Maize MYB transcription factor salt-tolerant gene and application thereof

Abstract

This application belongs to the field of molecular breeding technology. Specifically, this application provides a maize MYB-type transcription factor salt tolerance gene and its application. The nucleotide sequence of the salt tolerance gene is SEQ ID NO.1. This invention identifies a novel major maize salt tolerance gene MYB28 and its nucleotide sequence, which can be used to regulate maize salt tolerance. This invention also identifies a non-synonymous SNP site (SNP1409) within this gene that causes amino acid changes; the single nucleotide polymorphism at this site is closely related to maize salt tolerance. This application provides new materials and methods for accelerating the breeding process of salt-tolerant maize varieties.

Description

Technical Field

[0001] This application belongs to the field of molecular breeding technology. Specifically, this application provides a maize MYB-type transcription factor salt tolerance gene and its application. Background Technology

[0002] In saline-alkali soils, sodium ions (Na+) + Na+ is the most common and harmful cation; reducing Na+ + Entry into aboveground tissues is an important salt tolerance mechanism for most sweet soil plants (Yang et al., 2003; Munns and Tester, 2008). Maize (Zea mays ssp. mays) is one of the three major crops in my country. In recent years, my country's demand for maize-related products has been continuously increasing, and the self-sufficiency gap for maize has been widening. At the same time, the negative impact of environmental stresses such as salinity and alkalinity on maize production in my country has also shown a continuous upward trend, which brings new challenges to the sustainable development of maize production in my country (Egamberdieva et al., 2019; Zhang et al., 2019). Existing studies have shown that the Na+ content in the aboveground parts of different maize varieties... + Significant differences exist in the content and salt tolerance of these compounds (Liang et al., 2024). In recent years, a few studies have shown that the Na+ content in maize under salt stress varies significantly. + Genes related to homeostasis maintenance and salt tolerance were cloned, and they were found in different Na+ environments. + They play a role in maintaining homeostasis and provide important genetic resources for maize's salt and alkali tolerance. For example, through population Na... + Content-related QTL and GWAS analyses identified Na content-related components in maize xylem vessels. + Uninstalled ZmNC1 / ZmHKT1 and Na + Selective HAK / KUP / KT family transporters ZmNC2 / ZmHAK4 and ZmHAK11. These two proteins belong to different families and transport Na+ in xylem vessels. + Transported into surrounding parenchyma cells to reduce Na+ + Root transport to the aboveground parts reduces Na + Accumulation in the aboveground parts promotes salt tolerance in maize (Zhang et al., 2018; Zhang et al., 2019). Studies show that ZmqKC3 / ZmHKT2 encodes an HKT family transporter that negatively regulates potassium levels in the aboveground parts under salt stress. + Content and salt tolerance of maize (Cao et al., 2019). For example, Cao et al. found that the degradation of ZmNSA1 can lead to H+ content in the plasma membrane. +Increased transcriptional levels of ATPases (MHA2 and MHA4) enhance plasma membrane H... + - The function of ATPase is to promote root H + Efflux and transmembrane H + Gradient maintenance, thereby promoting Na + Homeostasis and salt tolerance in maize (Cao et al., 2020). These genes have elucidated the mechanism of Na homeostasis regulation to some extent, but many other regulatory mechanisms remain unknown.

