Corn grain size regulation gene X1 and SNP marker and application thereof

Abstract

The invention discloses a corn kernel size regulation gene X1 as well as an SNP (Single Nucleotide Polymorphism) marker and application thereof. The invention discovers that the X1 gene has the function of regulating and controlling the grain shape and the grain weight for the first time. The invention provides a novel SNP (Single Nucleotide Polymorphism) marker related to the corn grain size, the SNP marker has two alleles AA and GG, and the allele AA is clear to be regarded as an excellent / synergistic allele. The X1 gene provided by the invention can lead to increase of grain types such as the length of a corn grain of a mutant strain compared with that of a control grain; the length and other grain types of the X1 gene overexpression strain are reduced compared with those of the control grain. Precious gene resources are provided for corn breeding, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and plant genetic breeding, and specifically, to a maize grain size regulatory gene X1, its SNP markers and applications. Background Art

[0002] Maize (Zea mays L.) is an important food, economic and feed crop. With the continuous growth of the population, maize yield is becoming increasingly important for food security in China and the world. Increasing yield is one of the core objectives of maize breeding. Traditional maize breeding has a long cycle, low accuracy and efficiency, and it is difficult to break through the barriers of interspecific hybridization, restricting the utilization of genetic diversity resources. Molecular breeding technology has become an important means of modern breeding with its precision and efficiency, broadening the scope of utilization of genetic resources. Grain shape and grain weight are important components of maize yield. Through a large number of studies on maize grain mutants and genome-wide association analysis, it has been clarified that grain shape and grain weight are complex quantitative traits, and hundreds of genetic loci related to grain development have been identified. Due to the relatively dispersed effects of its genetic loci and the extensive genetic variation in maize, continuously excavating and identifying excellent allelic variations related to grain size, developing molecular markers for corresponding allelic variations, and screening grain size functional genes still have important production application value for maize breeding improvement.

[0003] Alternative splicing has an important impact on the growth and development of various species. Analyzing the genetic basis of alternative splicing helps to excavate genetic loci of complex traits. Although there is a large amount of research basis for the genetic analysis of grain size, most loci come from studies on artificially constructed populations, and more types of natural variation loci need to be excavated to analyze the complex genetic basis of grain size. With the rapid development of transcriptome sequencing, in-depth mining of alternative splicing variations in transcriptome data of natural populations helps to further improve the genetic basis analysis of grain size. Therefore, it is still necessary to excavate and analyze molecular markers and new genes that control grain size. Summary of the Invention

[0004] The object of the present invention is to provide a maize grain size regulatory gene X1, its mutants and applications.

[0005] Another object of the present invention is to provide SNP molecular markers for regulating maize grain size and their applications.

[0006] To achieve the object of the present invention, in the first aspect, the present invention provides the application of a maize grain size regulatory gene X1 in regulating plant grain shape, grain weight and yield.

[0007] The gene X1 is a gene encoding the following protein (A) or (B): (A)A protein consisting of the amino acid sequence shown in SEQ ID NO:5; or (B)A protein derived from (A), wherein one or several amino acids in the sequence shown in SEQ ID NO:5 are substituted, deleted or added, and which has the same function. Furthermore, the grain type is grain length.

[0008] Furthermore, the regulation is negative regulation.

[0009] Preferably, the plant of the present invention is a gramineous plant, more preferably maize.

[0010] In a second aspect, the present invention provides a method for increasing the grain length, grain weight and yield of maize, the method comprising: by means of genetic engineering, modifying the maize grain size regulation gene X1 so that the function of the gene is lost, thereby increasing the grain length, grain weight and yield of maize.

[0011] In a third aspect, the present invention provides a method for shortening the grain length, reducing the grain weight and yield of maize, the method comprising: by means of genetic engineering, overexpressing the maize grain size regulation gene X1 in maize.

[0012] Furthermore, the overexpression method may be selected from the following 1) to 5), or any optional combination: 1) By introducing a plasmid having the gene; 2) By increasing the copy number of the gene on the plant chromosome; 3) By changing the promoter sequence of the gene on the plant chromosome; 4) By operably linking a strong promoter to the gene; 5) By introducing an enhancer.

