Maize kernel size and amino acid content regulatory gene ZmCRK3, its encoding protein, SNP sites, functional markers and applications

Abstract

The application discloses a corn size and amino acid content regulating gene ZmCRK3 , its encoded protein, SNP site, its functional marker and application, aiming to provide excellent haplotypes and germplasm resources with excellent haplotypes for improving corn yield and quality. ZmCRK3 The kernel mutant of the gene was identified by genotyping and linkage analysis to be co-segregated with the kernel mutant phenotype. ZmCRK3 The mutation affects the kernel size and amino acid content of corn, and provides primers, amplification systems and PCR reaction procedures for detecting its mutant genotype; through association analysis, SNPs that affect kernel size and amino acid content were discovered, and haplotype analysis clarified ZmCRK3 The excellent haplotypes were identified ZmCRK3 Germplasm resources with excellent haploidy; utilizing the genes and molecular markers disclosed in this application can facilitate the creation of high-yield and high-quality breeding materials for corn, shorten the breeding cycle of new corn varieties, reduce breeding costs, and improve breeding efficiency.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and specifically to a gene that regulates corn kernel size and amino acid content. ZmCRK3、 and its encoded proteins, SNP sites, functional markers and applications. Background Art

[0002] corn( Zeamays L. ) is one of the most important food crops in the world, with many important biological and economic characteristics. Corn is the third largest food crop in the world, second only to wheat and rice. Corn is also one of the main sources of energy for humans, providing a large amount of calories and protein. Corn can also be used to make starch, alcohol, feed, cosmetics and industrial raw materials. In addition, corn can also be used as pet food. In short, corn plays an important role in agriculture, food processing, medicine, energy and other fields.

[0003] Grain is the main organ that determines corn yield and quality. The development and key regulatory genes of grain have an impact on grain quality. For example, key genes of amino acids, oil, carotenoids, etc. have been reported a lot. However, grain development and formation is an extremely complex biological process, which is regulated by key node genes in multiple pathways. Therefore, it is still necessary to further explore the key genes that affect corn yield and quality, identify their excellent haplotypes, and analyze their utilization pathways.

[0004] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0005] ZmCRK3 It's yeast Dbp5 and Arabidopsis thaliana LOS4 Studies have shown that Dbp5 and LOS4 affect the nuclear export of mRNA, regulating the growth and development of Arabidopsis and the response to adverse stress, while maize ZmCRK3 The mechanism of action and its regulatory mechanism on grain development have not been reported, and its utilization in corn yield and quality breeding also needs to be elucidated.

[0006] The present invention applies for ZmCRK3 CRISPR-Cas9 knockout mutants, grain phenotype and genotype analysis, and identification based on mutation site marker analysis ZmCRK3 Linked to the grain mutant phenotype, this gene encodes a DEAD-box helicase. ZmCRK3 Regulating the development of corn embryo and endosperm, and thereby affecting the accumulation of nutrients in the grain, is of great value to improving corn yield and quality.

[0007] In the first aspect disclosed in the present application, a gene regulating corn kernel size and amino acid content is identified. ZmCRK3 , its DNA sequence is shown in SEQ ID NO.1, and the sequences of its amplification primers CRK3-F / R are shown in SEQ ID NO.4 and SEQ ID NO.5; or its cDNA sequence is shown in SEQ ID NO.2.

[0008] The second aspect disclosed in the present application relates to a gene regulating corn kernel size and amino acid content ZmCRK3 The amino acid sequence of the encoded protein is shown in SEQ ID NO.3.

[0009] The third aspect disclosed in the present application relates to a corn kernel phenotypic mutation site, based on the presence of an AGA deletion / insertion in the 12336851-12336853 site region of chromosome 1 on the fourth edition of the corn reference genome B73.

