Application of GF14A gene in regulation and control of light protection capability of rice

Abstract

The invention relates to application of a GF14A gene in regulating and controlling the light protection capability of rice. By investigating the NPQ curve and field agronomic traits of a GF14A transgenic material under the condition of converting strong light into dark, the GF14A can accelerate the NPQ relaxation speed in dark, which indicates that the GF14A over-expressed rice can reduce non-photochemical quenching under weak light, optimizes the light protection regulation ability of the plant, and improves the yield of the rice. The effective panicle number, the weight, the number and the primary branch number of GF14A over-expressed rice are all higher than those of a wild type, and a molecular biology experiment shows that the gene optimizes the light protection regulation and control capability of the rice by influencing the transcriptional activation activity of an NPQ related transcription factor, which indicates that the gene has an important breeding value in high-photosynthetic-efficiency breeding. Meanwhile, the method has a wide application prospect in production.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of GF14A gene in regulating the light protection ability of rice. Background Art

[0002] Photosynthesis is the basis of all life activities of crops and the main source of crop yield. Most of the light energy absorbed by crops is used for photosynthesis, but it is still consumed in the form of heat dissipation and chlorophyll fluorescence. The amount of dissipation through chlorophyll fluorescence is very small and can be mostly ignored. When the light is too strong, the plant absorbs excess light energy, which will cause photodamage and poison the cells. At this time, the excess light energy must be dissipated through the non-photochemical quenching (NPQ) process. This is the photoprotection mechanism evolved by plants to consume excessive light energy in the form of heat dissipation when faced with excessive light absorption. Therefore, NPQ is an important indicator reflecting the photoprotection mechanism. By optimizing the NPQ process, the plant can be protected from light inhibition to the maximum extent, and the utilization efficiency of absorbed light energy can be increased, thereby increasing the biomass and yield of crops. Summary of the invention

[0003] The present invention found through yeast two-hybrid screening library experiments that non-photochemical quenching (NPQ)-related transcription factor OsBZIP72 interacts with GF14A protein, and further verified the interaction between GF14A and OsbZIP72 through molecular biological experiments, that is, GF14A negatively regulates the transcriptional activity of OsbZIP72. In addition, the present invention also comprehensively investigated the fluorescence induction curve and field agronomic traits of GF14A transgenic materials, and further explored the application value of GF14A transgenic materials in high-light-efficiency breeding.

[0004] The present invention provides the use of GF14A gene in any of the following:

[0005] A1) Regulate the photoprotection ability of plants;

[0006] A2) preparing products for regulating plant photoprotection ability;

[0007] A3) Cultivating plant varieties with high light protection regulation ability and / or high yield;

[0008] A4) Preparation and cultivation of high light protection regulation ability and / or high yield plant variety products.

[0009] Furthermore, the plant includes rice, the nucleotide sequence of the GF14A gene is shown as SEQ ID NO:1, the CDS sequence is shown as SEQ ID NO:2, and the protein sequence is shown as SEQ ID NO:3.

[0010] Furthermore, the high light protection regulation ability and / or high yield plant variety exhibits all or part of the following:

[0011] B1) Increased photosynthetic efficiency and enhanced photoprotection regulation ability;

[0012] B2) The effective ears, grain weight, number of filled grains and number of primary branches increased significantly.

[0013] Furthermore, the GF14A gene regulates the photoprotection ability of plants by negatively regulating the transcriptional activity of OsbZIP72.

[0014] The present invention also provides a method for increasing the light protection regulation ability and / or yield of a plant, by overexpressing the GF14A gene in the plant, wherein the nucleotide sequence of the GF14A gene is shown in SEQ ID NO: 1, the CDS sequence is shown in SEQ ID NO: 2, and the protein sequence is shown in SEQ ID NO: 3.

[0015] Further, the following steps are included:

[0016] (1) Construction of GF14A gene overexpression vector;

[0017] (2) introducing the recombinant vector of step (1) into rice callus through Agrobacterium-mediated method, and obtaining transgenic plants after culturing;

[0018] (3) Positive transgenic plants were obtained by screening using hygromycin markers, and positive plants overexpressing GF14A were further obtained using the RT-PCR method.