[0013] In a fourth aspect, the present invention provides the use of the transgenic maize obtained by the method in plant breeding.

[0014] Furthermore, the breeding methods include but are not limited to transgenic, hybridization, backcrossing, selfing or asexual reproduction.

[0015] In a fifth aspect, the present invention provides a mutant of the maize grain size regulation gene X1, which is a gene encoding the following protein (a) or (b): (a)A protein consisting of the amino acid sequence of positions 1 to 145 of the sequence shown in SEQ ID NO:5; or (b)A protein derived from (a), wherein one or several amino acids in the amino acid sequence of positions 1 to 145 of the sequence shown in SEQ ID NO:5 are substituted, deleted or added, and which has the same function.

[0016] In a sixth aspect, the present invention provides a biological material containing the gene mutant, and the biological material includes but is not limited to recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or non-renewable plant parts.

[0017] In a seventh aspect, the present invention provides any of the following applications of the gene mutant or the biological material containing the gene mutant: (1) For increasing the size of plant grains (increasing grain length); (2) For increasing plant yield (increasing grain weight); (3) For constructing transgenic plants; (4) For plant genetic breeding or improvement of plant germplasm resources.

[0018] In an eighth aspect, the present invention provides an SNP molecular marker for regulating the size of maize grains, and the SNP molecular marker contains a nucleotide sequence with a polymorphism of A / G at the 959th position of the sequence of the maize grain size regulatory gene X1 as shown in SEQ ID NO:1.

[0019] Furthermore, the grain length and grain weight of maize germplasm resources with the genotype AA at the locus with the polymorphism are greater than those of maize germplasm resources with the genotype GG.

[0020] In a ninth aspect, the present invention provides a primer for amplifying the SNP molecular marker.

[0021] In a tenth aspect, the present invention provides a detection reagent or kit containing the primer.

[0022] In an eleventh aspect, the present invention provides any of the following applications of the SNP molecular marker, the primer, or the detection reagent or kit containing the primer: 1) For identifying the size of maize grains; 2) For identification, improvement or molecular marker-assisted breeding of maize germplasm resources; 3) For early prediction of maize grain size and yield; 4) For screening maize with large grains and high grain weight.

[0023] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects: (1) The present invention provides a new SNP marker related to maize grain size, which has two alleles AA and GG, and it is clarified that the allele AA is regarded as an excellent / synergistic allele, which is represented by complementary bases TT and CC in specific embodiments.

[0024] (2) The present invention provides the gene X1 that controls the grain shape of maize, and clones its nucleotide sequence, laying a foundation for its application in maize breeding practice.

[0025] (3) The X1 gene provided by the present invention can cause an increase in grain type such as grain length in the mutant strain maize compared to the control; while the overexpression strain of the X1 gene has a decrease in grain type such as grain length compared to the control. Description of the Drawings

[0026] Figure 1 It is the alternative splicing structure diagram of the 5' UTR of the X1 gene (GRMZM2G160032) in the preferred embodiment of the present invention, and ATG is the translation initiation site.

[0027] Figure 2 It is the correlation between the gene X1 and grain size in the preferred embodiment of the present invention. A is the correlation between the intron splicing ratio PSI of the splicing event and gene expression. The X-axis is the expression of the X1 gene, and the Y-axis is the intron splicing ratio PSI of AS_eUTR and AS_tUTR respectively. B shows a negative correlation between the expression of the X1 gene and grain size. The X-axis from left to right is grain length KL, grain width KW, 100-grain volume KVPH, and 100-grain weight KWPH, and the Y-axis is the expression of the X1 gene. C shows a negative correlation between the expression level of the transcript T01 generated by the splicing event AS_eUTR and grain size. D shows a positive correlation between the expression level of the transcript T04 generated by the splicing event AS_tUTR and grain size.