[0010] The fourth aspect disclosed in the present application relates to a CAPS molecular marker for identifying corn kernel phenotypic mutants, comprising the following primers:

[0011] F: 5'-TATCGGCACATCTGGTACGC-3',

[0012] R: 5'-TAGCCAAGCACAGTCAGAGC-3';

[0013] It is designed based on the following sequence differences:

[0014] There is a 3 bp deletion (AGA) in the 12,336,851-12,336,853 site region of chromosome 1 of the maize reference genome B73, version 4, and the flanking sequences at both ends of the deletion are shown in SEQ ID NO.8-9.

[0015] In a fifth aspect disclosed in the present application, the corn kernel size and amino acid content regulating gene ZmCRK3 , the corn kernel phenotypic mutation site or the CAPS molecular marker is applied to the regulation of kernel size and / or corn amino acid content traits.

[0016] Another aspect disclosed in the present application relates to a SNP site that is significantly associated with corn kernel size and amino acid content. Based on the fourth version of the corn B73 reference genome, there are C / T mutations at chr1.s_12340784 and chr1.s_12340704, and there is a C / G mutation at chr1.s_12340707 (C / G).

[0017] Another aspect disclosed in the present application relates to a method for identifying a gene ZmCRK3 The dCAPS molecular markers of excellent haplotypes include primer pairs as shown in SEQ ID NO.10 and SEQ ID NO.11, SEQ ID NO. 12 and SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, and SEQ ID NO.16 and SEQ ID NO.10.

[0018] In another aspect disclosed in the present application, the SNP site or the dCAPS molecular marker is used to identify genes ZmCRK3 Among the excellent haplotypes.

[0019] In another aspect disclosed in the present application, the corn kernel development regulating gene ZmCRK3 , the application of the corn kernel phenotypic mutation site, the CAPS molecular marker, the SNP site or the dCAPS molecular marker in the breeding of varieties / lines for corn kernel size and / or amino acid content.

[0020] Another aspect disclosed in the present application is a method for breeding corn varieties / lines with high amino acid content, comprising the following steps:

[0021] (1) Detect and determine the genotypes of maize inbred line population materials at positions chr1.s_12340707 and chr1.s_12340784 based on the fourth version of the maize B73 reference genome;

[0022] (2) Selecting individuals with GT haplotype at positions chr1.s_12340707 and chr1.s_12340784 from the maize inbred line population material to increase the distribution frequency of the superior haplotypes generation by generation, thereby breeding varieties / lines with high amino acid content.

[0023] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0024] 1. Positional Cloning ZmCRK3 Genetic regulation of corn embryo and endosperm development affects the accumulation of grain storage materials and is of great value in improving corn yield and quality.

[0025] 2. ZmCRK3 The identification of excellent haplotypes and analysis of their utilization pathways provide technical support and excellent germplasm resources for the utilization of this gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 In the embodiment of this application ZmCRK3 Gene structure and amino acid sequence diagram; in the figure, A: ZmCRK3Gene structure and location of 3bp deletion; B: Alignment of amino acid sequences of wild-type and mutant ZmCRK3.

[0027] Figure 2 This is an analysis diagram of grain phenotypes and related indicators in the examples of the present application; in the figure, A: separation of wild-type and mutant grains in heterozygous ears; B: difference in light transmittance between wild-type and mutant grains on a light box and different proportions of floury endosperm; C: difference in accumulation of alcohol-soluble and non-alcohol-soluble proteins in wild-type and mutant grains; D: difference in amino acids between wild-type and mutant mature grains.

[0028] Figure 3 In the embodiment of this application ZmCRK3 Marker detection and linkage analysis of the mutation site of the gene; in the figure, A: 1-9 and 10-18 are the phenotypes of wild-type and mutant mature grains, respectively; B: 3bp deletion site detection: the upper band of 229bp is the wild type without deletion, the lower band of 184bp is the mutant with deletion, and the band in each lane corresponds to the grain in Figure A.

[0029] Figure 4 This is the result of the correlation analysis between grain length and amino acid content in one embodiment of the present application; in the figure, the upper ZmCRK3 Manhattan plot of the association analysis between SNP and grain length and amino acid content; in the middle is ZmCRK3 Schematic diagram of the gene structure; below is the LD block diagram of the SNP.