[0019] The present invention also provides a method for cultivating rice varieties with high light protection regulation ability and / or high yield, comprising the following steps:

[0020] (1) Construction of GF14A gene overexpression vector;

[0021] (2) introducing the recombinant vector of step (1) into rice callus through Agrobacterium-mediated method, and obtaining transgenic plants after culturing;

[0022] (3) Positive transgenic plants were obtained by marker screening, and positive plants overexpressing GF14A were further obtained by RT-PCR.

[0023] Furthermore, the nucleotide sequence of the GF14A gene is shown in SEQ ID NO: 1, the CDS sequence is shown in SEQ ID NO: 2, and the protein sequence is shown in SEQ ID NO: 3.

[0024] Furthermore, the obtained rice variety exhibits all or part of the following:

[0025] B1) Increased photosynthetic efficiency and enhanced photoprotection regulation ability;

[0026] B2) The effective ears, grain weight, number of filled grains and number of primary branches increased significantly.

[0027] The present invention examines the NPQ curve and field agronomic traits of GF14A transgenic materials under strong light-to-dark conditions, and finds that GF14A can accelerate the NPQ relaxation rate in the dark, thereby reducing non-photochemical quenching and enhancing the photoprotection regulation ability of the plant, and that the number of effective panicles, filled grain weight, filled grain number and primary branch number of GF14A overexpressing rice are all higher than those of the wild type, and that molecular biological experiments find that the gene improves the photoprotection regulation ability of rice by affecting the transcriptional activation activity of the NPQ-related transcription factor OsbZIP72, indicating that the gene has important breeding value in high light efficiency breeding, and at the same time has broad application prospects in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 :Yeast two-hybrid analysis demonstrated that GF14A interacts with OsbZIP72.

[0030] Figure 2 : BIFC and pull-down validate the interaction between OsbZIP72 and GF14A. (a) BIFC results, indicating that OsbZIP72 and GF14A interact in the nucleus; (b) GST pull-down results, proving that OsbZIP72 interacts with GF14A.

[0031] Figure 3 : GF14A inhibits the transcriptional activation ability of OsbZIP72. In the figure: The ratio of firefly luciferase to Renilla luciferase represents the relative activity of transcription factors. The values ​​in each column represent the average of three independent replicates, and the error bars represent the standard deviation.

[0032] Figure 4 : EMSA experiments showed that GF14A weakened the DNA binding activity of OsbZIP72 phosphorylated by SAPK1.

[0033] Figure 5:GF14A does not inhibit the transcriptional activation ability of exogenous fusion OsbZIP72. An exogenous GAL4 DNA binding domain was fused to the N-terminus of OsbZIP72 as an effector plasmid, and co-transformed into rice protoplasts with a firefly luciferase driven by a promoter consisting of five copies of the GAL4 binding element and a Renilla luciferase reporter gene driven by the Arabidopsis Ubiquitin promoter as an internal reference. It was found that the addition of 3ug of the plasmid expressing GF14A protein did not inhibit the transcriptional activity of OsbZIP72.

[0034] Figure 6 :Comparison of NPQ curves between GF14A overexpression materials and wild-type materials under strong light to dark conditions. In the figure: "*" indicates p value < 0.05 in t test, indicating significant difference. "**" indicates p value < 0.01 in t test, indicating extremely significant difference.

[0035] Figure 7 :(A) Number of effective ears. (B) Grain weight. (C) Number of filled grains. (D) Number of primary stalks. In the figure: Error bars represent SD, n=24, "*" indicates p value < 0.05 in t test, indicating significant difference. "**" indicates p value < 0.01 in t test, indicating extremely significant difference. DETAILED DESCRIPTION

[0036] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0037] Example 1: Using molecular biological methods to confirm that GF14A regulates the activation activity of OsbZIP72

