Transcription factor GPC6 for regulating and controlling protein content of rice grains and application of transcription factor GPC6

Abstract

The invention discloses a transcription factor GPC6 for regulating and controlling the protein content of rice grains and application of the transcription factor GPC6. The amino acid sequence of the transcription factor is shown as SEQ ID NO.1. The transcription factor comprises a genome region and a promoter, the nucleotide sequence of the genome region is shown as SEQ ID NO.2, the nucleotide sequence of the promoter is shown as SEQ ID NO.3, the coding sequence (CDS) is shown as SEQ ID NO.4, and the coding sequence (CDS) is shown as SEQ ID NO.3. The transcription factor GPC6 can be used for regulating and controlling the protein content of the rice grains. And comprises all alleles thereof. Whole genome association analysis is carried out on the glutelin content and the total protein content, and a site GPC6 capable of being repeatedly detected is identified on a No.6 chromosome. Gene cloning is carried out, gene resources for rice quality genetic improvement can be enriched, and help is provided for rice quality improvement, especially protein content improvement.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a transcription factor GPC6 for regulating the protein content of rice grains and an application thereof. Background Art

[0002] Rice is one of the most important crops in the world and the main food crop in daily life. The protein content in rice grains can significantly affect the cooking and nutritional quality of rice. There are many types of rice proteins, which can be divided into two categories according to their functions: structural proteins and storage proteins. There are many types of structural proteins, but they account for a small proportion of the total protein content; storage proteins are specifically synthesized and enriched in large quantities in the endosperm, so the content is relatively high, accounting for about 95% of the total protein. Therefore, the protein content of rice generally refers to the storage protein content. Storage proteins can be divided into four types according to their solubility: water-soluble albumin (about 12%), salt-soluble globulin (about 12%), alcohol-soluble alcohol-soluble protein (about 5%) and weakly alkaline or weakly acid-soluble glutenin (about 70%).

[0003] So far, some studies have been conducted on the genetic regulatory mechanisms that control the protein content of rice grains. Using positional cloning technology, predecessors have successfully cloned two genes that regulate the protein content of rice grains. One is OsAAP6, which encodes an amino acid transporter. Increasing the expression level of OsAAP6 leads to the upregulation of key storage protein genes and enhanced root absorption of a series of amino acids, which can be distributed to the grains through vascular tissues for the biosynthesis and accumulation of grain proteins, ultimately leading to larger and more protein bodies. The other is OsGluA2, which encodes a glutenin precursor protein. It positively regulates the content of total protein by affecting the glutenin content of rice and is a key site to explain the variation in rice protein content between indica and japonica subspecies. Generally speaking, the higher the GPC (Grain Protein Content), the worse the cooking and eating quality of rice, so reducing GPC has become one of the goals of rice breeding in my country. Studies have shown that knocking out glutenin family genes using CRISPR / Cas9 technology can reduce the glutenin content and thus improve the cooking and eating quality of rice. Meanwhile, during endosperm development, the expression of GPC-related genes is regulated by transcription factors, which regulate GPC gene expression by binding to the promoter of GPC genes. RISBZ1 is a homologous gene of the GPC gene regulatory factor O2 in maize. This gene can bind to and transactivate the GCN4 motif in rice, and can also bind to the ACGT element to regulate the endosperm-specific expression of GPC genes. OsNAC20 and OsNAC26 play important and redundant functions in the regulation of starch and storage protein synthesis. OsNAC20 and OsNAC26 can directly activate the expression of OsGluA1, GluB4 / 5 and some globulins and alcohol-soluble proteins to regulate the synthesis of storage proteins.

[0004] Although two genes that can regulate GPC have been cloned and some transcription factors that can regulate the expression of GPC-related genes have been identified, the genetic regulation mechanism of GPC is still unclear. Therefore, the present invention provides a transcription factor GPC6 that regulates the protein content of rice grains and its application. Summary of the invention

[0005] Technical problems solved: In view of the above technical problems, the present invention provides a transcription factor GPC6 for regulating the protein content of rice grains and its application. By performing genome-wide association analysis on glutenin content and total protein content, a reproducibly detectable locus GPC6 was identified on chromosome 6. Gene cloning can enrich the gene resources for genetic improvement of rice quality and provide assistance for improving rice quality, especially the protein content.

