Application of rice OsGLTF gene or its encoded protein in improving rice yield

Abstract

The present invention belongs to the technical field of rice genetic engineering, and specifically relates to the application of rice OsGLTF gene or the protein encoded by it in improving rice yield. The present invention discloses the application of rice OsGLTF gene or the protein encoded by it in improving rice yield, and relates to the technical field of bioengineering. The present invention increases the grain length and weight of transgenic rice and enlarges the panicle shape by overexpressing the OsGLTF gene in rice; while the grain length, thickness and grain weight of osgltf knockout mutant rice are reduced, indicating that the OsGLTF gene can regulate rice grain shape and yield, and has the potential for production application.

Description

Technical Field

[0001] The invention belongs to the technical field of rice genetic engineering, and in particular relates to the application of rice OsGLTF gene or protein encoded by the gene in improving rice yield. Background Art

[0002] The rice yield potential is directly determined by the number of panicles, the number of filled grains per panicle, and the thousand-grain weight. In addition, traits such as heading period, plant height, and plant type indirectly affect rice yield.

[0003] GATA transcription factors can specifically recognize and bind to the (T / A)GATA(G / A) sequence on the promoter of the target gene, so they are named GATA transcription factors (Arguello-Astorga and Herrera-Estrella 1998). A typical GATA transcription factor usually contains a special class IV zinc finger structure (C-X2-C-X17-20-C-X2-C). The first GATA transcription factor identified in plants is TNL1 of tobacco (Danielvedele and Caboche 1993). At present, several genes encoding GATA transcription factors have been cloned in rice. Among them, a base misplacement mutation on SNFL1 (Short and narrow flag leaf1) has been reported to cause severe defects in the development of rice flag leaves, resulting in short flag leaves (He, Wang et al. 2018). The functional loss of NECK LEAF 1 (NL1) can cause delayed flowering and reduced panicle shape in rice, proving that NL1 is involved in the regulation of rice organ development (Wang, Yin et al. 2009). The expression of CYTOKININ-RESPONSIVE GATATRANSCRIPTION FACTOR1 (CGA1) is induced by light, nitrogen and cytokinin, and inhibited by darkness and gibberellin GA signals. Functional analysis experiments have shown that CGA1 is involved in multiple biological pathways such as rice filling, chloroplast formation and organ senescence (Hudson, Guevara et al. 2013). OsGATA7 has been reported to be involved in the regulation of rice plant height, leaf angle, panicle shape and grain shape, and OsGATA7 is involved in the regulation of plant growth mediated by brassinosteroid signals (Zhang, Zhang et al. 2018). OsGATA8 is located at the saltol QTL locus related to salt tolerance. Subsequent analysis showed that the expression of OsGATA8 can be significantly induced by salt, drought and ABA. Overexpression of OsGATA8 can significantly improve the biomass accumulation and photosynthetic efficiency of rice under normal conditions and salt stress (Nutan, Singla-Pareek et al. 2020). Overexpression of OsGATA12 can significantly increase the number of chloroplasts in rice leaves, delay plant senescence and increase yield (Lu, Casaretto et al. 2017). It can be seen from the above-mentioned cloned rice GATA transcription factors that GATA transcription factors are widely involved in various biological processes in rice, such as photosynthesis, organ development, and hormone signal transduction. However, there are few reports on the detailed involvement of GATA transcription factors in rice grain development and yield regulation, and there are no reports on the function of OsGLTF in regulating rice grain shape and yield, and the mechanism is not yet clear. Summary of the invention

[0004] In order to explore new uses of the OsGLTF gene, the present invention discloses the following technical solutions:

[0005] The present invention discloses the application of rice OsGLTF gene or OsGLTF protein in improving rice yield. The nucleotide sequence of the rice OsGLTF gene is shown as SEQ ID NO.1 or SEQ ID NO.2; the amino acid sequence of the OsGLTF protein is shown as SEQ ID NO.3, and is encoded by the rice OsGLTF gene.

[0006] The present invention also discloses the application of rice OsGLTF gene or OsGLTF protein in regulating at least one of rice plant height, grain length and grain weight. The nucleotide sequence of the rice OsGLTF gene is shown as SEQ ID NO.1 or SEQ ID NO.2; the amino acid sequence of the OsGLTF protein is shown as SEQ ID NO.3, which is encoded by the rice OsGLTF gene.

