Application of gf14a gene in regulating light protection ability of rice

Abstract

The application relates to application of a GF14A gene in regulating light protection capability of rice. By investigating NPQ curves and field agronomic characters of GF14A transgenic materials under strong light and dark conditions, it is found that GF14A can accelerate the NPQ relaxation speed in the dark, indicating that overexpression of the GF14A rice under weak light can reduce non-photochemical quenching, optimize the light protection regulation capability of the plant, and the effective ear number, filled grain weight, filled grain number and primary branch number of the overexpression GF14A rice are higher than those of the wild type. Through a molecular biology experiment, it is found that the gene optimizes the light protection regulation capability of the rice by affecting the transcriptional activation activity of an NPQ related transcription factor, indicating that the gene has important breeding value in high light efficiency breeding, and has a wide application prospect in production.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the application of the GF14A gene in regulating the photosynthetic capacity of rice. Background Technology

[0002] Photosynthesis is the foundation of all life activities in crops and the main source of crop yield. Most of the light energy absorbed by crops is used for photosynthesis, but some is still consumed through heat dissipation and chlorophyll fluorescence. The amount dissipated through chlorophyll fluorescence is very small and largely negligible. When light is too intense, plants absorb excess light energy, causing photodamage and toxicity to cells. In this situation, they must dissipate the excess light energy through non-photochemical quenching (NPQ) processes. This is the photoprotective mechanism that plants have evolved to dissipate excess light energy through heat dissipation in the face of excessive light absorption. Therefore, NPQ is an important indicator of photoprotective mechanisms. Optimizing the NPQ process can maximize the protection of plants from photoinhibition, increase the efficiency of light energy utilization, and thus improve crop biomass and yield. Summary of the Invention

[0003] This invention, through yeast two-hybrid library screening experiments, discovered that the non-photochemical quenching (NPQ)-related transcription factor OsBZIP72 interacts with the GF14A protein. Molecular biology experiments further verified the interaction between GF14A and OsbZIP72, indicating that GF14A negatively regulates the transcriptional activity of OsbZIP72. Furthermore, this invention comprehensively examined the fluorescence induction curves and field agronomic traits of GF14A transgenic materials, further exploring the application value of GF14A transgenic materials in high-light-efficiency breeding.

[0004] This invention provides the application of the GF14A gene in any of the following:

[0005] A1) Regulates the plant's photoprotective capacity;

[0006] A2) Prepare products that regulate the photoprotective capacity of plants;

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

[0008] A4) Prepare and cultivate plant varieties with high light protection and / or high yield.

[0009] Furthermore, the plant includes rice, and 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.

[0010] Furthermore, the high light protection regulation capacity and / or high-yielding plant varieties exhibit all or some of the following:

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

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

[0013] Furthermore, the GF14A gene regulates plant photosensitivity by negatively regulating the transcriptional activity of OsbZIP72.

[0014] The present invention also provides a method for increasing the photosynthetic regulation capacity and / or yield of plants by overexpressing the GF14A gene in plants, 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] Furthermore, it includes the following steps:

[0016] (1) Construct a GF14A gene overexpression vector;

[0017] (2) The recombinant vector from step (1) was introduced into rice callus tissue by Agrobacterium-mediated transformation and transgenic plants were obtained after culture.

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

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

[0020] (1) Construct a GF14A gene overexpression vector;

[0021] (2) The recombinant vector from step (1) was introduced into rice callus tissue by Agrobacterium-mediated transformation and transgenic plants were obtained after culture.

[0022] (3) Positive transgenic plants were obtained by marker screening, and positive plants overexpressing GF14A were 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 resulting rice varieties exhibit all or some of the following characteristics:

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

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

[0027] This invention examines the NPQ curves and field agronomic traits of GF14A transgenic materials under strong light-to-dark conditions. It finds that GF14A accelerates NPQ relaxation in darkness, thereby reducing non-photochemical quenching and enhancing the plant's photosensitivity regulation. Furthermore, rice with GF14A overexpression exhibits higher effective panicle number, grain weight, grain number, and primary branch number than the wild type. Molecular biology experiments reveal that this gene enhances rice photosensitivity regulation by influencing the transcriptional activation activity of the NPQ-related transcription factor OsbZIP72. This indicates that the gene has significant breeding value in high-light-efficiency breeding and broad application prospects in agricultural production. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 Yeast two-hybrid assays demonstrated the interaction between GF14A and OsbZIP72.

[0030] Figure 2 : BIFC and pull-down assays confirmed the interaction between OsbZIP72 and GF14A. (a) BIFC results showed that OsbZIP72 and GF14A interact in the cell nucleus; (b) GST pull-down results confirmed the interaction between OsbZIP72 and GF14A.

[0031] Figure 3 GF14A inhibits the transcriptional activation of OsbZIP72. In the figure, the ratio of firefly luciferase to kidney luciferase represents the relative activity of transcription factors. Values ​​in each column represent the mean of three independent replicates, and 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 5GF14A 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 rice protoplasts with a firefly luciferase reporter gene driven by a promoter consisting of five copies of GAL4 binding elements and a Renilla luciferase reporter gene driven by an Arabidopsis thaliana Ubiquitin promoter. It was found that adding 3 μg of plasmid expressing GF14A protein did not inhibit the transcriptional activity of OsbZIP72.