[0006] Technical solution: In the first aspect, the present invention provides a transcription factor GPC6 for regulating the protein content of rice grains. The amino acid sequence of the transcription factor is shown in SEQ ID NO.1. Specifically:

[0007] SEQ ID NO.1:

[0008] MGRQPCCDKVGLKKGPWTAEEDQKLVAFLLTHGHCCWRVVPKLAGLLRCGKSCRLRWTNYLRPDLKRGLLSDDEERLVIDLHAQLGNRWSKIAARLPGRTDNEIKNHWNTHIKKKLRKMGIDPVTHQPLEPPRP PPREQDATPPPPPPPPQEIEESEEEEQEPSPLIEPHEITAPPPAAAAEAATSNCSVSPASVLSPSCSSSASAASAVDVAEWPEPMYMFGMDGIMDVGWNGLISGAGVDVDVDVDPFDHYYHDASFDDQDVWII.

[0009] Preferably, the transcription factor comprises a genomic region and a promoter, the nucleotide sequence of the genomic region is shown in SEQ ID NO.2, the nucleotide sequence of the promoter is shown in SEQ ID NO.3, the coding region sequence (CDS) of the GPC6 gene is shown in SEQ ID NO.4, and comprises all its alleles, specifically:

[0010] SEQ ID NO.2:

[0011]

[0012] SEQ ID NO.3:

[0013]

[0014] SEQ ID NO.4:

[0015] ATGGGGAGGCAGCCGTGCTGCGACAAGGTGGGGCTGAAGAAGGGGCCGTGGACGGCGGAGGAGGACCAGAAGCTCGTCGCCTTCCTCCTCACCCACGGCCACTGCTGCTGGCGCGTCGTCCCCAAGCTCGCAGGGTTGCTGAGGTGCGGGAAGAGCTGCAGGCTGAGGTGGACCAACTACCTGAGGCCCGACCTCAAGAGGGGCCTCCTCTCCGACGACGAGGAGCGGCTCGTCATCGACCTGCACGCGCAGCTCGGCAACAGGTGGTCCAAGATCGCGGCGCGGCTCCCCGGGAGGACGGACAACGAGATCAAGAACCACTGGAACACACACATCAAGAAGAAGCTCCGCAAGATGGGCATCGACCCCGTCACCCACCAGCCGCTGGAGCCGCCTCGTCCTCCGCCGCGCGAGCAGGATGCCACGCCGCCGCCGCCGCCGCCGCCGCCTCAGGAGATCGAGGAGAGCGAGGAGGAGGAGCAGGAGCCTTCGCCGTTGATCGAGCCGCACGAGATCACCGCGCCACCGCCGGCGGCCGCGGCCGAGGCCGCCACGAGCAACTGCTCTGTTTCCCCTGCTTCGGTGCTCTCCCCGTCCTGCTCCTCGTCGGCGTCGGCTGCCTCGGCGGTGGACGTGGCGGAGTGGCCGGAGCCCATGTACATGTTCGGCATGGATGGCATCATGGACGTCGGCTGGAACGGCCTCATCTCCGGCGCCGGCGTGGACGTCGACGTCGACGTCGACCCGTTCGACCATTACTACCACGACGCCAGCTTCGACGATCAAGACGTCTGGATCATCTGA。

[0016] Preferably, the primer pair used for amplifying the coding region of the transcription factor includes the forward primer SEQ ID NO.5 and the reverse primer SEQ ID NO.6. Specifically:

[0017] SEQ ID NO.5: ATGGGGAGGCAGCCGTGC;

[0018] SEQ ID NO. 6: TCAGATGATCCAGACGTCTTGATC.

[0019] In a second aspect, the present invention provides the use of the transcription factor GPC6 described in the first aspect in regulating the expression of the GPC synthesis gene of grain protein content in rice.