[0003] MYB transcription factors are one of the largest transcription factor families in plants, widely distributed across plant species and comprising a large number of members. Based on the number of conserved domains, they can be divided into four subfamilies. This family is prevalent in plant genomes and plays a crucial role in plant transcriptional regulatory networks (Dubos et al., 2010; Yanhui et al., 2006; Wang et al., 2023). The first MYB gene identified in plants was ZmMYBC1 in maize, which mediates anthocyanin synthesis in maize (Pazares et al., 1987). Subsequent studies have identified over 22,000 genes encoding MYB proteins in 24 plant species, including Arabidopsis, rice, wheat, and soybean. MYB transcription factors have a wide range of functions, participating in various aspects of plant growth and development, including cell differentiation, organ morphogenesis, cell cycle regulation, secondary metabolism regulation, reproductive process regulation, and hormone signaling responses. They also participate in plant responses to biotic and abiotic stresses. Although MYB transcription factors are characterized by their diverse functions and wide distribution, the molecular mechanism by which they regulate ion homeostasis under salt stress in maize remains unclear. Therefore, given the current situation of insufficient available gene resources and scarcity of salt-tolerant materials in maize breeding, identifying key salt-tolerant genes and developing their molecular markers has significant application value. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention discovers the MYB28 maize variety through positive genetic screening. W22 Larger fodder material MYB28 T8759 More salt-tolerant. Overexpression of MYB28 W22 Materials were salt-sensitive, while knockout materials obtained using CRISPR-Cas9 technology exhibited stronger salt tolerance. MYB28 negatively regulates salt tolerance in maize; its coding region is 1077 bp long and encodes 358 amino acids. Further research revealed that the MYB28 allele (MYB28) derived from teosinte T8759... T8759 The transcriptional repression activity was significantly stronger than that of the allele derived from maize W22 (MYB28). W22A non-synonymous SNP (SNP1409, W341G) within its coding region is the key variant leading to the difference in salt tolerance.

[0005] On the one hand, this application provides a maize MYB-type transcription factor salt tolerance gene, the nucleotide sequence of which is SEQ ID NO.1.

[0006] On the other hand, this application provides a maize MYB-type transcription factor encoded by the above-mentioned salt tolerance gene, wherein the amino acid sequence of the maize MYB-type transcription factor is SEQ ID NO.2.

[0007] On the other hand, this application provides the application of the above-mentioned salt tolerance genes or maize MYB-type transcription factors in regulating maize salt tolerance.

[0008] Furthermore, in the application, the above-mentioned salt tolerance gene is knocked out in maize to improve the salt tolerance of maize; or in the application, the above-mentioned salt tolerance gene is overexpressed to make maize more sensitive to salt stress.

[0009] Furthermore, the salt tolerance gene can be knocked out using the CRISPR-Cas9 system; or the salt tolerance gene can be overexpressed using the pBCXUN vector.

[0010] Furthermore, the target sequences SEQ ID NO.13 and SEQ ID NO.14 of the above-mentioned salt tolerance gene were knocked out using the CRISPR-Cas9 system.

[0011] On the other hand, this application provides a maize salt tolerance molecular marker SNP1409, which is located at position 1023 of the salt tolerance gene shown in SEQ ID NO.1. Its polymorphism is T or G. When it is T genotype, it is salt resistant, and when it is G genotype, it is salt sensitive.

[0012] On the other hand, this application provides a primer pair for detecting the above-mentioned corn salt-tolerant molecular marker SNP1409, the primer pair including qNaC3-F1 and qNaC3-R1, the nucleotide sequences of which are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0013] On the other hand, this application provides a method for detecting whether maize is salt-tolerant, comprising: using the maize genomic DNA to be tested as a template, performing PCR amplification using the above-mentioned primer pair, sequencing the amplification product, and detecting the genotype of the SNP1409 locus. If it is the T genotype, it is determined to be salt-tolerant; if it is the G genotype, it is determined to be salt-sensitive.

[0014] On the other hand, this application provides the application of the above-mentioned maize salt-tolerant molecular marker SNP1409, primer pairs or methods in salt-tolerant maize breeding.

[0015] On the other hand, this application provides the application of the above-mentioned salt tolerance genes or maize MYB-type transcription factors in regulating the transport of sodium ions from the roots to the aboveground parts of maize.

[0016] On the other hand, this application provides the application of the above-mentioned salt-tolerant genes or maize MYB-type transcription factors as maize transcriptional repressors.

[0017] The beneficial effects of this invention are:

[0018] (1) This invention provides a novel maize salt tolerance major gene MYB28 and its nucleotide sequence, and identifies a non-synonymous SNP site (SNP1409) within this gene that causes amino acid changes. The single nucleotide polymorphism at this site is closely related to maize salt tolerance and can be used as a molecular marker for maize salt tolerance. This invention also provides specific primer pairs and detection methods for detecting this molecular marker.

