA, pH=7.5. Autoclave at 121°C and store at room temperature.

1. Construction of recombinant vector

1. Acquisition of GmNF-YA20 gene

Primers GmNF-YA20-BHI and GmNF-YA20-XI were designed according to the sequence of GmNF-YA20 gene, and BamHI and XhoI restriction sites were introduced into the ends of the primers respectively.

GmNF-YA20–BHI: 5’-CGGGATCCATGACTACATCTACTCGTGACAT-3’;

GmNF-YA20-XI: 5'-CCGCTCGAGGGATGTTCTATCTGATGGT-3'.

Using the cDNA of the whole plant of soybean variety Tiefeng 8 as a template, PCR amplification was carried out with primers GmNF-YA20-BHI and GmNF-YA20-XI.

A PCR amplification product of about 618bp was obtained.

The PCR amplification product was recovered.

2. Construction of recombinant vector

① Digest the PCR amplification product recovered in step 1 with restriction enzymes BamHI and XhoI, and recover the 618bp digested product;

② Digest the expression vector YEP-GAP with restriction enzymes BamHI and XhoI, and recover the vector skeleton;

③ connecting the digested product of step ① to the carrier backbone of step ②;

④ Transform the ligation product of step ③ into JM109 strain (purchased from Clontech), culture overnight at 37°C, and pick positive clones to extract plasmids for sequencing;

Sequencing results show that the plasmid is a recombinant vector obtained by inserting the DNA fragment shown in the nucleotides 475 to 1089 at the 5' end of Sequence 2 of the sequence listing between the BamHI and XhoI restriction sites of the vector YEP-GAP, named is YEP-GAP-GmNF-YA20.

2. Validation of the in vivo binding specificity and activation properties of GmNF-YA20

1. Construction of yeast reporter

(1) Construction of normal dual yeast reporter

DNA fragment A (containing 4 CCAAT elements): 5'-GAATTC-CCAAT-CCAAT-CCAAT-CCAAT-GTCGAC-3' (core sequence of CCAAT: CCAAT).

The DNA fragment A was constructed into the upstream of the Pmin _HIS3 promoter of the pHis-1 vector (MATCHMAKER One-Hybrid System, Clontech Company) to obtain the recombinant vector pHis-1-CCAAT, and the pHis-1-CCAAT vector was transformed with XhoI and NcoI endonucleases. Cut into strands.

The DNA fragment A was constructed into the pLacZi vector (MATCHMAKER One-Hybrid System, Clontech Company) upstream of the P _CYCI promoter to obtain the recombinant vector pLacZi-CCAAT, and the pLacZi-CCAAT vector was cut into lines with XhoI and NcoI endonucleases, respectively.

First transform the linear pHis-1-CCAAT vector into yeast cells (YM4271 strain, MATCHMAKER One-Hybrid System, Clontech Company) to obtain yeast transformants (Yeast transformants) that can grow normally on SD/His-medium . Then, the yeast transformant was used as the host cell to continue to transform the linear pLacZi-CCAAT vector containing 4 repeated CCAAT elements. In this way, normal dual yeast reporters containing pHis-1-CCAAT and pLacZi-CCAAT were obtained by selection on the SD/His ^- /Ura ^- medium lacking both histidine and uracil.

(2) Construction of mutant double yeast reporter

DNA fragment B (containing 4 mCCAAT elements): 5'-GAATTC-mCCAAT-mCCAAT-mCCAAT-mCCAAT-GTCGAC-3' (MDRE: mutate the core sequence CCAAT of 4 CCAAT elements into TTTTA).

Using DNA fragment B to replace DNA fragment A, the method is the same as step (1) to obtain a mutant double yeast reporter.

2. Transformation of yeast by PEG/LiAc method and result analysis

(1) Inoculate the normal double yeast reporter and the mutant double yeast reporter containing pHis-1-CCAAT and pLacZi-CCAAT obtained in the above 1 into 1ml YPD liquid medium respectively, shake vigorously for 2 minutes, and dissolve the suspension after dispersing the clumps. Transfer to a Erlenmeyer flask containing 50ml of YPD liquid medium, shake overnight at 30°C/250rpm, measure OD600=1.7-1.8 (count about 4×10 ⁷ cells/mL);

(2) Take 30ml of the overnight culture from step (1) and transfer it to 300ml of fresh YPD medium, culture at 30°C/250rpm for about 3 hours (to OD600=0.5±0.1), centrifuge at 1000g at room temperature for 5min, collect the bacteria, and discard the supernatant , suspend with 1/2 volume 1×TE, centrifuge at 1000g/5min;