[0020] Preferably, the transcription factor knockout vector and the overexpression vector are transformed into rice varieties, and transgenic positive plants are screened.

[0021] Furthermore, the coding region of GPC6 was knocked out by the CRISPR / Cas9 system, and the coding region of the GPC6 gene was overexpressed by 1301U-Flag.

[0022] Further, the knockout line was tested for positive plants using forward primer SEQ ID NO.7 and reverse primer SEQ ID NO.8, and the overexpression line was tested for positive plants using forward primer SEQ ID NO.9 and reverse primer SEQ ID NO.10. Specifically:

[0023] SEQ ID NO.7:GATCGAGCCGCACGAGAT;

[0024] SEQ ID NO.8: TTCGGTGCATCAACCATGAC;

[0025] SEQ ID NO.9: AGAAGAAGCTCCCGCAAGATG;

[0026] SEQ ID NO. 10:ATGCCTGCAGGTCACTGGAT.

[0027] Furthermore, the transcription factor knockout vector is transformed into rice through Agrobacterium-mediated transformation.

[0028] In a third aspect, the present invention provides the use of the transcription factor GPC6 described in the first aspect in reducing the grain protein content GPC in rice.

[0029] Preferably, the transcription factor GPC6 is overexpressed in rice.

[0030] Beneficial effects: The present invention can inhibit the expression of alcohol-soluble proteins and glutenin-related genes in rice grains and reduce the protein content of the grains by overexpressing GPC6 in rice, thereby improving the quality of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Screening and identification of transcription factor GPC6, where A is the Manhattan plot of genome-wide association analysis of total protein and gluten (the upper figure is total protein, the lower figure is gluten); B is all candidate genes in the candidate region under the significant peak detected by chromosome 6 duplication; C is the gluten content of 8 high-protein varieties and 8 low-protein varieties; D is the expression levels of 21 candidate genes in 8 high-protein varieties and 8 low-protein varieties;

[0032] Figure 2 It is a haplotype analysis of the transcription factor GPC6, wherein A is a local Manhattan plot of the genome-wide association analysis of total protein and gluten in the GPC6 genomic region (including the promoter) (the upper figure is total protein, and the lower figure is gluten); B is the location and haplotype typing of four significant SNPs in the GPC6 genomic region; C is the total protein content between haplotypes; D is the gluten content between haplotypes; E is the expression level of GPC6 between haplotypes;

[0033] Figure 3 The gene structure and expression pattern of transcription factor GPC6, where A is GPC6 containing two Myb-DNA banding regions; B is the expression profile of GPC6 in rice;

[0034] Figure 4 is the component protein content of the knockout and overexpression lines of GPC6;

[0035] Figure 5 GPC6 inhibits the expression of genes in the alcohol-soluble protein family and the glutenin family, where A is the differentially expressed gene related to protein content in the RNA-seq results between the GPC6 knockout line and the wild type; B is the RT-PCR verification of the changes in the expression levels of some glutenin and alcohol-soluble protein genes in the knockout line and the overexpression line compared with the wild type;

[0036] Figure 6 The results of the effect of GPC6 on agronomic traits, where A is the plant type of ZH11, GPC6 knockout lines and overexpression lines; B is the grain type of ZH11, knockout lines and overexpression lines; C is the agronomic trait statistics of ZH11, knockout lines and overexpression lines. DETAILED DESCRIPTION

[0037] The present invention is described in detail below with reference to the accompanying drawings and specific examples: the experimental methods used in the examples are conventional methods unless otherwise specified; the raw materials and reagents used can all be obtained from commercial channels.

