Transcription factor gpc6 for regulating protein content of rice grains and application thereof

Abstract

The application discloses a transcription factor for regulating protein content of rice grains GPC6 and application, the amino acid sequence of the transcription factor is shown as SEQ ID NO. 1, the transcription factor comprises a genomic region and a promoter, the nucleotide sequence of the genomic 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 all alleles thereof are contained. The application identifies a repeatedly detectable locus on chromosome 6 by performing whole genome association analysis on gluten content and total protein content GPC6 . Gene cloning is carried out on the locus, so that the genetic resources for improving rice quality are enriched, and help is provided for improving rice quality, in particular, protein content.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a transcription factor GPC6 for regulating protein content of rice grains and application thereof. BACKGROUND

[0002] Rice is one of the most important crops in the world and is also a main food crop in daily life. The protein content in rice grains can significantly affect the cooking taste quality and nutritional quality of rice. Rice proteins are various in types, and can be divided into two categories according to their functions: structural proteins and storage proteins. The structural proteins are various in types, but account for a small proportion of the total protein content; the storage proteins are specifically synthesized and highly enriched in endosperm, and thus account for a high proportion, about 95% of the total protein. Therefore, the general rice protein content mainly refers to the storage protein content. The storage proteins can be further divided into four types according to their solubility: water-soluble albumin (about 12%), salt-soluble globulin (about 12%), alcohol-soluble prolamin (about 5%), and weak acid or weak base-soluble glutelin (about 70%).

[0003] So far, the genetic regulatory mechanism of controlling rice grain protein content has been partially studied. Using map-based cloning technology, two genes that can regulate the protein content of rice grain have been successfully cloned. One of them is OsAAP6, which encodes an amino acid transporter. Increasing the expression level of OsAAP6 leads to the up-regulation of storage protein key genes and the enhancement of the root absorption of a series of amino acids, which can be distributed to the grain through the vascular tissue for the biosynthesis and accumulation of grain protein, ultimately leading to the increase in the size and number of protein bodies. The other one is OsGluA2, which encodes a glutelin precursor protein and positively regulates the total protein content by affecting the glutelin content of rice, and is a key site for explaining the variation of rice protein content between indica and japonica subspecies. Generally speaking, the higher the GPC (Grain Protein Content), the worse the cooking taste quality of rice, so reducing GPC has become one of the breeding goals of rice in China. Previous studies have shown that knocking out the genes in the glutelin family using CRISPR / Cas9 technology can reduce the glutelin content and thus improve the cooking taste quality of rice. At the same time, the expression of GPC-related genes is regulated by transcription factors during endosperm development, and these transcription factors regulate the expression of GPC genes by binding to the promoters of GPC genes. RISBZ1 is a homologous gene of GPC gene regulatory factor O2 in maize, which can bind and trans-activate the GCN4 motif in rice, and also bind to the ACGT element to regulate the endosperm-specific expression of GPC genes. OsNAC20 and OsNAC26 play an important and redundant role in the regulation of starch and storage protein synthesis. OsNAC20 and OsNAC26 can directly activate the expression of OsGluA1, GluB4 / 5 and part of the globulin and prolamin 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 regulatory mechanism of GPC is still unclear. Therefore, the present application provides a transcription factor GPC6 for regulating the protein content of rice grain and its application. SUMMARY

[0005] The technical problem solved by the present application is that the present application provides a transcription factor GPC6 for regulating the protein content of rice grain and its application, which can enrich the genetic resources for improving the quality of rice and provide help for improving the quality of rice, especially the protein content, by identifying a repeatedly detected locus GPC6 on chromosome 6 through whole-genome association analysis of glutelin content and total protein content.

[0006] Technical solution: In a first aspect, the present invention provides a transcription factor GPC6 that regulates the protein content of rice grains, the amino acid sequence of which 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, and the coding sequence (CDS) of the GPC6 gene is shown in SEQ ID NO.4, containing 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] Secondly, the present invention provides the application of the transcription factor GPC6 described in the first aspect in regulating the expression of GPC synthesis genes in rice to regulate grain protein content.

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

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

[0022] Furthermore, positive plant detection was performed on the knockout lines using forward primer SEQ ID NO.7 and reverse primer SEQ ID NO.8, and positive detection was performed on the overexpression lines 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 was transformed into rice via Agrobacterium-mediated transformation.