[0028] Figure 3 It is the haplotype analysis of the splicing event PSI (A) and grain size (B - E) based on the SNP markers in the X1 gene region in the preferred embodiment of the present invention. In the TT genotype material, AS_eUTR is smaller, AS_tUTR is larger, and the grain type is larger.

[0029] Figure 4 It is the phenotype confirmation of the X1 gene overexpression material in the preferred embodiment of the present invention. A is the qRT-PCR of the X1 gene in the overexpression material. B is the grain type display of the control and overexpression (OE) materials. Scale bar = 1 cm. C is the phenotype statistics of the 100-grain weight, grain length, and grain thickness of the wild type and overexpression materials. The 100-grain weight, grain length, and grain thickness of the X1 overexpression material are all smaller than those of the control.

[0030] Figure 5 It is the phenotype confirmation of the X1 gene mutant material in the preferred embodiment of the present invention. A is the structure of the X1 gene in the EMS material, and the base mutation leads to premature termination of translation. B is the grain type display of the wild type and mutant (EMS) genotype materials. Scale bar = 1 cm. C is the phenotype statistics of the 100-grain weight, grain length, and grain thickness of the wild type and mutant materials. The 100-grain weight, grain length, and grain thickness of the X1 mutant material are all larger than those of the wild type.

[0031] Figure 6This is the expression pattern of the maize X1 gene in the preferred embodiment of the present invention. A shows the expression pattern of the X1 gene in the multi-tissue material of B73. The expression level of transcript T04 is significantly higher than that of T01, and it is mainly expressed in the endosperm. B shows the subcellular localization of X1. The X1 protein may be localized in the cytoplasm and nucleus. Detailed implementation mode

[0032] The present invention aims to provide a new molecular marker and functional gene for controlling maize kernel size, which helps to promote the development of molecular breeding technology for maize kernel phenotype.

[0033] The present invention adopts the following technical solutions: In the first aspect, the present invention provides a molecular marker for controlling maize kernel size, which contains a nucleotide sequence with a polymorphism of A / G at the 959th position of the sequence shown in SEQ ID NO:1.

[0034] The SNP (single nucleotide polymorphism) molecular marker locus related to maize kernel size provided by the present invention is derived from the maize gene X1 (GRMZM2G160032), located at the 959th nucleotide from the 5' starting end of the sequence shown in SEQ ID NO:1 (the flanking sequence of its locus) of the present invention. The nucleotide at the 959th bp of this sequence is A or G.

[0035] The present invention takes the association population composed of inbred lines with extreme kernel sizes and the association population composed of inbred lines from a wide range of sources as the research objects, identifies the genome-wide alternative splicing, and through correlation analysis, mines the alternative splicing genes related to kernel size. Combining with the genome-wide molecular markers, the general linear models of FastQTL and TASSEL software are respectively used to conduct association analysis on gene alternative splicing and kernel phenotypes, and a SNP locus significantly associated with maize kernel size is detected. The alleles of this locus are A and G, and there are two homozygous genotypes of AA and GG in the tested inbred lines, and its flanking sequence is shown in SEQ ID NO:1.

[0036] When the polymorphism locus of the molecular marker is A, it corresponds to large maize kernel shape; when the polymorphism locus of the molecular marker is G, it corresponds to small maize kernel shape.

[0037] The SNP molecular marker provided by the present invention is significantly correlated with maize kernel size. The 100-kernel weight of maize germplasm resources with the SNP locus genotype of AA is greater than that of maize germplasm resources with the locus genotype of GG.

[0038] Second aspect, the present invention provides a gene capable of negatively regulating the size of maize kernels. The gene is the X1 gene, and its DNA sequence is as shown in SEQ ID NO:1, the RNA sequence of its corresponding transcript T01 is as shown in SEQ ID NO:2, and the RNA sequence of its corresponding transcript T04 is as shown in SEQ ID NO:3.

[0039] The amplification primers for the above-mentioned gene X1 include NcoI primers and BstEII primers, and their nucleotide sequences are as shown in SEQ ID NO:6 and SEQ ID NO:7 respectively.