[0007] A method for increasing rice yield comprises over-expressing or over-expressing an OsGLTF gene in a rice crop, wherein the nucleotide sequence of the OsGLTF gene is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] A method for increasing rice yield comprises the following steps:

[0009] (1) connecting the OsGLTF gene to a plasmid to construct an overexpression or overexpression vector containing the OsGLTF gene;

[0010] (2) Introducing the overexpression or overexpression vector of the OsGLTF gene constructed in step (1) into rice cells, and obtaining transgenic plants after culturing.

[0011] Preferably, the plasmid vector in step (1) is a binary expression vector.

[0012] Preferably, in step (2), after the overexpression or over-expression vector containing the OsGLTF gene is transferred into the genetically engineered Agrobacterium, the vector is then infected into rice cells.

[0013] Preferably, the genetically engineered Agrobacterium is Agrobacterium EHA105 strain.

[0014] Preferably, the source of rice cells infected by the genetically engineered Agrobacterium in step (2) is callus tissue of rice seeds.

[0015] Beneficial effects of the present invention:

[0016] The present invention overexpresses or overexpresses the OsGLTF gene, and compared with wild-type plants, the grain length and 1000-grain weight of transgenic rice are increased; while when the OsGLTF gene is knocked out in rice, the grain length, grain width and 1000-grain weight of the rice are reduced, indicating that the OsGLTF gene can regulate rice grain shape and yield and has the potential for production application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Construction and identification of OsGLTF transgenic and knockout mutants. (A) Schematic diagram of knockout vector and overexpression vector; (B) Quantitative qPCR results of overexpression strains; (C) Sequencing of knockout vector.

[0018] Figure 2 Plant height phenotypes of OsGLTF transgenic materials, mutants and wild type, (A) is a photo of plant phenotypes, (B) is the statistical results of plant height. Among them, ** indicates p < 0.01, and * indicates p < 0.05.

[0019] Figure 3 The ear phenotypes of OsGLTF transgenic materials, mutants and wild types, (A) is the ear phenotype photo, (B) is the statistical result of ear length. Among them, ** indicates p < 0.01, and * indicates p < 0.05.

[0020] Figure 4 The grain shape and weight phenotypes of OsGLTF transgenic materials, mutants and wild type. (A) is a statistical photo of thousand-grain weight, (B) is a picture of grain length phenotype, (C) is a picture of grain thickness phenotype, and (D) is the statistical result of grain width phenotype. ** indicates p < 0.01, and * indicates p < 0.05. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1

[0023] 1. Construction of CRISPR / Cas9 knockout vector

[0024]

[0025] The primer sequences are as follows:

[0026] OsGLTF-Cas1F:gagtttgtgataattatagc

[0027] OsGLTF-Cas1R:gctataattatcacaaactc

[0028] OsGLTF-Cas2F:ctttagatctaaactatccg

[0029] OsGLTF-Cas2R:cggatagtttagatctaaag

[0030] 2. Construction of OsGLTF overexpression vector

[0031] The plant binary expression plasmid pCAMBIA1300-UBI was used to construct the OsGLTF overexpression vector. Primers OsGLTF-OX-F / R were designed, and the cDNA of Zhonghua 11 was used as a template to amplify the full-length CDS sequence of the OsGLTF gene (nucleotide sequence as SEQ ID Shown in NO.2: atggaacctgagtcctccctccacaaggttgttgttcctttagatctaaactatccgaggatttgttcccactgccaaaccagcaaaacatcggtatggcg caacggcccctttggccccaagtctttgtgcaatgcttgtggggatccgatatcacagaaagggaattgatgctcttgagttagaaggtaagaggagcaaagacaagaag aggaagacgagtagaaacgaggttccactccggaggggattgaggaaaaataataagaatgcaaaggaggtagattttggagtgaggatgatgatggagggatgccaat cagaaccgatgctaacacagggccaacaatacgaagatgatgtgaagaaggctgctatccaactaatgtacctttcaagggccgcaaacacttaa; the amino acid sequence of the encoded protein is such as SEQ IDNO.3: mepesslhkvvvpldlnypricshcqtsktsvwrngpfgpkslcnacgiryhrkgidalelegkrskdkkrktsrnevplrrglrknnknak evdfgvrmmmegcqsepmltqgqqyeddvkkaaiqlmylsraant). The pCAMBIA1300-UBI vector was double-digested with KpnI and BamHI at the same time, and the linearized fragment was obtained after recovery. The full-length CDS of OsGLTF and the linearized vector were connected with T4 ligase. Enzyme digestion and sequencing verified the correct overexpression vector: pCAMBIA1300-UBI-OsGLTF. pCAMBIA1300-UBI-OsGLTF was transformed into Agrobacterium tumefaciens EHA105 competent cells to obtain pCAMBIA1300-UBI-OsGLTF Agrobacterium bacterial solution.