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

[0035] Figure 7 (A) Number of effective ears. (B) Grain weight. (C) Number of filled grains. (D) Number of primary branches. In the figure: the error bar represents SD, n=24, "*" indicates that the p value in the t-test is <0.05, indicating a significant difference. "**" indicates that the p value in the t-test is <0.01, indicating an extremely significant difference. Detailed Implementation

[0036] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0037] Example 1: Molecular biology methods were used to confirm the activation activity of OsbZIP72 regulated by GF14A.

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

[0039] The interaction between GF14A and OsbZIP72 was then verified through in vivo and in vitro experiments. First, using a BiFC (bimolecular fluorescence complementation) assay, OsbZIP72 was cloned into the pVYNE(R) vector, and GF14A was cloned into the pVYCE vector. 5 μg of each vector was mixed and transformed into rice protoplasts. After 16 h of dark incubation, the fluorescence signal of each transformed protoplast was detected using confocal microscopy (Leica Microsystems, Wetzlar, Germany). The results showed that when GF14A-pVYCE and OsbZIP72-pVYNE(R) were co-transformed into rice protoplasts, a yellow fluorescent protein (YFP) signal was observed in the cell nucleus. Figure 2 a). The GST pull-down experiment showed 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 investigated using a dual-luciferase activity assay with rice protoplasts. Rice protoplasts were prepared using 13-day-old Zhonghua 11 seedlings and transformed. The constructed effect plasmid, reporter plasmid, and internal control plasmid containing Renilla luciferase (rLUC) were co-transfected into rice protoplasts in a ratio of 6:6:1. The co-transfected protoplast cells were cultured in the dark at 24°C for 12 h, and luciferase activity was measured using a dual-luciferase reporter gene detection system (Promega, Madison, WI, USA). The results showed that the transcriptional activity of OsbZIP72 was inhibited by GF14A, and this inhibition became more severe with increasing GF14A levels. Figure 3 This indicates that GF14A negatively regulates the transcriptional activity of OsbZIP72.

[0040] To investigate the specific inhibitory effect of GF14A on OsbZIP72, we examined both DNA-binding and transcriptional activation activities. For DNA-binding activity, the relevant protein was first expressed in prokaryotes. Subsequently, FAM-labeled single-stranded DNA was synthesized and annealed to double strands, and binding and competitive reactions were performed using an EMSA / Gel-Shift kit (Beyotime, Shanghai, China). The FAM-labeled signal was captured using an FLA-5100 imaging system (Fuji, Aichi, Japan). We used SAPK1-phosphorylated OsbZIP72 and the unphosphorylated protein in our EMSA experiments. The results showed that GF14A attenuated the DNA-binding activity of SAPK1-phosphorylated OsbZIP72 protein without affecting the unphosphorylated protein. Figure 4 Furthermore, regarding transcriptional activation activity, we found that GF14A could not alter the transcriptional activation activity of the OsbZIP72 protein fused with the exogenous GAL4 DNA binding domain. Figure 5 In other words, when we artificially add a DNA-binding domain to OsbZIP72, its transcriptional activity will not be negatively regulated by GF14A.

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

[0042] Example 2: Demonstrating the regulatory effect of the GF14A gene on the photosynthetic capacity of rice using transgenic materials.

[0043] GF14A-OX is a transgenic material overexpressing GF14A developed in our laboratory. Based on the GF14A gene information in the Rice Genome Annotation Project database (http: / / rice.plantbiology.msu.edu / ), full-length cDNA was amplified using pU1301-GF14A-F and pU1301-GF14A-R. Homologous recombination of the fragment was performed into the overexpression vector pU1301 using ABclonal's 2X seamless cloning premix (catalog number: RK21020). After confirmation by sequencing, genetic transformation was carried out using Agrobacterium-mediated transformation.

[0044] pU1301-GF14A-F:

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

[0046] pU1301-GF14A-R:

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

[0048] gf14a-cri is a GF14A knockout transgenic material cultivated in our laboratory. The target gene knockout site was selected using the CRISPR-design website developed by the Bioinformatics Center of Huazhong Agricultural University (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR), and targeting primers were designed based on the vector system from 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 target was assembled into the expression vector pYLCRISPR / Cas9-MH in a single-gene, dual-target configuration. After sequencing confirmation, it was used for genetic transformation. The recipient rice variety was Nipponbare japonica. GF14A-WT is a wild-type Nipponbare material.

[0054] This invention measured the chlorophyll fluorescence parameters of the GF14A transgenic rice line. The specific steps are as follows:

[0055] (1) Place the rice leaves in 50 mL of 0.1‰ agarose solution, repeat 3 times for each material, bring them back to the laboratory, place the leaves in a dark room, and treat them in the dark 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 10 minutes to warm up 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 levels of photoluminescence parameters.