[0040] For the overexpression vector of the above-mentioned gene X1, its positive detection primer pair includes Bar-F and Bar-R, and their nucleotide sequences are as shown in SEQ ID NO:9 and SEQ ID NO:10 respectively.

[0041] Third aspect, the present invention provides a biological material carrying the X1 gene, and the biological material is an expression cassette, an expression vector, a host cell or a host bacterium.

[0042] The plant expression vector of the present invention is pCambia3301.

[0043] The present invention constructs the X1 gene into the expression vector pCambia3301 and propagates it in Escherichia coli. Through the agrobacterium-mediated transformation method, the X1 gene carried by pCambia3301 is transferred into maize Zong 31 (zong31), and transgenic maize overexpressing X1 is obtained. The results show that X1 has the function of inhibiting the size of maize kernels.

[0044] The present invention also provides a cloning vector or various expression vectors containing the X1 nucleotide sequence or its fragment, a host cell containing the vector, a transformed plant cell containing the nucleotide sequence or its specific fragment, and a transgenic plant.

[0045] Fourth aspect, the present invention also provides a maize X1 gene mutant, in which the base C at the 436th position downstream of the start codon ATG in the coding region of the maize X1 gene is mutated to T, and this mutation leads to premature termination of translation. Specifically, a base mutation occurs at the 436th position in the coding sequence (CDS) of the X1 gene of the maize genome (B73 V3, https: / / maizegdb.org / gene_center / gene / GRMZM2G160032), resulting in the loss of the function of the X1 gene.

[0046] The ems mutant of the above-mentioned gene X1 has a coding region sequence as shown in SEQ ID NO:8, and its genotype detection primer pair includes EMS-F and EMS-R, and the nucleotide sequences of the two are respectively as shown in SEQ ID NO:19 and SEQ ID NO:20.

[0047] The present invention provides the following applications of the above-mentioned maize X1 gene mutant or biological material containing the mutant: (1) Application in increasing grain size; (2) Application in plant genetic breeding for increasing grain size or yield; (3) Application in the construction of transgenic plants for increasing grain size or yield; (4) Application in the improvement of plant germplasm resources for increasing grain size or yield; The biological material is an expression cassette, a vector, a host cell or a host bacterium.

[0048] In the above applications, by reducing the expression level of the X1 gene, the grain size or yield of the plant is increased.

[0049] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art, and the raw materials used are all commercially available products.

[0050] Example 1 Alternative splicing analysis confirms that maize X1 gene is significantly correlated with grain size Based on the transcriptome data of 141 maize inbred lines (Li et al., 2020, cis-Regulatory variation affecting gene expression contributes to the improvement of maize kernel size), the grain alternative splicing map was mined, and the correlation analysis was performed between the intron splicing ratio (PSI) of the gene and the grain size phenotype data (grain length, grain width, grain thickness, hundred-grain volume and hundred-grain weight), with p-value < 1.0×10 -5 as the significance threshold, and a total of 38 genes with alternative splicing related to grain size were obtained. Among them, the alternative splicing in the 5’ UTR of the X1 gene (GRMZM2G160032) is significantly correlated with grain size. The intron splicing variation in the 5’ UTR region results in two transcripts of the X1 gene: T01 with a shorter 5’UTR sequence (the name of the splicing event is AS_eUTR) and T04 with a longer 5’UTR sequence (the name of the splicing event is AS_tUTR), and the coding region sequences of these two transcripts are exactly the same (Figure 1 ).

[0051] Comparing the splicing event PSI with gene expression, it was found that AS_eUTR was positively correlated with the expression level of gene X1, and AS_tUTR was negatively correlated with the expression level of gene X1 ( Figure 2 , A).

[0052] Comparing gene expression with grain size data, it was found that there was a weak correlation between the expression of gene X1 and grain size, which was easily overlooked in the study of gene expression at the whole genome level ( Figure 2 , B).