[0028] Thirdly, the present invention provides the application of the transcription factor GPC6 described in the first aspect in reducing the protein content of grains in rice.

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

[0030] Beneficial effects: This invention can inhibit the expression of gliadin and glutenin-related genes in rice grains by overexpressing GPC6 in rice, thereby reducing the protein content of the grains and improving rice quality. Attached Figure Description

[0031] Figure 1 For the screening and identification of transcription factor GPC6, A is the Manhattan plot of genome-wide association analysis of total protein and glutenin (top plot is total protein, bottom plot is glutenin); B is all candidate genes in the candidate region under the significant peak detected by repeats on chromosome 6; C is the glutenin content of 8 high-protein and 8 low-protein varieties; D is the expression level of 21 candidate genes in 8 high-protein and 8 low-protein varieties.

[0032] Figure 2 For haplotype analysis of transcription factor GPC6, A is a local Manhattan plot of genome-wide association analysis of total protein and glutenin in the GPC6 genomic region (including the promoter) (top plot shows total protein, bottom plot shows glutenin); B shows the location and haplotype classification of four significant SNPs in the GPC6 genomic region; C shows the total protein content among haplotypes; D shows the glutenin content among haplotypes; E shows the GPC6 expression level among haplotypes.

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

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

[0035] Figure 5 To investigate the inhibition of gliadin and glutenin family gene expression by GPC6, A represents differentially expressed genes related to protein content in RNA-seq results between GPC6 knockout and wild-type lines; B represents changes in the expression levels of some glutenin and gliadin genes in knockout and overexpression lines compared to wild-type lines, verified by RT-PCR.

[0036] Figure 6 The results show the effects of GPC6 on agronomic traits. In this table, A represents the plant type of ZH11, the GPC6 knockout line, and the overexpression line; B represents the grain type of ZH11, the knockout line, and the overexpression line; and C represents the statistical analysis of agronomic traits of ZH11, the knockout line, and the overexpression line. Detailed Implementation

[0037] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments: Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the raw materials and reagents used are all commercially available.

[0038] Example 1

[0039] 367 rice accessions were collected and sequenced to determine the content of component proteins, and genome-wide association analysis was performed. The results are as follows: Figure 1As shown in Figure AB, significant loci were found on chromosome 6 that were consistently detectable in both total protein (sum of the four protein components) and glutenin analysis. Based on these significant loci (glutenin: Chr06: 8235272, total protein: Chr06: 8248648) and the 115kb region before and after them, 21 potential candidate genes were screened, as shown in Table 1 below.

[0040] Table 1. 21 candidate genes within the candidate region of chromosome 6

[0041]

[0042]

[0043] To further identify functional genes among the candidate genes, 16 varieties were randomly selected, such as... Figure 1 As shown in Figure C, this includes 8 high-protein varieties and 8 low-protein varieties. The expression levels of 21 candidate genes in the grains of these varieties 15 days after flowering were analyzed. The results are as follows... Figure 1 As shown in Figure D, the expression level of gene XVII showed a 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 XVII may be a key functional gene regulating protein content, and this gene was named GPC6.

[0044] To further determine the function of GPC6, the results of genome-wide association analysis (GWAS) of total protein and glutenin were further analyzed. The results are as follows: Figure 2 As shown in Figure A: Within the GPC6 genomic region (including the promoter region), four single nucleotide polymorphisms (SNPs) have significantly lower p-values ​​than other SNPs, and all four SNPs are located in the promoter region. Therefore, it can be inferred that these four SNPs are key SNPs regulating GPC6 expression.

[0045] Haplotype analysis was performed on four key SNPs. The results are as follows: Figure 2 As shown in Figure B: The population used for association analysis can be divided into three haplotypes, with haplotypes I and III mostly consisting of japonica rice, and haplotype II mostly consisting of indica rice. For example... Figure 2 As shown in C and D, there are significant differences in glutenin and total protein content among the three haplotypes, mainly manifested in haplotype I having significantly lower glutenin and total protein content than haplotypes II and III. The expression levels of GPC6 were analyzed in a randomly selected group of varieties across the three haplotypes, and the results are as follows... Figure 2 As shown in Figure E, the expression level of GPC6 in haplotype I was significantly higher than that in haplotypes II and III.