[0038] Example 1

[0039] 367 rice samples were collected for sequencing, and the content of component proteins was determined, and genome-wide association analysis was performed. Figure 1As shown in AB: On chromosome 6, significant sites that can be stably detected in the analysis results of total protein (the sum of four component proteins) and gluten were found; based on these significant sites (gluten: Chr06: 8235272, total protein: Chr06: 8248648) and the 115kb region before and after, 21 potential candidate genes were screened, as shown in Table 1 below:

[0040] Table 1 21 candidate genes in the candidate interval of chromosome 6

[0041]

[0042]

[0043] In order to further identify the functional genes among the candidate genes, 16 varieties were randomly selected, such as Figure 1 As shown in Figure C, there are 8 varieties with high protein content and 8 varieties with low protein content. The expression levels of 21 candidate genes in the grains of these varieties 15 days after flowering were analyzed. Figure 1 As shown in D, the expression level of gene number XVII showed significant difference between high-protein and low-protein varieties, while the expression levels of other genes did not differ significantly. Based on this finding, it is speculated that gene number XVII may be a key functional gene regulating protein content, and the gene is named GPC6.

[0044] To further define the function of GPC6, the results of genome-wide association studies of total protein and glutenin were further analyzed. Figure 2 As shown in A: In the genomic region of GPC6 (including the promoter region), there are four single nucleotide polymorphisms (SNPs) with significantly lower p-values ​​than other SNPs, and these four SNPs are all located in the promoter region, which further infers that these four SNPs are key SNPs for regulating GPC6 expression.

[0045] Haplotype analysis was performed on four key SNPs. Figure 2 As shown in Figure B: The population used for association analysis can be divided into three haplotypes, of which haplotype I and haplotype III are mostly japonica rice, and haplotype II is mostly indica rice. Figure 2 As shown in Figures C and D, there are significant differences in the gluten content and total protein content of the three haplotypes, mainly in that the gluten and total protein content of haplotype I are significantly lower than those of haplotype II and haplotype III. Some varieties were randomly selected among the three haplotypes to analyze the expression of GPC6. The results are shown in Figure 3. Figure 2 As shown in E: The expression level of GPC6 in haplotype I is significantly higher than that in haplotype II and haplotype III.

[0046] By comprehensively analyzing the results of haplotype analysis, it was observed that the expression of the GPC6 gene was negatively correlated with the protein content, that is, the higher the GPC6 expression, the lower the protein content in the grain. This finding suggests that the GPC6 gene may be involved in the regulation of protein content as an inhibitory factor. In addition, it was noted that there were genetic differences in the GPC6 gene between indica and japonica rice, indicating that GPC6 may be involved in the inheritance of differences between indica and japonica rice.

[0047] Through the prediction of the online plant transcription factor database (PlantTFDB), such as Figure 3 As shown in Figure A: GPC6 was found to be a MYB family transcription factor containing two MYB binding domains. To explore the expression pattern of GPC6, RT-PCR analysis was performed on different tissues of rice. The results are shown in Figure A. Figure 3 As shown in B: The expression of GPC6 starts at 5 days after flowering (5DAF) and gradually increases over time, reaching a peak at 20 days after flowering (20DAF), and then the expression level begins to decline. In addition, the expression of GPC6 in rice roots, stems, leaves, sheaths and panicles is almost negligible.

[0048] In order to further verify the gene function of GPC6, the gene was knocked out using CRISPR / Cas9 technology, and an overexpression vector was constructed to transform ZH11. The protein content of the transgenic strain and the wild-type component was measured. The results are as follows Figure 4 As shown in the figure, the globulin, alcohol-soluble protein and glutenin in the knockout line were significantly increased compared with the wild type, and the overexpression line was significantly reduced compared with the wild type, while there was no difference in albumin between the knockout line and the overexpression line. In order to analyze the reasons for the changes in protein content in the transgenic lines, the expression levels of GPC-related genes were analyzed in the grains taken 15 days after flowering. Figure 5 As shown in A: RNA-seq results show that compared with the wild type, some genes of the glutenin family and alcohol-soluble protein family in the GPC6 knockout strain were significantly upregulated; Figure 5 As shown in B, RT-PCR also verified the results of RNA-seq. It can be inferred that GPC6 can participate in the regulation of GPC genes as a transcription factor in agronomic traits, such as Figure 6 As shown, there were no significant differences in heading date, plant height, flag leaf length, tiller number, ear length, number of grains per ear and fruit setting rate between the transgenic lines and the wild type.