[0030] Figure 5 It is a molecular marker for identifying haplotypes in an embodiment of the present application; in the figure, A: dCAPS-F1R1 PCR product in agarose gel electrophoresis band; B: dCAPS-F2R2 (chr1.s_12340704) PCR product in 12% polyacrylamide gel band; C: dCAPS-F2R2 PCR product MwoI After enzyme digestion, the electrophoresis bands in 12% polyacrylamide gel; D: dCAPS-F3R3 (chr1.s_12340707) PCR product bands in 12% polyacrylamide gel; E: dCAPS-F3R3 PCR product after EcoRV After enzyme digestion, the electrophoresis bands in 12% polyacrylamide gel; F: dCAPS-F4R1 (chr1.s_12340784) PCR product in 12% polyacrylamide gel; G: dCAPS-F4R1 PCR product after MwoIElectrophoresis bands in 12% polyacrylamide gel after enzyme digestion; Note: Lane 1 (CIMBL96) and 2 (CIMBL98) are Hap1; Lane 3 (B11), 4 (CIMBL10) and 5 (647) are Hap2; Lane 6 (CIMBL113) and 7 (CIMBL11) are Hap3; Lane 8 (CIMBL25), 9 (CIMBL18), 10 (CIMBL101) and 11 (CIMBL13) are Hap4. DETAILED DESCRIPTION

[0031] The specific implementation modes of the present invention are described below in conjunction with the accompanying drawings and examples. However, the following examples are only used to illustrate the present invention in detail and do not limit the scope of the present invention in any way.

[0032] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents and raw materials involved are all commercially available conventional products unless otherwise specified; the tests and detection methods involved are all conventional methods unless otherwise specified.

[0033] Embodiment 1: ZmCRK3 Phenotypic identification and genotypic analysis of genes

[0034] Utilizing the target gene knocked out by the inventor's research group during the grain proteomics research ZmCRK3 The resulting grain mutants (collected by Tang Jihua in Yuanyang, Henan in the summer of 2019) were identified through phenotypic analysis and genotypic identification. ZmCRK3 is a gene that regulates grain development and storage material accumulation (see Figure 1 ).

[0035] The mutant kernels were wrinkled at the top and lighter in color. The mutant kernels were opaque and had a higher proportion of powdery endosperm than the wild-type kernels. The detection of alcohol-soluble and non-alcohol-soluble proteins revealed that the γ-alcohol-soluble protein in the mutant endosperm was significantly reduced. The contents of most amino acids in mature kernels of the wild-type and mutant kernels were different (see Figure 2 ).

[0036] Embodiment 2: ZmCRK3 Molecular marker design and linkage analysis

[0037] A PCR molecular marker was developed for the 3bp deletion, which is easy to detect the functional nucleotide sequence.

[0038] B73V4: Chr1: 12336851-12336853, indicating the actual position of the 3bp deletion marker product on the fourth version of the reference genome B73.

[0039] Based on this, the following CAPS primers were designed (as shown in SEQ ID NO.6-7):

[0040] F: 5'-TATCGGCACATCTGGTACGC-3' (SEQ ID NO.6),

[0041] R: 5'-TAGCCAAGCACAGTCAGAGC-3' (SEQ ID NO.7);

[0042] The detection of CAPS molecular markers includes the following steps:

[0043] (1) The reaction system is:

[0044] DNA template (30-50 ng / μL) 1 μL

[0045] Left primer (10 μM) 1 μL

[0046] Right primer (10 μM) 1 μL

[0047] ddH2O 2 μL

[0048] 2×Taq Plus Master Mix II 5 μL.

[0049] (2) The PCR amplification procedure for CAPS is:

[0050] 1 cycle 95℃, 3 min;

[0051] 35 cycles of 95°C denaturation for 15 s, 60°C annealing for 20 s, and 72°C extension for 30 s;

[0052] Delay 72℃ 5 min;

[0053] Soak 25℃.

[0054] (3) The enzyme digestion system of CAPS is:

[0055] PCR product 1 μL

[0056] ddH2O 7.8 µL

[0057] 10× Buffer R 1 µL

[0058] Van91I (ER0711, Thermo Scientific™) 0.2 µL.