[0019] (2) Validation results using 115 maize inbred lines showed that the molecular marker was significantly associated with the Na⁺ content in the aboveground parts, and the detection results were reliable. Since maize salt tolerance is a quantitative trait inherited trait, phenotypic analysis is time-consuming and laborious. The above-mentioned major maize salt tolerance gene MYB28, molecular marker SNP1409, specific primer pairs, and detection methods can all be applied to maize salt tolerance breeding. Genotyping can be performed at the seedling stage or even the seed stage, which is time-saving and accurate, and can accelerate the breeding process of maize salt-tolerant varieties. Attached Figure Description

[0020] Figure 1A The results of QTL analysis are presented for a BC2S3 recombinant inbred line (RIL) population using the teosinte material T8759 and the maize inbred line W22.

[0021] Figure 1B This shows the distribution of Na⁺ content in the aboveground parts of recombinant inbred lines (RILs) carrying the T8759 and W22 alleles at the qNaC3 locus.

[0022] Figure 1C This shows the growth phenotypes of recombinant inbred lines T211 carrying the T8759 and W22 alleles at the qNaC3 locus, under control and 100 mM NaCl conditions.

[0023] Figure 1D This shows the biomass of the recombinant inbred line T211 carrying the T8759 and W22 alleles at the qNaC3 locus, under control and 100 mM NaCl conditions.

[0024] Figure 1EThis shows the Na⁺ content of recombinant inbred lines T211 carrying the T8759 and W22 alleles at the qNaC3 locus, under control and 100 mM NaCl conditions.

[0025] Figure 1F The results show the results of fine localization of qNaC3 using recombinant organisms.

[0026] Figure 2A This is a schematic diagram of the pBUE411-MYB28 carrier structure.

[0027] Figure 2B This diagram shows the structure of the ZmMYB28 gene, the target sites of myb28-1 and myb28-2 knockout materials, and the comparison of the knockout gene and protein sequences.

[0028] Figure 3 Display MYB28 W22 Construction of overexpression materials; Part A is a schematic diagram of the PbCXUN-HA-FLAG-MYB28 vector structure; Part B is the detection of overexpression level of the overexpression lines.

[0029] Figure 4 Phenotypic analysis of ZmMYB28 genetic materials: wild-type, myb28 mutant and overexpression materials, growth phenotype (part A), biomass (part B) and percentage of biomass reduction (part C), and aboveground parts (part D) under control and 100 mM NaCl conditions.

[0030] Figure 5 Functional analysis of ZmMYB28 genetic materials: wild-type, myb28 mutant, and overexpression materials were analyzed under control and 100 mM NaCl conditions, including the analysis of xylem sap (part A) and root (part B) NaCl. + content.

[0031] Figure 6A This is a schematic diagram of the conserved domains of different MYB proteins.

[0032] Figure 6B ZmMYB28 was observed in maize protoplasts. W22 and ZmMYB28 W22 Subcellular localization, DAPI staining to label the cell nucleus.

[0033] Figure 6C Showing the control and MYB28-NIL under 100 mM NaCl conditions, W22 and MYB28-NIL T8759 The relative transcription level of MYB28 in plants.

[0034] Figure 6DImages from a luciferase reporter gene experiment.

[0035] Figure 6E Display MYB28 T8759 Its inhibitory activity was significantly higher than that of MYB28. W22 Transcriptional repression activity.

[0036] Figure 6F The results of a comparative experiment on the luciferase reporter gene of wild-type and mutant MYB28 protein.

[0037] Figure 7 Sequencing peak diagram of SNP1409 in maize inbred line amplified by molecular markers.

[0038] Figure 8 The statistical analysis results of HapT and HapA salt ion contents at SNP1409 of 115 maize inbred lines are presented. Detailed Implementation

[0039] The following embodiments are provided to better understand the present invention, but are not limited thereto. These embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention in any way.

[0040] Unless otherwise specified, the equipment and reagents used in each embodiment are all commercially available.