[0046] A comprehensive analysis of haplotype analysis results revealed a negative correlation between GPC6 gene expression and protein content; that is, higher GPC6 expression levels were associated with lower protein content in the grains. This finding suggests that the GPC6 gene may act as a repressor in the regulation of protein content. Furthermore, genetic differences in the GPC6 gene were observed between indica and japonica rice varieties, indicating that GPC6 may be involved in the inheritance of these differences.

[0047] Predictions from 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 investigate the expression pattern of GPC6, RT-PCR analysis was performed on different rice tissues. The results are as follows... Figure 3 As shown in Figure B, GPC6 expression begins 5 days after flowering (5DAF) and gradually increases over time, reaching a peak at 20 days after flowering (20DAF), after which the expression level begins to decline. Furthermore, GPC6 expression in the roots, stems, leaves, sheaths, and young panicles of rice is almost negligible.

[0048] To further verify the gene function of GPC6, the gene was knocked out using CRISPR / Cas9 technology, and an overexpression vector was constructed and transformed into ZH11 cells. The protein content of the transgenic lines and wild-type components was measured, and the results are as follows: Figure 4 As shown: the knockout lines exhibited significantly higher levels of globulin, prolamins, and glutenin compared to the wild type, while the overexpression lines showed significantly lower levels. Albumin levels, however, did not differ between the knockout and overexpression lines. To analyze the reasons for the changes in protein content in the transgenic lines, the expression levels of GPC-related genes were analyzed from seeds taken 15 days after flowering. Figure 5 As shown in Figure A: RNA-seq results showed that, compared with the wild type, some genes in the glutenin and prolamins families were significantly upregulated in the GPC6 knockout lines; Figure 5 As shown in Figure B, RT-PCR also verified the results of RNA-seq. Therefore, it can be inferred that GPC6 can act as a transcription factor involved in the regulation of GPC genes, and in agronomic traits, such as... Figure 6 As shown, there were no significant differences between the transgenic lines and the wild type in heading date, plant height, flag leaf length, number of tillers, ear length, number of grains per ear, and seed setting rate.

[0049] In conclusion, GPC6 can act as a repressor 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 in rice components

[0052] The rice was ground into rice flour using a wind mill. After passing the ground rice flour through a 100-mesh sieve, the moisture content was balanced again in an oven. 100±0.5mg of rice flour was weighed into a 2mL centrifuge tube using a balance with a strength of 0.01%.

[0053] Then, extract albumin, globulin, prolyl, and glutenin from the rice flour in 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 globulins;

[0056] ③. Ethylene glycol is used as the extraction solvent to extract alcohol-soluble proteins;

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

[0058] Each time, the appropriate extraction buffer was added and the mixture was shaken thoroughly. The centrifuge tubes were then subjected to extraction on a shaker at 30°C and 200 rpm for 2 hours to ensure sufficient contact between the rice flour and the extraction buffer. Afterward, the mixture was centrifuged at 12,000 rpm for 15 minutes to separate the supernatant. The above process was repeated three times for each protein component, and the resulting supernatants were combined. Finally, the protein content in the combined supernatant was quantitatively analyzed using the BCA method.

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

[0060] 2) Genome-wide association analysis

[0061] Genome-wide association analysis was performed on each trait using a mixture linear model (mlm) in EMMAX software. A 1.0 × 10⁻⁶ m² model was used. -5 Significant loci were screened using a threshold, and candidate genes were selected within a 115kb interval upstream and downstream of the significant locus. Results: In the genome-wide association analysis, the influence of population structure and phylogenetic relationships was comprehensively considered; in the association analysis of glutenin content and total protein content, a significant locus that was repeatedly detected was identified on chromosome 6. Genes within the candidate region of this locus were screened, and a total of 21 candidate genes were identified; after validation, GPC6 was successfully identified.

[0062] 3) Haplotype analysis

[0063] Haplotype analysis was performed using Shapeit software. Results: Haplotype analysis of four significant SNPs in the GPC6 promoter region revealed three haplotypes. Haplotypes 1 and 3 were predominantly Japonica rice, while haplotype 2 was indica rice. The glutenin and total protein content of haplotype 1 was significantly lower than that of haplotype 2. Conversely, the GPC6 expression level in haplotype 1 was significantly higher than that in haplotypes 2 and 3. Based on the comprehensive analysis of the differences in glutenin, total protein content, and GPC6 expression levels among different haplotypes, GPC6 was preliminarily identified as a repressor involved in the regulation of GPC.