[0059] (4) The enzyme digestion procedure of CAPS is:

[0060] 37℃ for 10 h, 65℃ for 20 min.

[0061] The test results of CAPS are as follows: Figure 3 shown.

[0062] CAPS markers were designed to identify the differences in 3bp deletion fragments. ZmCRK3 The product lengths are 229 bp and 184 bp respectively, of which 229 bp represents the wild-type individual and 184 bp represents the material with 3 bp deletion.

[0063] Embodiment three: ZmCRK3 Candidate gene association analysis

[0064] To clarify ZmCRK3 The utilization pathway of the gene was to use 182 SNPs (single nucleotide polymorphism sites) (B73V4: 12332451-12340928) of this gene in 507 inbred lines as genotypes, and candidate gene association analysis was performed based on the grain phenotype, population structure (Q) and kinship (K) of the population (P<1 / 182=5.49E-03).

[0065] A total of 43 SNPs were detected that were significantly associated with grain length, amino acid composition, and total amino acid content (see Tables 1 to 7). ZmCRK3 The chr1.s_12340704, chr1.s_12340707 and chr1.s_12340784 of the 3'UTR of the gene were significantly higher in amino acid composition and total amino acid content (p<0.05 / 182=2.75E-04, see Figure 4 ).

[0066] Table 1 SNPs significantly associated with grain length and amino acid content

[0067]

[0068] Table 2 SNPs significantly associated with grain length and amino acid content (continued from Table 1)

[0069] .

[0070] Table 3 SNPs significantly associated with grain length and amino acid content (continued from Table 1)

[0071] .

[0072] Table 4 SNPs significantly associated with grain length and amino acid content (continued from Table 1)

[0073] .

[0074] Table 5 SNPs significantly associated with grain length and amino acid content (continued from Table 1)

[0075] .

[0076] Table 6 SNPs significantly associated with grain length and amino acid content (continued from Table 1)

[0077] .

[0078] Table 7 SNPs significantly associated with grain length and amino acid content (continued from Table 1)

[0079] .

[0080] Embodiment 4: ZmCRK3 Excellent haplotype identification

[0081] Among the loci significantly associated with amino acid content, chr1.s_12340704 and chr1.s_12340707 were completely LD. Therefore, only chr1.s_12334562 located in the 5'UTR and chr1.s_12340704, chr1.s_12340707 and chr1.s_12340784 located in the 3'UTR were used for haplotype analysis. Among the 444 inbred lines, there were 4 combinations with extremely significant differences in amino acid content ( p <9.99E-04) (see Table 8): Hap1 (CC 15 copies), Hap2 (GC 84 copies), Hap3 (CT 334 copies), Hap4 (GT 57 copies). Among them, the content of most amino acids of the superior haplotype Hap4 is higher than that of other haplotypes, and its distribution frequency in inbred lines is low (57 / 444), which has the potential for use in breeding improvement; the content of all amino acids of the unfavorable haplotype Hap3 is lower than that of other haplotypes, and its distribution frequency in inbred lines is high (334 / 444); the distribution frequencies of other haplotypes Hap1 and Hap2 are also relatively low (15 / 444, 84 / 444), and the amino acid content is between Hap4 and Hap3 (see Table 8).

[0082] Table 8 Haplotype analysis

[0083] .

[0084] Embodiment five: ZmCRK3 Development of molecular markers for superior haplotypes

[0085] Based on the identification of superior amino acid haplotypes, simple and easy-to-operate PCR markers dCAPS / CAPS were developed for these three SNPs. Their actual positions in the fourth version of the maize B73 reference genome are 12340704 bp, 12340707 bp and 12340784 bp on chromosome 1, respectively.

[0086] dCAPS-F1:5'-GCAATTTACAGTGTAAAATAGTATTTTCCA-3' (SEQ ID No. 10);

[0087] dCAPS-R1:5'-TCCACCAACCATGATGTCAT-3' (SEQ ID No. 11).