[0049] In summary, GPC6 can act as an inhibitory factor to directly participate in the regulation of GPC-related genes and inhibit the protein content in rice grains.

[0050] Example 2

[0051] 1) Determination of protein content of rice components

[0052] The polished rice was ground into rice flour using a pneumatic mill. The ground rice flour was passed through a 100-mesh sieve and then the moisture content was balanced again in an oven. 100±0.5 mg of rice flour was weighed into a 2 mL centrifuge tube using a 1 / 10,000 balance.

[0053] Then, albumin, globulin, alcohol-soluble protein and glutenin in rice flour are extracted in sequence according to the following steps:

[0054] ①. Use 1 mL of deionized water (ddH2O) as the extraction solution to extract albumin;

[0055] ②. Use 1M sodium chloride (NaCl) solution to extract globulin;

[0056] ③. Use ethylene glycol as the extracting liquid to extract alcohol-soluble protein;

[0057] ④. Use 0.05M sodium hydroxide (NaOH) solution to extract gluten;

[0058] Each time the corresponding extract is added and shaken to mix, the centrifuge tube is shaken on a shaker at 30°C and 200rpm for 2 hours to ensure that the rice flour is fully in contact with the extract. After that, centrifuge at 12000rpm for 15 minutes to separate the supernatant. For the extraction of each component protein, repeat the above process three times and combine the obtained supernatants. Finally, the protein content in the combined supernatant is quantitatively analyzed using the BCA method.

[0059] Results: After measuring the content of component proteins in 367 resequenced rice germplasms, a genome-wide association analysis was performed, and a significant locus was successfully identified on chromosome 6. Then, by measuring the content of component proteins in mutant strains, the role of GPC6 in rice GPC regulation was successfully identified.

[0060] 2) Genome-wide association analysis

[0061] The mixed linear model (MLM) in EMMAX software was used to perform genome-wide association analysis for each trait. -5 The threshold was used to screen significant loci, and candidate genes were screened in the interval of 115 kb upstream and downstream of the significant loci. Results: In the genome-wide association analysis, the effects of population structure and kinship were comprehensively considered; in the association analysis of gluten content and total protein content, a significant locus that was repeatedly detected was screened on chromosome 6, and the genes in the candidate segment were screened, and a total of 21 candidate genes were screened; after verification, GPC6 was successfully identified.

[0062] 3) Haplotype analysis

[0063] Shapeit software was used for haplotype analysis. Results: The four significant SNPs in the promoter region of GPC6 were analyzed for haplotypes, which can be divided into three haplotypes, of which haplotype 1 and haplotype 3 are mostly japonica rice, and haplotype 2 is indica rice. The glutenin and total protein contents of haplotype 1 were significantly lower than those of haplotype 2. On the contrary, the expression level of GPC6 in haplotype 1 was significantly higher than that of haplotype 2 and haplotype 3. The differences in glutenin, total protein content and expression level of GPC6 in different haplotypes were comprehensively analyzed, and GPC6 was preliminarily identified as a repressor factor involved in the regulation of GPC.

[0064] 4) Cloning of GPC6 gene

[0065] GPC6 was cloned from endosperm cDNA 15 days after anthesis using GPC6 amplification primers (forward primer: SEQ ID NO.5: ATGGGGAGGCAGCCGTGC, reverse primer: SEQ ID NO.6: TCAGATGATCCAGACGTCTTGATC). The CDS sequence is shown in SEQ ID NO.4, and the amino acid sequence is translated from the CDS sequence and shown in SEQ ID NO.1. GPC6 is encoded by 267 amino acids, of which MYB_DNA-binding is 14-61aa and 71-111aa.