[0053] Comparing the expression levels of transcripts generated by alternative splicing with grain size data, it was found that T01 generated by AS_eUTR was significantly negatively correlated with grain size, and T04 generated by AS_tUTR was significantly positively correlated with grain size ( Figure 2 , C).

[0054] The correlation between gene X1 and grain size identified by alternative splicing analysis was supported by the transcriptome data of 236 independent maize inbred lines (Fu et al., 2013, RNA sequencing reveals the complex regulatory network in the maize kernel). Above, through the relationship between population alternative splicing variation and grain size, gene X1 was found to be significantly correlated with grain size.

[0055] The DNA sequence of the said gene X1 is shown as SEQ ID NO:1 (from https: / / www.maizegdb.org / ); the cDNA sequence corresponding to T01 is shown as SEQ ID NO:2 (from https: / / www.maizegdb.org / ); the cDNA sequence corresponding to T04 is shown as SEQ ID NO:3 (from https: / / www.maizegdb.org / ); the DNA sequence corresponding to CDS is shown as SEQ ID NO:4 (from https: / / www.maizegdb.org / ); the amino acid sequence is shown as SEQ ID NO:5 (from https: / / www.maizegdb.org / ).

[0056] The above materials are all common maize materials and can be obtained by conventional means; the genotype and grain size phenotype data were obtained from published articles.

[0057] Example 2 A SNP molecular marker significantly correlated with grain size of maize X1 gene Using whole-genome SNP markers of 141 and 368 maize inbred line materials (Li et al., 2020, cis-Regulatory variation affecting gene expression contributes to the improvement of maize kernel size; Fu et al., 2013, RNA sequencing reveals the complexregulatory network in the maize kernel) as genotypes and intron splicing ratio PSI as molecular phenotypes, the general linear model of the association analysis software FastQTL was used to identify the lead SNP of the sQTL of the cis-regulatory site within 10 kb upstream and downstream of the 5'UTR of the X1 gene in both populations: S3_216434043 (located at the 216434043rd nucleotide on chromosome 3 of B73 V3 and the 959th nucleotide of the X1 gene), which had the highest significance of association. This SNP has two genotypes, CC and TT. The haplotype distribution of PSI based on this SNP in the 141 materials is shown in the following figure. Figure 3 As shown in A.

[0058] The genotype and grain phenotype data of 337 inbred lines from the two populations were combined, and the general linear model (LM) of the association analysis software TASSEL was used to identify SNP markers associated with grain size with a significant threshold of p-value < 0.001. Among them, the lead SNP of the sQTL was significantly associated with 100-grain weight, 100-grain volume, grain length, and grain width.

[0059] Based on the haplotype analysis of grain size based on this SNP marker, it was found that the 100-grain weight, 100-grain volume, grain length, and grain width of inbred lines with different haplotypes were significantly different. The grain size of the TT genotype was larger ( Figure 3 , BE). Allele TT is considered as superior / enhancing allele.

[0060] Example 3 Confirmation of the overexpression phenotype of corn kernel size gene X1 Using the cDNA of maize inbred line CL012 (small grain) as a template, the coding region of the X1 gene was amplified using SEQ ID NO:6 and SEQ ID NO:7, and an HA-tag was added to its end. The vector was ligated to the overexpression vector (CaMV35S promoter, pCambia3301 vector backbone), and the vector was transformed into maize inbred line 31 (zong31) by the Agrobacterium method and named OE.

[0061] Positive detection of the OE strain using SEQ ID NO:9 and SEQ ID NO:10 yielded two independent T1 generation positive transgenic ears. Identification of the transgenic vector in the T1 generation plants confirmed that the inserted fragment was a single copy.

[0062] RNA was extracted from the grains of T2 generation negative and positive transgenic materials and the receptor material Zong 31 at 15 days after pollination, respectively. The expression levels of the X1 gene in the transgenic materials were determined using SEQ ID NO:11 and SEQ ID NO:12. The results showed that the expression level of the X1 gene in the OE2 transgenic material was significantly increased by 30-fold compared to the control Zong 31, while OE1 was only increased by 1.6-fold compared to the control Zong 31 ( Figure 4 , A).