[0038] The present invention constructs the full-length cDNA of GF14A into pGBKT7 as a bait vector, and uses the full-length cDNA of OsbZIP72 as a prey vector. The interaction between GF14A and OsbZIP72 is confirmed by yeast two-hybridization point-to-point verification ( Figure 1). The specific process is as follows: Matchmaker Gold yeast two-hybrid system (Clontech) was used to verify the interaction of the two proteins in yeast. The cDNA sequences of the two proteins required for the experiment were constructed into pGBKT7 containing BD and pGADT7 containing AD, respectively, and verified by sequencing to be fused with GAL4-BD and GAL4-AD, respectively. The constructed plasmids were co-transformed into Y2H gold yeast (Saccharomyces cerevisiae) using PEG / LiAc, and the pGBKT7-T vector was co-transformed with pGADT7-p53 or pGADT7-Lam as positive and negative controls, respectively. Single colonies expressing fusion proteins were selected on -Leu / -Trp plates, diluted in sterile water, and 12 μl of the dilution was spotted on SD / -Leu / -Trp / -His / -Ade / Xa-Gal / 200 ng ml-1AbA medium and grown at 28°C for 3 days. The growth conditions are used to determine whether two proteins interact in yeast.

[0039] Subsequently, the interaction between GF14A and OsbZIP72 was verified by in vivo and in vitro experiments. First, OsbZIP72 was cloned into the pVYNE(R) vector and GF14A was cloned into the pVYCE vector by BiFC (bimolecular fluorescence complementation) experiment. 5 μg of each vector was mixed to transform rice protoplasts. After 16 hours of dark culture, the fluorescence signal of each transformed protoplast was detected by confocal microscopy (Leica Microsystems, Wetzlar, Germany). The results showed that when GF14A-pVYCE and OsbZIP72-pVYNE(R) were co-transformed into rice protoplasts, yellow fluorescent protein (YFP) signal was observed in the nucleus ( Figure 2 a). GST pull down experiments revealed that OsbZIP72-His was pulled down by beads containing GF14A-GST, but not by GST alone, indicating that OsbZIP72 interacts with GF14A in vitro ( Figure 2b). The effect of GF14A on the transcriptional activity of OsbZIP72 was then studied using a dual-luciferase activity analysis experiment in rice protoplasts. 13-day-old Zhonghua No. 11 seedlings were used to prepare rice protoplasts for protoplast transformation. The constructed effect plasmid, reporter plasmid, and internal reference plasmid carrying Renilla luciferase (rLUC) were co-transfected into rice protoplasts, and the ratio of effect plasmid, reporter plasmid, and internal reference plasmid was 6:6:1. The co-transfected protoplast cells were cultured at 24°C in the dark for 12 h, and then the luciferase activity was determined using the dual-luciferase reporter gene assay system (Promega, Madison, WI, USA). The results showed that the transcriptional activity of OsbZIP72 was inhibited by GF14A, and this inhibitory effect became more severe with the increase in the amount of GF14A ( Figure 3 ), indicating that GF14A negatively regulates the transcriptional activity of OsbZIP72.

[0040] In order to study the specific inhibitory effect of GF14A on OsbZIP72, we still studied it from the two aspects of DNA binding activity and transcriptional activation activity. For DNA binding activity, the relevant protein was first expressed in prokaryotes. Subsequently, the single-stranded DNA labeled with FAM was synthesized and annealed into double strands, and the binding and competition reactions were carried out using the EMSA / Gel-Shift kit (Beyotime, Shanghai, China). The FAM-labeled signal was captured by the FLA-5100 imaging system (Fuji, Aichi, Japan). We used OsbZIP72 phosphorylated by SAPK1 and non-phosphorylated proteins in the EMSA experiment. The results showed that GF14A can weaken the DNA binding activity of OsbZIP72 protein phosphorylated by SAPK1 without affecting the non-phosphorylated protein ( Figure 4 ). In addition, regarding transcriptional activation activity, we found that GF14A could not alter the transcriptional activation activity of OsbZIP72 protein fused to the exogenous GAL4 DNA binding domain ( Figure 5 ), that is to say, when we artificially add a DNA binding domain to OsbZIP72, its transcriptional activity will not be negatively regulated by GF14A.

[0041] In summary, we conclude that GF14A does not directly affect the transcriptional activation activity of OsbZIP72, but negatively regulates the transcriptional activity of OsbZIP72 by weakening its DNA binding activity, and it negatively regulates OsbZIP72 phosphorylated by SAPK1.