[0066] 5) CRISPR / Cas9 vector construction

[0067] First, the target was designed on the website (http: / / skl.scau.edu.cn / targetdesign / ). The GPC6 knockout target primers are shown in Table 2 below:

[0068] Table 2 GPC6 knockout target primer information

[0069]

[0070] According to the steps provided by the website developer, the target was connected to the corresponding gRNA, and then connected to pYLCRISPR / Cas9-MH(B) by the cutting and ligation method. The plasmid was sent to Boyuan Company to obtain transformed seedlings. Results: Mutants were screened from T0 using SEQ ID NO.7 and SEQ ID NO.8, and homozygous mutant strains were identified after further self-pollination and named GPC6-KO. After the component protein of GPC6-KO was determined, it was found that the content of globulin, alcohol-soluble protein and glutenin was significantly increased compared with the wild type.

[0071] 6) Overexpression vector construction

[0072] The coding region of GPC6 was amplified using SEQ ID NO.5 and SEQ ID NO.6, and connected to 1301U-flag by homologous recombination. The plasmid was sent to Boyuan Company to obtain transformed seedlings. Results: The mutant was screened from T0 using SEQ ID NO.9 and SEQ ID NO.10, and self-pollination was continued until no separation occurred. It was named GPC6-OE. After determining the component proteins of GPC6-OE, it was found that the content of globulin, alcohol-soluble protein and glutenin was significantly reduced compared with the wild type.

[0073] 7) RNA-seq

[0074] The seeds were collected 15 days after flowering and stored in liquid nitrogen. RNA extraction and data analysis were completed by Kidio. Results: After screening the differentially expressed genes, the expression of genes in the alcohol-soluble protein family and the glutelin family was significantly upregulated in GPC6-KO.

[0075] 8) RT-PCR

[0076] The seeds were collected 15 days after flowering, ground into powder, and RNA was extracted using the Tiangen kit (DP419), and quantified using reverse transcription (AE341) and SYBR (AQ621) from Quanshijin. Results: RT-PCR also verified the results of RNA-seq. The expression levels of glutelin and alcohol-soluble protein-related genes increased in the GPC6-KO strain and decreased in the GPC6-OE strain.

[0077] Conclusion: GPC6 was identified as a key gene regulating rice grain protein content through genome-wide association analysis and haplotype analysis. In-depth research found that GPC6, as a transcriptional repressor, can inhibit the expression of glutenin family and alcohol-soluble protein family genes to participate in the regulation of rice grain protein content.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A transcription factor that regulates protein content in rice grains GPC6 , characterized in that: The amino acid sequence of the transcription factor is shown in SEQ ID NO.

1.

2. A transcription factor for regulating rice grain protein content according to claim 1 GPC6 , characterized in that: The transcription factor comprises a genomic region and a promoter, the nucleotide sequence of the genomic region is shown in SEQ ID NO.2, and the nucleotide sequence of the promoter is shown in SEQ ID NO.3; GPC6 The coding region sequence of the gene is shown in SEQ ID NO.4, and includes all its alleles.

3. A transcription factor for regulating rice grain protein content according to claim 1 GPC6 , characterized in that: The primer pair used to amplify the coding region of the transcription factor includes a forward primer SEQ ID NO.5 and a reverse primer SEQ ID NO.

6.

4. The transcription factor according to any one of claims 1 to 3 GPC6 Application in regulating the expression of GPC synthesis genes in rice grain protein content.

5. The use according to claim 4, characterized in that: The transcription factor knockout vector and the overexpression vector are transformed into rice varieties, and transgenic positive plants are screened.

6. The use according to claim 5, characterized in that: pass CRISPR / Cas9 System knockout GPC6 The coding region of the gene was overexpressed by 1301U-Flag GPC6 The coding region of a gene.

7. The use according to claim 5, characterized in that: The knockout line was tested for positive plants using forward primer SEQ ID NO.7 and reverse primer SEQ ID NO.8, and the overexpression line was tested for positive plants using forward primer SEQ ID NO.9 and reverse primer SEQ ID NO.

10.

8. The use according to claim 5, characterized in that: The transcription factor knockout vector is transformed into rice through Agrobacterium-mediated transformation.

9. The transcription factor according to any one of claims 1 to 3 GPC6 Application of GPC in reducing grain protein content in rice.

10. The use according to claim 9, characterized in that: Overexpression of transcription factors in rice GPC6 .

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