[0032] The primer sequences are as follows:

[0033] OsGLTF-OX-F:gaggatccATGGAACCTGAGTCCTCCCTCC;

[0034] OsGLTF-OX-R:ttgggtaccAGTGTTTGCGGCCCTTGAA.

[0035] 3. Construction of OsGLTF overexpression strain by Agrobacterium transformation

[0036] In order to obtain knockout and overexpression materials of OsGLTF, the Agrobacterium-mediated genetic transformation method was used to transform the OsGLTF overexpression vector into the wild-type Zhonghua 11 variety, and the OsGLTF knockout vector into the wild-type Zhonghua 11 (ZH11) variety. The specific operation steps are as follows:

[0037] 1) Rice callus culture: Select mature and plump rice seeds, about 20 grams, and shell them. On the clean bench, first wash them twice with sterile water. Then, soak the seeds in 75% alcohol for 2 minutes, and then rinse them twice with sterile water again. Sterilize them after each operation. Then, soak the seeds in sodium hypochlorite disinfectant, seal them, and shake them in a shaker for 20 minutes. On the clean bench, rinse the seeds with sterile water until there is no foam. To ensure sterility, place the treated seeds on sterile filter paper and blow dry the surface moisture in the clean bench. Use sterilized tweezers to evenly spread the sterilized seeds on the N6B5 culture medium, make sure to do it on the clean bench, and place the culture dish in a dark room at 28°C for culture. To reduce the risk of seed contamination, be sure to avoid bacterial contamination of the culture medium. During the culture process, ensure that good sterile conditions are maintained, check and record the status of the culture medium, and pay attention to prevent bacterial contamination.

[0038] 2) Agrobacterium infection of callus: The plasmids containing knockout, overexpression and mCherry fusion tags were transferred into Agrobacterium respectively, and then the cultured bacterial solution was collected for centrifugation. The bacteria were resuspended with PHI suspension and the concentration was adjusted to OD600 between 0.08 and 0.1. Subsequently, acetosyringone (As) was added at a ratio of 0.2% (v / v). Next, callus tissue with good growth was collected, placed in a sterile culture bottle, and about 40 ml of pre-prepared PHI Agrobacterium suspension was added, and then allowed to stand at room temperature for 20 minutes, shaking every 5 minutes. Pour out the suspension, place the callus tissue on sterile paper, and dry it on a clean bench. Finally, the callus tissue was transferred to a culture dish with new sterile paper, sealed with a sealing film, and placed in a dark room environment at 19°C for 3 days.

[0039] 3) Screening of resistant callus: The co-cultured callus tissues were transferred to N6B5 screening medium containing 500 mg / L cefotaxime, 400 mg / L carbenicillin and 50 mg / L hygromycin. Most of the callus tissues turned brown about 10 days after screening. The browned callus tissues were removed and the resistant callus tissues were retained for subculture.

[0040] 4) Differentiation and rooting: The positive callus was transferred to a differentiation medium containing 50 mg / L hygromycin B and 250 mg / L carbenicillin on a sterile clean bench. Then, the seedlings growing about 2 cm on the differentiation medium were transferred to a rooting medium containing 50 mg / L hygromycin B and 200 mg / L carbenicillin.

[0041] 5) Seedling strengthening and transplanting: After about two weeks of cultivation, select seedlings about 10 cm tall with well-developed root systems, wash off the culture medium with warm water, and transplant them to the greenhouse.

[0042] The OsGLTF overexpression strain was tested by PCR using hygromycin primers (hygF / hygR). The results showed that among the 17 randomly selected transgenic plants, 8 were positive for hygromycin, which preliminarily indicated that the pCAMBIA1300-UBI-OsGLTF overexpression vector had been successfully transferred into rice plants ( Figure 1 ).

[0043] The primer sequences are as follows:

[0044] hygF: 5′-GTGCTTGACATTGGGGAGTT-3′;

[0045] hygR: 5′-GATGTTGGCGACCTCGTATT-3′.

[0046] Twenty-eight OsGLTF knockout transgenic lines were obtained by CRISPR / Cas9 technology. A total of 27 positive lines were detected by amplifying the hygromycin gene fragment (primers hygF / hygR). After multiple generations of self-pollination, the target fragment containing the target site was amplified by primers Cr-OsGLTF-F / R for knockout type detection. Two homozygous OsGLTF knockout lines were obtained after removing the transgenic background: one with a 4-base deletion (named cr-OsGLTF-4) and the other with a 77-base deletion (named cr-OsGLTF-77) ( Figure 1 ).