[0058] (4) In Slow.Kin, adjust the induction time to 360s at AL-width. Adjust 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 if the yellow line segment in the figure is vertical. If it is vertical, you can clamp the blade. Turn on Bal. in the lower left corner. If the green button appears, it is in the on state. Confirm or turn on 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) Record the Fv / Fm value (which reflects leaf activity and should generally be >0.78) during measurement. Record the NPQ value at the end of the measurement.

[0061] (7) Change the blade, clamp it, click Bal., check the voltage, click start. If a prompt box appears, select No to continue the measurement.

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

[0063] The results are as follows Figure 6 As shown, 0-360s represents the NPQ induction phase under strong light, and 360s-720s represents the dark relaxation phase. At 520s, the NPQ value of GF14A-OX was 0.963±0.07, while that of GF14A-WT was 1.064±0.09, with a significant difference between the two. Furthermore, between 520s and 720s, the NPQ value of GF14A-OX was significantly lower than that of GF14A-WT. These phenotypic results of the transgenic materials indicate that the GF14A gene can accelerate the NPQ relaxation rate in darkness, thereby promoting photosensitivity in rice.

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

[0065] This invention investigates mature plants of the GF14A transgenic material. Mature rice was harvested, placed in a net bag, and dried for 15 days. The dry weight was measured as plant biomass. Measurements included total panicle number, effective panicle number, total grain number, filled grain weight, filled grain number, empty grain number, 100-grain weight, panicle length, number of primary branches, and number of secondary branches. Figure 7 As shown, the number of effective ears in GF14A-WT ranged from 7.04 to 18.64, while the number of effective ears in GF14A-OX increased to 12.92 to 25.80, a highly significant increase of 50.77% compared to GF14A-WT (P = 0.00013729). Regarding kernel weight, the kernel weight of GF14A-WT ranged from 5.71 to 11.26, while the kernel weight of GF14A-OX increased to 11.95 to 19.39, a highly significant increase of 84.63% compared to GF14A-WT (P = 1.15755E-11), while the kernel weight of GF14A-CRI decreased by 27.1% compared to GF14A-WT. The percentage of positive granules was 3% (P = 0.05931012), with the particle weight ranging from 4.40 to 8.95. In terms of particle count, GF14A-OX also showed a highly significant increase, reaching 642.48-335.45, while the particle count of GF14A-WT was 261.46-515.83. GF14A-OX significantly increased by 25.81% compared to GF14A-WT (P = 0.00812181), while the particle count of gf14a-cri decreased to 218.50-387.86, a significant decrease of 28.19% compared to GF14A-WT (P = 0.04557789).

[0066] Overall, the agronomic traits of GF14A-OX were 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 detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. GF14A The application of genes in any of the following: A1) Cultivating high-yielding plant varieties; A2) Preparation and cultivation of high-yield plant varieties; By overexpressing in plants GF14A Genes to increase plant yield; the plant is rice, and the... GF14A The nucleotide sequence of the gene is shown in SEQ ID NO:1; The GF14A Genes through negative regulation OsbZIP72 The transcriptional activity was enhanced, thereby increasing plant yield; the high-yielding plant varieties exhibited a significant increase in effective panicles, grain weight, number of grains, and number of primary branches.

2. The application according to claim 1, characterized in that, The high-yielding plant varieties also exhibited increased photosynthetic efficiency and optimized photosynthetic regulation capabilities.

3. A method for increasing plant yield, characterized in that, Overexpression in plants GF14A Gene, the plant is rice, the GF14A The nucleotide sequence of the gene is shown in SEQ ID NO: 1; GF14A Genes through negative regulation OsbZIP72 It enhances transcriptional activity and increases plant yield; the yield increase is manifested by a significant increase in effective panicles, grain weight, number of grains, and number of primary branches.

4. The method according to claim 3, characterized in that, Includes the following steps: (1) Construction GF14A Gene overexpression vectors; (2) The recombinant vector from step (1) was introduced into rice callus tissue by Agrobacterium-mediated transformation and transgenic plants were obtained after culture. (3) Positive transgenic plants were obtained by screening with hygromycin markers, and overexpression was further obtained by RT-PCR. GF14A Positive plants.

5. A method for cultivating high-yield rice varieties, characterized in that, Includes the following steps: (1) Construction GF14A Gene overexpression vectors; (2) The recombinant vector from step (1) was introduced into rice callus tissue by Agrobacterium-mediated transformation and transgenic plants were obtained after culture. (3) Positive transgenic plants were obtained by marker screening, and overexpression was further obtained by RT-PCR. GF14A Positive plants; The above GF14A The nucleotide sequence of the gene is shown in SEQ ID NO:

1. GF14A Genes through negative regulation OsbZIP72 The transcriptional activity is increased to improve yield and thus obtain high-yielding rice varieties; the high-yielding rice varieties are characterized by a significant increase in effective panicles, grain weight, number of grains and number of primary branches.

6. The method according to claim 5, characterized in that, The high-yielding rice varieties obtained also showed increased photosynthetic efficiency and enhanced photosynthetic regulation capabilities.

Images