[0064] 4) Cloning of the GPC6 gene

[0065] GPC6 was cloned from endosperm cDNA 15 days after flowering 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 was obtained by translating the CDS sequence and is 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) Construction of CRISPR / Cas9 vectors

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

[0068] Table 2. Primer information for GPC6 knockout target sites.

[0069]

[0070] Following the steps provided by the website developer, the target site was ligated to the corresponding gRNA, and then ligated to pYLCRISPR / Cas9-MH(B) using a cut-and-ligate method. The plasmid was then 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 after further self-crossing, a homozygous mutant line was identified and named GPC6-KO. Protein analysis of GPC6-KO revealed a significant increase in the content of globulin, prolamins, and glutenin compared to the wild type.

[0071] 6) Construction of overexpression vectors

[0072] The coding region of GPC6 was amplified using SEQ ID NO.5 and SEQ ID NO.6, and ligated to the 1301U-flag using homologous recombination. The plasmid was then sent to Boyuan Company to obtain transformed seedlings. Results: A mutant was screened from T0 using SEQ ID NO.9 and SEQ ID NO.10, and self-crossing continued until no further segregation occurred; this mutant was named GPC6-OE. Protein analysis of GPC6-OE revealed a significant decrease in the content of globulin, prolamins, and glutenin compared to the wild type.

[0073] 7) RNA-seq

[0074] Fifteen days after harvesting, the seeds were cryopreserved in liquid nitrogen. RNA extraction and data analysis were performed by Gedio Corporation. Results: After screening for differentially expressed genes, the expression levels of prolamins and glutenin family genes were significantly upregulated in GPC6-KO.

[0075] 8) RT-PCR

[0076] Fifteen days after flowering, the seeds were collected, ground into powder, and RNA was extracted using the Tiangen reagent kit (DP419). Quantification was performed using TransGen reverse transcription (AE341) and SYBR (AQ621). Results: RT-PCR also validated the RNA-seq results. Glutenin and prolamin-related genes showed increased expression in the GPC6-KO line and decreased expression in the GPC6-OE line.

[0077] Conclusion: Genome-wide association analysis and haplotype analysis identified GPC6 as a key gene regulating rice grain protein content. Further investigation revealed that GPC6, as a transcriptional repressor, participates in the regulation of rice grain protein content by inhibiting the expression of glutenin and gliadin family genes.

[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 within the protection scope of the present invention.

Claims

1. Transcription factors GPC6 Application of GPC synthesis gene expression in regulating grain protein content in rice, wherein the amino acid sequence of the transcription factor is shown in SEQ ID NO.1, and the synthesis gene is OsGluA1, OsGluA2, OsGluA3, OsGluB1, OsGluB2, OsGluB4 / 5, OsGluB6, OsGluB7, OsGluC, OsGluD, RISBZ1, RP6 and RM1.

2. The application according to claim 1, characterized in that: Transform rice varieties with the knockout or overexpression vectors of the transcription factors and screen for transgenic positive plants.

3. The application according to claim 2, characterized in that: pass CRISPR / Cas9 System knockout GPC6 The coding region of the gene is overexpressed via 1301U-Flag. GPC6 The coding region of a gene.

4. The application according to claim 2, characterized in that: The knockout lines were tested for positive results using the forward primer SEQ ID NO.7 and the reverse primer SEQ ID NO.8, and the overexpression lines were tested for positive results using the forward primer SEQ ID NO.9 and the reverse primer SEQ ID NO.

10.

5. The application according to claim 2, characterized in that: The transcription factor knockout vector was transformed into rice via Agrobacterium-mediated transformation.

6. Transcription factors GPC6 Application of GPC in reducing grain protein content in rice, wherein the amino acid sequence of the transcription factor is shown in SEQ ID NO.1, and the grain protein is globulin, prolysin, and glutenin.

7. The application according to claim 6, characterized in that: Overexpression of transcription factors in rice GPC6 .

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