(3) Discard the supernatant, suspend with 1.5ml freshly prepared 1×TE/LiAc solution, shake and mix well for later use;

(4) Take out 0.1ml of yeast competent for transformation, and add the following solutions in sequence: 0.1μg of the recombinant vector YEP-GAP-GmNF-YA20 prepared by 1, 0.1mg of ssDNA (salmon sperm DNA, SiTaa), 0.6ml of PEG/LiAc high-speed shaking for 1 minute , 30°C/200rpm shaking culture for 30 minutes;

(5) Add 70ul DMSO (siTaa#D8779), invert and mix gently, heat shock at 42°C for 30 minutes, shake gently during this period, bathe in ice for 2 minutes, and centrifuge at 1000g for 5 minutes at room temperature;

(6) Discard the supernatant, add 0.5ml 1×TE buffer to suspend the cells;

(7) Dip the suspension with an inoculation loop, and culture it as a line on SD/His ^- /Ura ^- /Trp ^- selective medium containing 0, 15mmol/L3-AT respectively.

(8) Half of the plate was cultured with normal double yeast reporter, and the other half was cultured with mutant double yeast reporter for control analysis.

⑼ Place it upside down in the incubator, and incubate at 30°C for 3-4 days.

⑽ It was found that both the normal yeast reporter and the mutant yeast reporter grew on the SD/His ^- /Ura ^- /Trp ^- medium plate of 0mmol/L3-AT, but the diameter of the mutant yeast reporter was significantly smaller ; On the SD ^/ His-/ ^Ura- /Trp ^- medium plate of 15mmol/L3-AT, the normal yeast reporter can grow normally, but the mutant yeast reporter is suppressed and does not grow.

3. Detection of galactosidase activity

(1) Pick normal yeast reporter colonies and mutant yeast reporter colonies from SD/His ^- /Ura ^- /Trp ^- media plates of 0mmol/L3-AT respectively. Transfer to YPD liquid medium, shake culture at 30°C, and wait for the growth to the late logarithmic growth stage, take 1.5ml of bacterial liquid, and centrifuge at 3000rpm for 30s;

(2) Discard the supernatant, drain the liquid in the tube, place the centrifuge tube in liquid nitrogen for 10 minutes, take it out and let it thaw naturally, add 50ul Z/X-gal solution, and incubate at 30°C. It was found that the normal yeast reporter was in the It turned blue within 6-8 hours, while the mutant yeast reporter did not change within 12 hours and remained white.

It shows that the transcription factor GmNF-YA20 can combine with the CCAAT cis-acting element, and has an activation function, activates the Pmin promoter, and promotes the expression of the reporter gene. Thus, the in vivo binding specificity and activation function of GmNF-YA20, which is a transcription factor, was demonstrated.

Embodiment 3, the application of GmNF-YA20 in improving the stress tolerance of plants

1. Obtaining transgenic GmNF-YA20 Arabidopsis

1. Construction of recombinant vector

1) Cloning of GmNF-YA20 gene

A pair of primers (GmNF-YA20-121F and GmNF-YA20-121R) were designed according to the sequence of the GmNF-YA20 gene, and SmaI and SacI restriction sites were introduced at the ends of the primers,

GmNF-YA20-121F: 5'-TCCCCCGGGATGACTACATCTACTCGTGACAT-3';

GmNF-YA20-121R: 5'-CGAGCTCTTAGGATGTTCTATCTGATGGT-3'.

Using the cDNA of Glycine max L. Tiefeng 8 plant as a template, GmNF-YA20-121F and GmNF-YA20-121R were used for PCR amplification, and the PCR product (GmNF-YA20 gene) with a size of about 618bp was obtained.

The above PCR product was recovered.

2) Construction of recombinant vector

① Digest the PCR product recovered in step 1 with restriction enzymes SmaI and SacI, and recover the 618bp digested product;

② Digest pBI121 (purchased by Clontech) with restriction enzymes smaI and SacI, and recover the 14000bp vector backbone;

③ connecting the digested product of step ① to the carrier backbone of step ②;

④ Transform the ligation product of step ③ into TOP10 strain (purchased from Beijing Tiangen Company) by electroporation, culture overnight at 37°C, and pick positive clones to extract plasmids for sequencing.

Sequencing results show that the plasmid is a recombinant vector obtained by inserting the DNA fragment shown in the 475th-1092nd nucleotides shown in Sequence 2 of the sequence table between the SmaI and SacI restriction sites of the vector pBI121, named pBI121- GmNF-YA20.