[0042] Example 2: Using transgenic materials to confirm the regulatory effect of GF14A gene on rice photoprotection ability

[0043] GF14A-OX is a transgenic material that overexpresses GF14A cultivated in our laboratory. According to the gene information of GF14A in the Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu / ) of the rice Nipponbare database, pU1301-GF14A-F and pU1301-GF14A-R were used to amplify the full-length cDNA, and the fragments were homologously recombined into the overexpression vector pU1301 using ABclonal's 2X seamless cloning premix (Cat. No.: RK21020). After sequencing confirmation, genetic transformation was performed using the Agrobacterium-mediated method. Among them,

[0044] pU1301-GF14A-F:

[0045] (The underlined part is the Kpn1 linker, and the bold part is the Kpn1 sequence);

[0046] pU1301-GF14A-R:

[0047] (The underlined part is the BamH1 linker, and the bold part is the BamH1 sequence).

[0048] gf14a-cri is the GF14A knockout transgenic material cultivated in our laboratory. The knockout target site of the target gene was selected by referring to the CRISPR-design website (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR) developed by the Bioinformatics Center of Huazhong Agricultural University, and the targeting primers were designed according to the vector system of Professor Liu Yaoguang's laboratory at South China Agricultural University.

[0049] GF14A-cri-U3F(ggcaAGAGAGAGAGACGATGGCGG)

[0050] GF14A-cri-U3R(aaacCCGCCATCGTCTCTCTCCT);

[0051] GF14A-cri-U6aF(gccgGAGCAGAAGGAGGAAGGCCG)

[0052] GF14A-cri-U6aR(aaacCGGCCTTCCTCCTTCTGCTC)

[0053] The targets were assembled into the expression vector pYLCRISPR / Cas9-MH in the form of single gene and dual targets, and used for genetic transformation after sequencing confirmation. The transformation receptor was selected from japonica rice Nipponbare. GF14A-WT is the wild-type Nipponbare material.

[0054] The present invention measures the chlorophyll fluorescence parameters of GF14A transgenic rice. The specific steps are:

[0055] (1) Place the rice leaves in 50 mL of 0.1‰ agarose solution. Repeat this process three times for each material. Bring the leaves back to the laboratory and place them in a dark room overnight.

[0056] (2) Install the instrument, connect it to the computer, open the DUAL-PAM application, select the program in Open User Settings on the left, click Settings, select Mode, select Fluo+P700, select SP Analysis in Analysis, click the balance icon in Meas.light, wait for 10 minutes, and preheat the P700 measurement light source so that the P700 value is stable between 0 and 0.2. If it is unstable, click the small triangle to manually adjust it to between 0 and 0.2.

[0057] (3) In Actinic.Light, select 14-stop actinic light parameters.

[0058] (4) In Slow.Kin, adjust the induction time to 360s at AL-width and the relaxation time to 300s at Recovery Times.

[0059] (5) Click the horizontal main menu in the lower left corner and select Slow.Kin. Change Manual to Ind+Rec in the box above Start. Click Calib to calibrate. Check whether the yellow line in the figure is vertical. If it is vertical, you can clamp the blade. Open the green button of Bal. in the lower left corner to turn it on. Confirm or open the PAL button to see if it is red. Confirm that Fluo on the right is 0 and P700 is still between 0-0.2. If it is not within the range, click the triangle to adjust manually. Click start. If a prompt box appears, select No to start the measurement.

[0060] (6) During the measurement, record the value of Fv / Fm (reflects leaf activity, generally > 0.78). At the end of the measurement, record the NPQ value.

[0061] (7) Replace the blade, clamp it, click Bal., check the voltage, click Start, if a prompt box appears, select No and continue measuring.

[0062] (8) After measuring all the leaves, click report, click Export Report to csv File, export and analyze the data.

[0063] The results are as follows Figure 6 As shown, 0-360s is the NPQ induction stage under strong light, and 360s-720s is the dark relaxation stage. At 520s, the NPQ value of GF14A-OX was 0.963±0.07, and the NPQ value of GF14A-WT was 1.064±0.09, and there was a significant difference between the two. And between 520s-720s, the NPQ value of GF14A-OX was significantly lower than that of GF14A-WT. The phenotypic results of the above transgenic materials show that the GF14A gene can accelerate the NPQ relaxation rate in the dark, thereby promoting the light protection of rice.