[0063] Phenotypic identification of the mature grains of T3 generation negative and positive transgenic materials of OE2 was carried out. The statistical results showed that the 100-grain weight of the grains overexpressing the X1 gene was significantly decreased, only 74.27% of the wild type, and the grain length was significantly decreased by 9.44% compared to the wild type ( Figure 4 , B and C), indicating that the X1 gene has a negative regulatory effect on the grain length and grain weight traits of grains.

[0064] Example 4 Confirmation of grain phenotypes of EMS mutants of maize grain genes To further determine the negative effect of the X1 gene on grain size and grain weight, EMS mutants related to the X1 gene were screened from the B73 background in this study. The DNA sequence of the X1 gene in the mutant was as shown in SEQ ID NO:8. The base C at position 436 downstream of the ATG in X1-EMS was mutated to T, which led to premature termination of translation, and the encoded protein was truncated from 628 amino acids to 145 amino acids ( Figure 5 , A).

[0065] To observe the regulation of X1 on grain phenotypes in different background materials, X1-EMS was hybridized with large-grain materials in the association population: 9508, BAIHE43, HUANGLV12, and HAI9_21, and small-grain materials: RW64A, HUANGJIN63, and CL012, and then self-crossed continuously to the F3 generation. SEQ ID NO:19 and SEQ ID NO:20 were used to detect whether the genotypes of the F3 generation materials were wild type or mutant.

[0066] Phenotypic analysis showed that the grain weight of the F3 generation of the cross between the small-grain material CL012 and X1-EMS was significantly up-regulated (37.02%), and the grain length and grain thickness of the F3-segregating mutant grains were also significantly increased by 11.59% and 16.30% respectively compared to the F3-segregating wild type ( Figure 5, B-C). Combining the above results, it indicates that the X1 gene has a negative regulatory effect on the traits of grain length and grain weight, and this regulatory effect is affected by different backgrounds.

[0067] Example 5 Expression pattern of maize X1 gene To study the expression pattern of the X1 gene, grains at different days after pollination of B73 plants, as well as embryos and endosperms at some stages, were selected to extract RNA. Using qRT-PCR technology, with two pairs of primers (SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, SEQ ID NO:16), the expression patterns of two different spliced transcripts of the X1 gene at the maize grain development stage were detected. The results showed that both of the two different spliced transcripts of the X1 gene were expressed in grains, but the expression level of the T04 transcript was significantly higher than that of the T01 transcript, and it was mainly expressed in the endosperm, reached the peak 12 days after pollination, and then began to decline ( Figure 6 , A).

[0068] The subcellular localization of the X1 protein was predicted online using TargetP (http: / / www.cbs.dtu.dk / services / TargetP / ) and Predotar (https: / / urgi.versailles.inra.fr / predotar / predotar.html). The results showed that no obvious signal peptide was found, and it was speculated to be located in the cytoplasm. To determine the subcellular localization of X1, yellow fluorescent protein (YFP) was fused with the 5′ end and 3′ end of the full-length CDS fragment of X1 respectively. The fusion vector was injected into Nicotiana benthamiana leaves by Agrobacterium tumefaciens, and the X1 protein was transiently expressed. The fluorescence signal was detected by a confocal laser microscope 48 hours after injection. The results showed that YFP signals were observed in both the cytoplasm and the nucleus whether YFP was fused at the 5′ end or the 3′ end, indicating that the X1 protein might be localized in the cytoplasm and the nucleus ( Figure 6 , B). The primers for subcellular localization were SEQ ID NO:17 and SEQ ID NO:18.

