CN104231063A - Chlamydomonas reinhardtii protein F6 capable of improving photosynthetic efficiency of plants and encoding gene and application of chlamydomonas reinhardtii protein F6 - Google Patents

Abstract

The invention discloses a Chlamydomonas reinhardtii protein F6 capable of improving plant photosynthetic efficiency, its coding gene and application. The protein is a protein of the following (a) or (b): (a) a protein consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing; (b) the amino acid sequence of Sequence 2 in the Sequence Listing through one or several amino acids The substitution and/or deletion and/or addition of residues can improve the photosynthetic efficiency of plants. The results show that: F6 protein has an important function in photosynthesis, which provides a good target gene for the transformation of high light efficiency engineering algae strains, and also provides a good candidate target gene for crop improvement.

Description

A kind of Chlamydomonas reinhardtii protein F6 capable of improving plant photosynthetic efficiency and its coding gene and application

technical field

The invention relates to the field of biotechnology, in particular to a Chlamydomonas reinhardtii protein F6 capable of improving plant photosynthetic efficiency, its coding gene and its application.

Background technique

Photosynthesis is the basis for the survival of the biological world, and is closely related to food and energy issues faced by human beings. With the increase of the world's population, food, energy and resource problems are becoming more and more severe, and it is imminent to improve the light energy utilization efficiency of photosynthetic organisms.

Chlamydomonas reinhardtii is a model species for the study of photosynthesis and photosynthetic hydrogen production. Uncovering important new genes that can improve light energy utilization efficiency will not only lay the foundation for the design of high hydrogen-releasing engineering strains, but also help discover Chlamydomonas with high efficiency and low efficiency. New ideas and new methods for low-cost hydrogen production are of great value, and also provide alternative target genes for molecular breeding of crops with high light efficiency. The discovery of new genes through scientific research has become an important force driving the development of crop breeding.

Contents of the invention

One object of the present invention is to provide a Chlamydomonas reinhardtii protein F6 and its coding gene which can improve the photosynthetic efficiency of plants.

The protein provided by the present invention is the protein of the following a) or b):

a) a protein consisting of the amino acid sequence shown in Sequence 2 in the sequence listing;

b) A protein in which the amino acid sequence shown in Sequence 2 in the sequence listing has been substituted and/or deleted and/or added by one or several amino acid residues and is related to plant photosynthetic efficiency.

The protein-related biological material provided by the present invention is any one of the following B1) to B5):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette containing the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

B4) a recombinant plasmid containing the nucleic acid molecule described in B1), or a recombinant plasmid containing the expression cassette described in B2);

B5) A recombinant algal strain containing the nucleic acid molecule described in B1), or a recombinant algal strain containing the expression cassette described in B2), or a recombinant algal strain containing the recombinant vector described in B3).

Among the above-mentioned related biological materials, the nucleic acid molecules described in B1) are shown in the 1st-435th nucleotide molecules of Sequence 1 in the sequence listing.

The application of the above-mentioned proteins or related biological materials in improving the photosynthetic efficiency of plants is also within the protection scope of the present invention.

In the above application, the plants are algae, specifically Rhine green algae.

Another object of the present invention is a method for constructing transgenic plants with improved photosynthetic capacity.

The method for constructing a transgenic plant with improved photosynthetic capacity provided by the present invention comprises the following steps: introducing the coding gene of the above-mentioned protein into a starting plant to obtain a transgenic plant; compared with the starting plant, the photosynthetic capacity of the transgenic plant is improved.

In the above method, the gene encoding the protein is the DNA molecule of nucleotides 1-435 of Sequence 1 in the Sequence Listing.

In the above method, the improvement of the photosynthetic ability is the improvement of photosynthetic efficiency.

In the above method, the plant is algae, specifically Chlamydomonas reinhardtii.

Experiments show that the F6 gene is related to the photosynthetic phenotype of F6, and the F6 gene has an important function in photosynthesis, which provides a good target gene for the transformation of high-efficiency engineering algae strains, and also provides a good preparation for crop improvement. Select the target gene.

Description of drawings

Figure 1 is the identification of Fv/Fm phenotypes of wild type, mutant F6 and complementary algal strains.

Figure 2 is the DNA level identification of wild type, mutant F6 and complementary algal strains.

Figure 3 is the identification of protein levels of wild type, mutant F6 and complementary algae strains.

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.

Plant material: Chlamydomonas reinhardtii species CC400 (mt+), purchased from the Chlamydomonas reinhardtii Center of Duke University, USA (Chlamy center, http://www.chlamy.org/).