[0064] Example 3: Field performance of agronomic traits of GF14A transgenic material

[0065] The present invention investigates the mature plants of GF14A transgenic materials, puts the harvested mature rice into a net bag, and airs it for 15 days, weighs the dry weight as the plant biomass, and measures the total number of panicles, effective panicles, total number of grains, full grain weight, full grain number, empty grain number, 100-grain weight, panicle length, primary stalk number, and secondary stalk number. Figure 7 As shown in the figure, the effective ear number of GF14A-WT was 7.04-18.64, while that of GF14A-OX increased to 12.92-25.80, which was a very significant increase of 50.77% (P=0.00013729) compared with GF14A-WT; in terms of filled grain weight, the filled grain weight of GF14A-WT was 5.71-11.26, while that of GF14A-OX increased to 11.95-19.39, which was a very significant increase of 84.63% (P=1.15755E-11) compared with GF14A-WT, while the filled grain weight of gf14a-cri decreased by 27.1% compared with GF14A-WT. 3% (P=0.05931012), and the filled grain weight ranged from 4.40 to 8.95. In terms of the filled grain number, GF14A-OX also had a very significant increase, reaching 642.48-335.45, while the filled grain number of GF14A-WT was 261.46-515.83, and GF14A-OX was significantly increased by 25.81% compared with GF14A-WT (P=0.00812181), while the filled grain number of gf14a-cri decreased to 218.50-387.86, which was significantly decreased by 28.19% compared with GF14A-WT (P=0.04557789).

[0066] Judging from the overall results, the agronomic traits of GF14A-OX are better than those of GF14A-WT, indicating that GF14A can positively regulate rice yield traits and has great potential for utilization in production.

[0067] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. Use of GF14A gene in any of the following: A1) Regulate the photoprotection ability of plants; A2) preparing products for regulating plant photoprotection ability; A3) Breeding plant varieties with high light protection ability and / or high yield; A4) Preparation and cultivation of plant variety products with optimized light protection regulation ability and / or high yield.

2. The use according to claim 1, characterized in that: The plant includes rice, and the nucleotide sequence of the GF14A gene is shown as SEQ ID NO:

1.

3. The use according to any one of claims 1-2, characterized in that: The photoprotection regulation ability optimized and / or high-yield plant varieties exhibit all or part of the following: B1) Increased photosynthetic efficiency and optimized photoprotection regulation ability; B2) The effective ears, grain weight, number of filled grains and number of primary branches increased significantly.

4. The use according to claim 1, characterized in that: The GF14A gene regulates the photoprotection ability of plants by negatively regulating the transcriptional activity of OsbZIP72.

5. A method for increasing plant light protection regulation ability and / or yield, characterized in that: The GF14A gene is overexpressed in the plant, and the nucleotide sequence of the GF14A gene is shown in SEQ ID NO:

1.

6. The method according to claim 5, characterized in that The following steps are involved: (1) Construction of GF14A gene overexpression vector; (2) introducing the recombinant vector of step (1) into rice callus through Agrobacterium-mediated method, and obtaining transgenic plants after culturing; (3) Positive transgenic plants were obtained by screening using hygromycin markers, and positive plants overexpressing GF14A were further obtained using the RT-PCR method.

7. A method for cultivating rice varieties with high light protection regulation ability and / or high yield, characterized in that: The following steps are involved: (1) Construction of GF14A gene overexpression vector; (2) introducing the recombinant vector of step (1) into rice callus through Agrobacterium-mediated method, and obtaining transgenic plants after culturing; (3) Positive transgenic plants were obtained by marker screening, and positive plants overexpressing GF14A were further obtained by RT-PCR.

8. The method according to claim 7, characterized in that The nucleotide sequence of the GF14A gene is shown in SEQ ID NO:

1.

9. The method according to any one of claims 7-8, characterized in that: The resulting rice varieties exhibit all or part of the following: B1) Increased photosynthetic efficiency and enhanced photoprotection regulation ability; B2) The effective ears, grain weight, number of filled grains and number of primary branches increased significantly.

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