[0088] dCAPS-F2: 5'-CAAAGTTTTGCTATGGCTTTCTGTG -3' (SEQ ID No. 12);

[0089] dCAPS-R2:5'-GTAATTTTCTGCCATGAAGATAGCTCC-3' (SEQ ID No. 13).

[0090] dCAPS-F3: 5'-TTTATTTGCCGTGGAGACGC-3' (SEQ ID No. 14);

[0091] dCAPS-R3: 5'-GCATGTCACATGACGCGATAT-3' (SEQ ID No. 15);

[0092] dCAPS-F4: 5'-GGCAGAAAATTACACGGACA-3' (SEQ ID No. 16).

[0093] dCAPS-F1R1 is a first-round PCR primer pair for chr1.s_12340704, chr1.s_12340707, and chr1.s_12340784. The PCR product of dCAPS-F1R1 diluted 10 times was used as a template for the second round of amplification. dCAPS-F2R2 is a second-round PCR primer pair for chr1.s_12340704. The restriction endonuclease MwoIThe sequence with T base at this site cannot be cut and is a larger band, while the sequence with C base at this site can be cut and is a smaller band (GcTTTCTGTGc / t, the first c is the introduced mismatch site, and c / t is the difference in the site of the inbred line itself); dCAPS-F3R3 is the second round PCR primer pair of chr1.s_12340707, and the restriction endonuclease EcoRV The sequence of the C base at this site cannot be cut and is a larger band, while the sequence of the G base at this site can be cut and is a smaller band (g / cATAtC, g / c is the difference in the site of the inbred line itself, and t is the introduced mismatch site); dCAPS-F4R1 is the second round PCR primer pair of chr1.s_12340784, and the restriction endonuclease MwoI After enzyme digestion, the DNA was detected using 12% polyacrylamide gel electrophoresis. The sequence with T base at this site could not be cut and was a larger band, while the sequence with C base at this site could be cut and was a smaller band (Gc / tGGGCGAAGC, c / t is the difference in the site of the inbred line itself, and this site is a CAPS marker).

[0094] Molecular marker detection for excellent haplotypes includes the following steps:

[0095] (1) The reaction system is:

[0096] DNA template (30~50 ng / μL) 1μL; Left primer (10μM) 0.5μL; Right primer (10 μM) 0.5μL; ddH2O 2 μL; 2×PCR Mix 5μL.

[0097] (2) The PCR amplification program for dCAPS-F1R1, dCAPS-F2R2, dCAPS-F3R3 and dCAPS-F4R1 was as follows: 1 cycle of pre-denaturation at 95℃ for 3 min; 35 cycles of denaturation at 95℃ for 15 s, annealing at 57℃ for 20 s, and extension at 72℃ for 30 s; Delay at 72℃ for 5 min; Soak at 25℃.

[0098] (3) The PCR amplification program of dCAPS-F1R1 was as follows: 1 cycle of pre-denaturation at 95℃ for 3 min; 35 cycles of denaturation at 95℃ for 15 s, annealing at 60℃ for 20 s, and extension at 72℃ for 30 s; Delay at 72℃ for 5 min; Soak at 25℃.

[0099] (4) The enzyme digestion procedure of dCAPS is: 37℃, 10 h; 65℃, 20 min.

[0100] Test results such as Figure 5 shown.

[0101] Although some preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0102] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations to the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for breeding corn varieties / lines with high amino acid content, comprising the following steps: (1) Detect and determine the genotypes of maize inbred line population materials at positions chr1.s_12340704, chr1.s_12340707, and chr1.s_12340784 based on the fourth version of the maize B73 reference genome; (2) Selecting individuals with haplotypes T, G, and T at positions chr1.s_12340704, chr1.s_12340707, and chr1.s_12340784, respectively, from the maize inbred line population material, and increasing the distribution frequency of superior haplotypes generation by generation through self-pollination, thereby breeding varieties / lines with high amino acid content; the amino acid is at least one of Arg, Asp, Gly, His, Ile, Lys, Thr, and Val.

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