Strains and plasmids: The pSI103 plasmid was purchased from the Chlamydomonas reinhardtii Center of Duke University (Chlamy center http://www.chlamy.org/); the pJR38 plasmid was provided by Professor Ralph Bock of the Max Planck Institute of Molecular Physiology, in the literature "Generation of Chlamydomonas strains that efficiently express nuclear transgenes, Neupert et al., The Plant Journal, 2009, 57, 1140-1150", the public can obtain from the Institute of Botany, Chinese Academy of Sciences. Both pSI103 plasmid and pJR38 plasmid contain paromomycin resistance gene.

Preparation of medium:

1) Normal TAP culture medium: NH ₄ Cl 0.4g/L; MgSO ₄ 7H ₂ O 0.1g/L; CaCl ₂ 2H ₂ O 0.05g/L; K ₂ HPO ₄ 0.108g/L; KH ₂ PO ₄ 0.056g/L; Trisbase 2.423g/L; Hunter's traceelements 1ml/L; glacial acetic acid 1ml/L; the rest is water.

2) Solid TAP medium: add 1.5% agar powder to normal TAP medium.

3) Hunter's trace elements: H ₃ BO ₄ 11.4g/L; ZnSO ₄ 7H ₂ O 22.0g/L; MnCl ₂ 4H ₂ O 5.06g/L; CoCl ₂ 6H ₂ O 1.61 g/L; CuSO ₄ ·5H ₂ O 1.57g/L; (NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O 1.10g/L; FeSO ₄ ·7H ₂ O 4.99g/L; the rest is water.

4) Cultivate Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) CC400 (mt+), F6 mutants and complementary algae strains with TAP solid medium: prepare TAP liquid culture medium, autoclave at 121°C for 20 minutes, and wait for the temperature of the culture medium to drop to room temperature , using an inoculation needle to pick a single clone of Chlamydomonas reinhardtii from the solid medium into the liquid TAP culture medium, and place it in a constant temperature light incubator on a shaker for continuous light culture (25°C, 60rpm, 100μEs ^-1 m ^-2 ), Suspension culture cells, solid culture, the monoclonal transfer to TAP solid medium for streak culture.

Embodiment 1, the acquisition of F6 gene

1. Extraction of Chlamydomonas reinhardtii total RNA:

Take Chlamydomonas reinhardtii CC400 (mt+) cells (4-6) x 10 ⁶ cells/ml in the exponential growth phase, centrifuge at 5000rpm for 5min, add 1mL of lysis solution RZ, shake and mix well. After standing at room temperature for 5 minutes, centrifuge at 12,000 rpm for 5 minutes at 4°C, take the supernatant, and transfer it to a new RNase-free centrifuge tube. Add 200 μL of chloroform, cover the tube cap, shake vigorously for 15 s, and place at room temperature for 3 min. Centrifuge at 12000rpm for 10min at 4°C. Transfer the upper aqueous phase to a new RNase-free centrifuge tube, slowly add 0.5 times the volume of absolute ethanol, and mix well. The resulting solution and precipitate were all transferred to an adsorption column. Centrifuge at 12000rpm at 4°C for 30s, discard the waste liquid in the centrifuge tube, add 500 μL protein-free solution, centrifuge at 12000rpm at 4°C for 30s, discard the waste liquid. Add 700μL of washing solution, let stand at room temperature for 2min, centrifuge at 12000rpm for 30s at 4°C, and discard the waste solution. Add 500 μL of washing solution, let stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 30 seconds at 4°C, and discard the waste solution. Centrifuge at 12,000 rpm for 2 minutes at 4°C to remove residual liquid, and open the lid to dry. Transfer the adsorption column to a new RNase-free centrifuge tube, add 50 μL RNase-free dd-H ₂ O, place at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes at 4°C. The supernatant was discarded to obtain the total RNA of Chlamydomonas reinhardtii.

2. cDNA first-strand synthesis:

Using the Chlamydomonas reinhardtii total RNA obtained above as a template, the first strand of cDNA was synthesized using the cDNA first strand synthesis kit from Quanshijin Co., Ltd. Reaction system: 2μL 10x TSII mix, 1ul 10μmol OligodT20, 1-5μg total RNA, add sterilized double distilled water to 20μL. Warm at 50°C for 30 minutes, then heat at 70°C for 15 minutes to terminate the reaction. First-strand cDNA of Chlamydomonas reinhardtii was obtained.

3. Amplification of F6 gene:

Using the first strand of cDNA of Chlamydomonas reinhardtii obtained above as a template, the primer sequences in Table 1 were used to amplify the F6 gene by PCR. Reaction program: pre-denaturation at 98°C for 2min, denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 30s, 35 cycles, extension at 72°C for 5min. Reaction system: 12.5 μL of 2×HS GC buffer I, 2 μL of dNTP mixture (2.5 mM each), 1 μL of template cDNA, 0.5 μL of PRIMERSTAR (5U/μL), 1 μL of each F/R, supplemented with ddH ₂ O to 25 μL. The above primers were synthesized at Sangon Bioengineering Co., Ltd. The primer sequences are shown in Table 1 below.