3. Obtaining recombinant Agrobacterium

Use the recombinant plasmid pBI121-GmNF-YA20 to transform Agrobacterium C58C1 (purchased by Beijing Baierdi Biotechnology Co., Ltd.) to obtain recombinant Agrobacterium C58C1/pBI121-GmNF-YA20 (extract the plasmid and send it for sequencing, it is pBI121-GmNF-YA20, then Recombinant bacteria containing the plasmid are positive).

4. Obtained from Arabidopsis transgenic GmNF-YA20

1) Inoculate the recombinant Agrobacterium obtained in 3 in LB (containing 50mg/ml rifampicin, 100mg/ml kanamycin, 50mg/ml gentamicin) liquid medium, and culture at 28°C and 3000rpm for about 30 hours , to obtain the bacterial solution;

2) Transfer the bacterial solution to LB (containing 50mg/ml rifampicin, 100mg/ml kanamycin, 50mg/ml gentamycin), and culture at 28°C and 300rpm for about 14 hours (the OD600 of the bacterial solution reaches 1.5- 3.0);

3) Collect the bacteria, centrifuge at 4000g for 10min at 4°C, and dilute with MS liquid medium containing 10% sucrose (containing 0.02% silwet) to an OD600 of about 0.8-1.0;

4) Put the whole plant of Arabidopsis thaliana (Colombia ecotype Col-0, purchased by SALK company, also known as wild-type Arabidopsis thaliana) upside down together with the flowerpot in the container containing the bacterial solution in step 4, and soak the flowers After soaking for about 50 seconds, take out the flower pot, put it on the side of the tray, cover it with a black plastic sheet, uncover the plastic sheet after 24 hours, place the flower pot upright, carry out normal light cultivation, and harvest _T1 generation seeds, kanamyces The positive plants selected by element selection (concentration of 50 μg/L kanamycin) were regenerated plants of the T ₁ generation, which were subcultured until the regenerated plants of the T ₃ generation were obtained.

The _T2 generation represents the seeds produced by the selfing of the _T1 generation and the plants grown from it, and the _T3 generation represents the seeds produced by the _T2 generation selfing and the plants grown from it.

The RNA of the whole plant of the regenerated plants of the T ₃ generation was extracted, and the cDNA obtained by reverse transcription was used as a template, and the primer pair F: 5'-ATGACTACATCTACTCGTGACAT-3'; R: 5'-TTAGGATGTTCTATCTGATGGT-3'; was used for PCR amplification.

The results of some samples are shown in Figure 4, M is Marker, col-0 is Columbia ecotype wild type Arabidopsis, and L1-L9 are T ₃ generation regenerated plants.

L1, L3-L9 obtained fragments with a size of 618bp were positive, that is, Arabidopsis thaliana transformed with GmNF-YA20 in the T ₃ generation.

Using the same method, the plasmid pBI121 was transformed into wild-type Arabidopsis thaliana to obtain the empty vector Arabidopsis, and the T ₃ generation empty vector Arabidopsis was cultivated. The same method was used to identify the 618bp fragment.

2. Stress tolerance identification of transgenic plants

1. Drought tolerance identification

The _three lines GmNF-YA20-3, GmNF-YA20-4, GmNF-YA20-5 of T 3 generation transformed into GmNF-YA20 Arabidopsis, the T ₃ generation transformed into empty vector Arabidopsis and wild type Arabidopsis respectively After 25 seeds have been sterilized, use a toothpick to evenly spot a single seed on MS medium (MSO) and MS medium (4% PEG+MSO) containing 4% (mass percentage) PEG, and seal with a parafilm , placed at 4°C for 3 days, moved to 23°C, 12h light/8h dark, 60% relative humidity in a culture room for 7 days and counted the length of the main root. Three replicates were set for each treatment, and the results were averaged.

The results are shown in Figure 5. It can be seen that on the MS medium without PEG, there was no significant difference in the length of the main root between the wild-type Arabidopsis and the T ₃ generation transgenic GmNF-YA20 Arabidopsis; while under PEG stress, T The main root length of Arabidopsis transgenic to GmNF-YA20 _{in the third} generation was longer than that of wild-type Arabidopsis.

Count the taproot length,

Cultured in MS medium (MSO), wild-type Arabidopsis Col-0, T ₃ generation Arabidopsis line GmNF-YA20-3, T ₃ generation Arabidopsis line GmNF-YA20 The main root lengths of GmNF-YA20-4 and GmNF-YA20 Arabidopsis line GmNF-YA20-5 of the T ₃ generation were 3.61cm, 3.43cm, 3.82cm, and 3.94cm, respectively;

Under PEG stress, wild-type Arabidopsis Col-0, the line GmNF-YA20-3 of Arabidopsis transformed into GmNF-YA20 in _T3 generation, and the GmNF- _YA20-4 line of Arabidopsis transformed into GmNF-YA20 in T3 generation The main root lengths of the line GmNF-YA20-5 of T ₃ generation GmNF-YA20 Arabidopsis were 1.95cm, 2.62cm, 2.58cm, and 2.61cm, respectively;

After drought stress treatment, the length of the main root of the transgenic plants was significantly longer than that of the wild type, and there was no significant difference in the results of _T3 transgenic Arabidopsis and wild type Arabidopsis.