[0069] Example 6 Application of maize grain length gene X1 in the improvement breeding of maize grain shape For the T3 and T4 generations of 2 independent positive events each of the maize kernel size gene X1 overexpression lines and EMS lines, they were all planted in Beijing and Sanya City, Hainan Province in November 2021. Leaves of the corresponding materials were taken at the seedling stage, and DNA was extracted using the CTAB method. The transgenic overexpression materials were positively detected using the universal primers Bar-F and Bar-R. The DNA sequence of the universal primer Bar-F is as shown in SEQ ID NO:8, and the DNA sequence of Bar-R is as shown in SEQ ID NO:9. Using the PCR product sequencing method, the EMS hybrid materials were genotyped using the universal primers EMS-F and EMS-R. The DNA sequence of the universal primer EMS-F is as shown in SEQ ID NO:19, and the DNA sequence of EMS-R is as shown in SEQ ID NO:20. Two independent and homozygous transformation events were selected for the combination of testcross materials with different inbred line materials. Currently, 1 new germplasm of overexpression materials for detecting kernel phenotypes has been created, and the relevant germplasm has been introgressed into the backgrounds of the other four backbone inbred lines; and 1 new germplasm of EMS for detecting kernel phenotypes has been tested.

[0070] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. Use of maize grain size regulatory gene X1 in regulating plant grain shape, grain weight and yield; The gene X1 is a gene encoding the following protein (A) or (B): (A) a protein consisting of the amino acid sequence shown in SEQ ID NO:5; or (B) a protein derived from (A) with one or several amino acids substituted, deleted or added in the sequence shown in SEQ ID NO:5 and having the same function; The grain shape is grain length; The regulation is negative regulation; Preferably, the plant is a gramineous plant, more preferably maize.

2. Method for increasing corn kernel length, kernel weight and yield, characterized in that, The method includes: using genetic engineering means to modify the maize grain size regulatory gene X1 so that the gene function is lost, thereby increasing the maize grain length, grain weight and yield; The gene X1 is the same as the gene described in claim 1.

3. A method for shortening the length of corn kernels, reducing the grain weight and yield, characterized in that, The method includes: using genetic engineering means to overexpress the maize grain size regulatory gene X1 in maize; the gene X1 is the same as the gene described in claim 1; The mode of overexpression is selected from the following 1) to 5), or an optional combination: 1) By introducing a plasmid with the gene; 2) By increasing the copy number of the gene on the plant chromosome; 3) By changing the promoter sequence of the gene on the plant chromosome; 4) By operably linking a strong promoter to the gene; 5) By introducing an enhancer.

4. Use of the transgenic maize obtained by the method according to claim 2 or 3 in plant breeding; The breeding methods include transgenic, hybridization, backcross, self-cross or asexual reproduction.

5. A mutant of the maize grain size regulatory gene X1, characterized in that, It is a gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence from the 1st to the 145th amino acid sequence shown in SEQ ID NO:5; or (b) a protein derived from (a) with one or several amino acids substituted, deleted or added in the amino acid sequence from the 1st to the 145th amino acid sequence shown in SEQ ID NO:5 and having the same function.

6. A biological material containing the gene mutant described in claim 5, and the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector or engineering bacteria.

7. Any of the following applications of the gene mutant described in claim 5 or the biological material described in claim 6: (1) For increasing plant grain size; (2) For increasing plant yield; (3) For constructing transgenic plants; (4) For plant genetic breeding or plant germplasm resource improvement.

8. SNP molecular markers for regulating maize kernel size, characterized in that, The SNP molecular marker contains a nucleotide sequence with a polymorphism of A / G at the 959th position of the sequence shown in SEQ ID NO:1 of the maize grain size regulatory gene X1; The grain length and grain weight of the maize germplasm resource with the genotype of AA at the locus with the polymorphism are greater than those of the maize germplasm resource with the genotype of GG.

9. Primers for amplifying the SNP molecular marker described in claim 8 or a detection reagent or kit containing the primers.

10. Any of the following applications of the SNP molecular marker described in claim 1 or the primers described in claim 9 or a detection reagent or kit containing the primers: 1) For identifying maize grain size; 2) For the identification, improvement or molecular marker-assisted breeding of maize germplasm resources; 3) For the early prediction of maize grain size and yield; 4) For screening maize with large grains and high grain weight.

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