[0047] The detection primer sequences are as follows:

[0048] Cr-OsGLTF-F: 5′-ACACATAGAAACAAGAAAGCCAGGC-3′;

[0049] Cr-OsGLTF-R: 5′-CAAATCAACAAGCTCTGAGCAACA-3′.

[0050] Example 2

[0051] Analysis of rice planting and yield phenotypes of OsGLTF gene overexpression and mutant strains

[0052] The CRISPR-Cas9 knockout mutants used the two homozygous mutant strains (cr-OsGLTF-4 and cr-OsGLTF-77) constructed in Example 1, and the OsGLTF overexpression strains used the two strains with the highest expression levels (OE-OsGLTF-3 and OE-OsGLTF-8) ( Figure 1 ). Rice seeds were washed with 70% alcohol and then soaked in pure water for germination. After being raised on a flat tray for a week, they were transplanted to the field and grown until the grains matured. The comparison of seed size and 1000-grain weight used fully mature and dried seeds. All statistical data are the average of more than 30 rice seedlings.

[0053] Figure 2 This is a comparison of the whole plant of the wild type and mutant strains, where Figure A shows from left to right the mutant strain with OsGLTF knocked out by CRISPR-Cas9 technology, the wild type, and the transgenic plant with OsGLTF overexpression. Figure B shows the statistical data of the plant height of the wild type, knockout mutant, and wild type, indicating that the mutant strain with OsGLTF knocked out by CRISPR-Cas9 technology is shorter than the wild type and overexpressed plants, and the plant height of the overexpressed OsGLTF gene is taller than the wild type and mutant materials.

[0054] Figure 3 Figure 1 is a comparison of the ear size between the wild type and the mutant, where Figure A shows the overexpression, wild type and mutant materials from left to right. Figure B shows the statistical data of ear length of the wild type, overexpression materials and mutant materials. These show that the overexpression OsGLTF mutant has more primary and secondary branches than the WT and a larger ear shape.

[0055] Figure 4 This is a statistical comparison of grain length, grain width, grain thickness and 1000-grain weight between the wild type and the CRISPR-Cas9 knockout OsGLTF mutant overexpression material. It can be clearly seen from the figure that OsGLTF overexpression increases rice grain length but has no effect on grain width and thickness, but increases 1000-grain weight. There is no significant difference between the mutant and the wild type in grain length and width, but the grain thickness is reduced and the 1000-grain weight is reduced.

[0056] above Figures 2 to 4 The results showed that overexpression of the OsGLTF gene could increase rice plant height, grain length and grain weight, and significantly improve rice yield.

Claims

1. Rice OsGLTF The application of the gene or OsGLTF protein in increasing rice yield is characterized in that: Overexpression or overexpression of rice in rice plants OsGLTF Gene or OsGLTF protein, the rice OsGLTF The nucleotide sequence of the gene is shown in SEQ ID NO.1 or SEQ ID NO.2; the amino acid sequence of the OsGLTF protein is shown in SEQ ID NO.3, and the rice OsGLTF Genetically encoded.

2. Rice OsGLTF The use of a gene or an OsGLTF protein in increasing at least one of rice plant height, grain length and grain weight, characterized in that: Overexpression or overexpression of rice in rice plants OsGLTF Gene or OsGLTF protein, the rice OsGLTF The nucleotide sequence of the gene is shown in SEQ ID NO.1 or SEQ ID NO.2; the amino acid sequence of the OsGLTF protein is shown in SEQ ID NO.3, and the rice OsGLTF Genetically encoded.

3. A method for increasing rice yield, characterized in that: Overexpression or overexpression in rice plants OsGLTF Gene, OsGLTF The nucleotide sequence of the gene is shown in SEQ ID NO.1 or SEQ ID NO.

2.

4. The method according to claim 3, characterized in that The following steps are involved: (1) OsGLTF Genes were connected to plasmids to construct OsGLTF Gene overexpression or overexpression vector; (2) Convert the OsGLTF The gene overexpression or overexpression vector is introduced into rice cells, and transgenic plants are obtained after cultivation.

5. The method according to claim 4, characterized in that In step (1), the plasmid is a binary expression plasmid.

6. The method according to claim 4, characterized in that In step (2), OsGLTF After the gene overexpression or overexpression vector is transferred into Agrobacterium genetic engineering bacteria, it infects rice cells.

7. The method according to claim 6, characterized in that The Agrobacterium genetic engineering bacteria is Agrobacterium EHA105 strain.

8. The method according to claim 7, characterized in that In step (2), the cell source of rice infected by the genetically engineered Agrobacterium is the callus tissue of rice seeds.

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