2. Identification of salt tolerance

_Three lines (GmNF-YA20-3, GmNF-YA20-4 and GmNF-YA20-5) of Arabidopsis thaliana transfected with GmNF-YA20 in the third generation of T, transfected with empty vector Arabidopsis and wild-type Arabidopsis in _{the third} generation of T After the seeds were sterilized, they were planted on MS medium, sealed with a parafilm, placed at 4°C for 3 days, then moved into a culture room at 23°C, 12h light/8h dark, and 60% relative humidity for 5 days, and separated with tweezers. Transplanted to MS medium (MSO) and MS medium (150mM NaCl+MSO) containing 150mM NaCl, each treatment set three replicates, and the results were averaged.

The results are shown in Figure 6. It can be seen that there is no significant difference in the growth status of the 3 strains of Arabidopsis thaliana transformed into GmNF-YA20 in the T ₃ generation on the MS medium and the wild-type Arabidopsis; while under 150mM NaCl stress , the main root length of _T3 transgenic Arabidopsis GmNF-YA20 was higher than that of wild type Arabidopsis.

Count the taproot length,

Cultured in MS medium (MSO), wild-type Arabidopsis Col-0, T ₃ generation Arabidopsis line GmNF-YA20-3, T ₃ generation Arabidopsis line GmNF-YA20 The main root lengths of GmNF-YA20-4 and GmNF-YA20 Arabidopsis line GmNF-YA20-5 of the T ₃ generation were 3.61cm, 3.43cm, 3.82cm, and 3.94cm, respectively;

Under 150mM NaCl stress, wild-type Arabidopsis Col-0, the line GmNF-YA20-3 of Arabidopsis thaliana transformed from _T3 generation to GmNF-YA20, and the line _GmNF -YA20- 4. The main root lengths of the line GmNF-YA20-5 transformed into GmNF-YA20 Arabidopsis in _{the third} generation of T are 1.55cm, 1.68cm, 1.63cm, and 1.65cm, respectively;

After 150mM NaCl stress treatment, the length of the main root of the transgenic plants was significantly longer than that of the wild type, and there was no significant difference in the results between the _T3 generation of empty vector Arabidopsis and wild type Arabidopsis.

It can be seen from the above experiments that the gene GmNF-YA20 can improve the drought tolerance and salt tolerance of plants.

Claims (7)

1. a kind of application of protein or its encoding gene in plant stress tolerance is adjusted；The resistance of reverse for drought tolerance and/or Salt tolerance；

The protein is made of the amino acid sequence shown in sequence in sequence table 1.

2. application according to claim 1, it is characterised in that：The encoding gene is following 1) -3) in it is any：

1) DNA molecular in sequence table shown in sequence 2；

2) DNA molecular in sequence table shown in sequence 2 from the nucleotide of 5 ' end 475-1092；

3) DNA molecular in sequence table shown in sequence 2 from the nucleotide of 5 ' end 475-1089.

3. application according to claim 1 or 2, it is characterised in that：The plant is dicotyledon or monocotyledon.

4. application according to claim 3, it is characterised in that：The dicotyledon is arabidopsis.

5. a kind of method for cultivating genetically modified plants is to import a kind of DNA molecular in purpose plant, resistance of reverse is obtained higher than institute State the genetically modified plants of purpose plant；

The DNA molecular is following 1) -3) in it is any：

1) DNA molecular in sequence table shown in sequence 2；

2) DNA molecular in sequence table shown in sequence 2 from the nucleotide of 5 ' end 475-1092；

3) DNA molecular in sequence table shown in sequence 2 from the nucleotide of 5 ' end 475-1089.

The resistance of reverse is drought tolerance and/or salt tolerance.

6. according to the method described in claim 5, it is characterized in that：The DNA molecular imports the purpose by recombinant vector Plant；

The recombinant vector is that the DNA molecular shown in sequence in sequence table 2 is inserted into expression vector, obtains sequence in expressed sequence table The carrier of the protein of amino acid sequence composition shown in row 1；

The purpose plant is dicotyledon or monocotyledon.

7. according to the method described in claim 6, it is characterized in that：The dicotyledon is arabidopsis.