Table 1. Primer sequences used in PCR amplification

5. Cloning and identification of PCR products:

Use Tiangen Agarose Gel DNA Recovery Kit (DP209-03) to recover and purify the target band, and connect the target fragment to the pEasy-blunt vector (purchased from Beijing Quanshijin Company) to obtain the recombinant vector. The recombinant vector was transformed into Escherichia coli (Escherichia coli) Trans-T1 competent cells (purchased from Beijing Quanshijin Company), and the positive clones were screened by the blue-white method. Single clones were picked and cultured for PCR identification, and the positive clones were sent to Beijing Sanbo Biotechnology Co., Ltd. for sequencing. Sequencing results show that: the gene sequence inserted into the pEasy-blunt vector is shown in the 1st-435th nucleotide molecule in SEQ ID No.1, and the amino acid sequence of the protein encoded by the gene is shown in the 1st-144th position in SEQ ID No.2 shown.

Embodiment 2, the acquisition of F6 mutant

1. Obtaining recombinant Chlamydomonas reinhardtii

After linearizing the plasmid pSI103 with KpnI, the pChlamiRNA3 plasmid was obtained. Weigh 0.1g of glass beads (425-600um, Sigma) into a 1.5ml centrifuge tube, sterilize at 121°C for 20 minutes, and store at room temperature for use. Add 100 ul of CC400 (mt+) algae solution and 10 ul of pChlamiRNA3 plasmid into the above-mentioned centrifuge tube containing glass beads, shake on vortex (Genie 2) at speed 7 for 15 seconds, and place at room temperature 25°C for 4 minutes. Use a pipette to transfer the algae solution to fresh TAP culture solution, and place it in a constant temperature light incubator for continuous light culture on a shaker for 24 hours (25°C, 60rpm, 110μEs ^-1 m ^-2 ), centrifuge at 2500rpm, and collect After the precipitate is suspended with TAP culture medium, it is coated on a medium plate containing paromomycin (10ug/ml), and the algal strain capable of growing is recombinant Chlamydomonas reinhardtii.

2. Obtaining F6 mutants

Screen F6 mutants from the recombinant Chlamydomonas reinhardtii obtained above by means of chlorophyll fluorescence assay: when carrying out fluorescence assay, Chlamydomonas is first scratched from the plate, inoculated into a 50ml Erlenmeyer flask and cultivated to the exponential growth phase, then transferred to In a 250ml Erlenmeyer flask with 100ml TAP medium, adjust the initial OD750 to 0.05. Afterwards, it was placed on a shaker in a constant temperature light incubator for continuous light cultivation. After growing to the exponential growth phase, the chlorophyll concentration was adjusted to 12ug/ml. After 20 minutes of dark adaptation, the Fv/Fm value was measured by IMAGING-PAM.

The Fv/Fm value of the wild type was 0.74, and the recombinant algae strains with a Fv/Fm value less than 0.5 were screened. When the Fv/Fm value of the recombinant algal strain is 0.44, the recombinant algal strain is the F6 mutant.

The F6 mutant was sequenced, and the results showed that the sequence shown in SEQ ID No.3 on the wild type was replaced by the sequence on the plasmid pSI103 shown in SEQ ID No.4 to obtain the F6 mutant. The rest of the genome sequence of the F6 mutant is consistent with the wild-type genome sequence, and positions 156-1026 in the sequence shown in SEQ ID No. 3 are the sequence of the F6 gene.

Embodiment 3, the acquisition of complementary algal strains

The clones sequenced correctly in Example 1 were extracted with plasmids (purchased from Beijing Suolaibao Company). The above plasmid and the pJR38 vector were digested with restriction endonucleases NdeI and EcoRI (purchased from Takara Company), respectively, and after digesting at 37°C for 2 hours, Tiangen (Beijing, China) Agarose Gel DNA Recovery Kit (DP209- 03) Recover and purify the target band and the pJR38 vector, and connect the purified target band and the pJR38 vector with T4 DNA ligase at 25°C for 3 hours to obtain the pJR38-F6 plasmid. Then the pJR38-F6 plasmid was transformed into Escherichia coli (Escherichia coli) Trans-T1 competent cells (purchased from Beijing Quanshijin Company). Pick a single clone, and perform PCR amplification with the above primers F/R to obtain positive clones, and send them to Beijing Sanbo Biotechnology Co., Ltd. for sequencing. Sequencing results show that: the gene sequence inserted into the pEasy-blunt vector is shown in the 1-435th nucleotide molecule in SEQ ID No.1, and the amino acid sequence of the protein encoded by the gene is shown in the 1-144th in SEQ ID No.2 bit shown.

Plasmids (purchased from Beijing Suolaibao Company) were extracted from clones with correct sequencing. It was transformed into the F6 mutant obtained in Example 2, and the specific primer sequences shown in Table 1 were used to screen and verify the complementary algal strains.

Embodiment 4, chlorophyll fluorescence assay

F0: initial fluorescence, initial fluorescence is the fluorescence output when the photosystem II (PSII) reaction center is fully open, and it is related to the concentration of chlorophyll in leaves. Fm: maximum fluorescence output (maximal fluorescence), which is the fluorescence output when the PSⅡ reaction center is completely closed, which can reflect the electron transfer through PSⅡ, and is usually measured after the leaves are dark-adapted for 20 minutes. Fv=Fm-F0: variable fluorescence, reflecting the reduction of QA. Fv/Fm: It is the maximum photochemical quantum yield of PSⅡ, which reflects the fluorescence energy conversion efficiency in the reaction center of PSⅡ, or the light energy conversion efficiency of the maximum PSⅡ.

In order to determine the relationship between the phenotype of the F6 mutant and the F6 gene, we measured the fluorescence parameter Fv/Fm values of the wild type, F6 mutant and complementary strains (comp16, comp20, comp26). When performing fluorescence measurement, Chlamydomonas was first scratched from the plate, inoculated into a 50ml Erlenmeyer flask and cultured to the exponential growth phase, then transferred to a 250ml Erlenmeyer flask containing 100ml of TAP medium, and the initial OD750 was adjusted to 0.05. Afterwards, it was placed on a shaker in a constant temperature light incubator for continuous light cultivation. After growing to the exponential growth phase, the chlorophyll concentration was adjusted to 12ug/ml. After 20 minutes of dark adaptation, the Fv/Fm value was measured by IMAGING-PAM.

As can be seen from Figure 1, under normal conditions, the Fv/Fm value of the wild type is 0.74, while the Fv/Fm value of the F6 mutant is 0.44, which is only about 60% of the wild type, which shows that the mutant Photosystem II was affected. In a series of complementary algal strains screened, the Fv/Fm values were restored to varying degrees (85%-95%), and the results of Western blot and PCR detection also proved that the expression of F6 gene in the complementary algal strains was comparable to that in the wild The phenotypes are similar (Figure 2, Figure 3), which shows that the F6 gene is related to the photosynthetic phenotype of F6, and the F6 gene has an important function in photosynthesis, and the deletion of this gene will seriously affect photosynthesis.

Claims (9)

1.F6 protein is following protein a) or b):

A) protein be made up of the aminoacid sequence shown in sequence in sequence table 2;

B) by the aminoacid sequence shown in sequence in sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and the protein relevant to plant photosynthesis efficiency.

2. the biomaterial relevant to protein described in claim 1 is following B1) to B5) in any one:

B1) nucleic acid molecule of protein described in coding claim 1;

B2) containing B1) expression cassette of described nucleic acid molecule;

B3) containing B1) recombinant vectors of described nucleic acid molecule or containing B2) recombinant vectors of described expression cassette;

B4) containing B1) recombinant plasmid of described nucleic acid molecule or containing B2) recombinant plasmid of described expression cassette;

B5) containing B1) the restructuring algae strain of described nucleic acid molecule or containing B2) the restructuring algae strain of described expression cassette or containing B3) the restructuring algae strain of described recombinant vectors.

3. relevant biological material according to claim 2, is characterized in that: B1) described in nucleic acid molecule as shown in the 1-435 position nucleic acid molecule of sequence in sequence table 1.

4. relevant biological material described in protein described in claim 1 or Claims 2 or 3 is improving the application in plant photosynthesis efficiency.

5. application according to claim 4, is characterized in that: described plant is algae, is specially Lay mattress green alga.

6. build a method for the transgenic plant that photosynthetic capacity improves, comprise the steps: the encoding gene of protein described in claim 1 to import to set out in plant, obtain transgenic plant; Transgenic plant are compared with the plant that sets out, and photosynthetic capacity improves.

7. method according to claim 6, is characterized in that: the encoding gene of described protein is the DNA molecular of the 1-435 position Nucleotide of sequence 1 in sequence table.

8. the method according to claim 6 or 7, is characterized in that: described photosynthetic capacity rises to photosynthetic efficiency and improves.

9., according to the arbitrary described method of claim 6-8, it is characterized in that: described plant is algae, is specially Chlamydomonas reinhardtii.