[0041] Example 1: Cloning and Functional Analysis of Maize Salt Tolerance Gene

[0042] To identify the major genetic loci associated with differences in salt tolerance between teosinte and maize, QTL analysis was performed using BC2S3 recombinant inbred line (RIL) populations of teosinte T8759 and maize inbred line W22. The analysis revealed that qNaC3 was the major QTL locus leading to the difference in aboveground Na⁺ content between maize inbred line W22 and teosinte T8759. Figure 1A ), and qNaC3 W22 Allele-positive lines have lower Na⁺ content in the aboveground parts ( Figures 1B-1E Using 10 molecular markers, the qNaC3 site was located within a 282 kb region. This region contains two genes: Zm00001d050400 (MYB28) and Zm00001d050401. Figure 1F Furthermore, the functions of these two genes had not been previously studied. The study showed that qKC3 encodes the transcription factor ZmMYB28, which negatively regulates Na+. + Translocation from roots to above-ground parts and salt tolerance.

[0043] To obtain a mutant of ZmMYB28, the inventors (1) designed a CRSPR-Cas9 knockout target, such as Figure 2BAs shown (using a dual-target scheme, the target sequences are TGGACTGAAGAAAGGTCCA (SEQ ID NO.13) and TGGCGGGCATTACCTGCGA (SEQ ID NO.14); (2) The PCR fragment containing the target sequence and the pBUE411 vector were digested and ligated with BsaI, and the ligation product was transformed into E. coli. Colony PCR amplification was performed using universal primers FD3 / RD. Colonies with the target size band were detected by electrophoresis as positive clones. The corresponding bacterial solution was sent to Beijing Sanbo Yuanzhi Biotechnology Co., Ltd. for sequencing to obtain the correct pBUE411-MYB28 vector ( Figure 2A (3) Transgenic plants were obtained by infecting immature embryos; (4) Transgenic positive plants were identified by PCR amplification and sequencing of gene fragments including the target site. Finally, two candidate MYB28 gene knockout materials (named myb28-1 and myb28-2) were obtained. Figure 2B ).

[0044] To obtain maize MYB28 overexpressing plants, the inventors (1) based their prediction of the MYB28 gene structure on the MaizeGDB website. The full-length gene (from start codon to stop codon) is 1476 bp, containing 3 exons and 2 introns. The coding region is 1077 bp long and encodes 358 amino acids, such as... Figure 3 As shown in Figure A, the coding region's nucleotide and amino acid sequences are as follows:

[0045] SEQ ID NO.1:

[0046] T GGGTCCTTGACAGAGTAAACTCAGAGATGACAGCACAGTCGCCTTCCTTGGTCTAA

[0047] SEQ ID NO.2:

[0048] MGRSPCCEKIGLKKGPWTPEEDEKLLAFVEEHGHGSWRALPAKAGLQRCGKSCRLRWTNYLRPDIKRGKLSLQEEQTIIQLHALLGNRWSAIATHLPNRTDNEIKNHWNTHLKKRLAKIGIDPVTHKSTCGTLTGTTNDRSAKAAASLSHMAQWENARLEAEARLARESKTRTATPTPSALHAQPMDLPASAASPWLDVLHAWQGAKIDLESPTSTLTFTGSNGGMLPTPRTNGPEVSESNSVMSHYQMSDELEGEETYWQIFSKHQVPEVDSKESEDDFIGCEEPWFSGMAGVGAGFTGMLLDVSYEHELSECWGESSSGQTVEHSKQASDKEDKDYWN W VLDRVNSEMTAQSPSLV-

[0049] (2) In order to clone the full-length coding sequence of MYB28, salt-tolerant maize inbred line B73 seedlings grown for 10 days were used as material. Total RNA was extracted using the plant total RNA extraction kit (Cat.#DP432) of Tiangen Biotech (Beijing) Co., Ltd., and cDNA was obtained by reverse transcription using M-MLV reverse transcriptase of Promega (Beijing) Biotechnology Co., Ltd. PCR amplification was performed using MYB28-specific primers (forward primer MYB28-gene-F: AATGGGGCGATCACCATGCTGTGA (SEQ ID No. 3); reverse primer MYB28-gene-R: TTAGACCAAGGAAGGCGACTGTG (SEQ ID No. 4), and a product with the expected size (1077 bp) was obtained. The PCR product was recovered and purified using the gel recovery kit (Cat.#DP105-3) of Tiangen Biotech (Beijing) Co., Ltd.; (3) The recovered PCR fragment and XcmⅠ The digested pBCXUN-HA-Flag vector was ligated, and the ligation product was transformed into E. coli. Colony PCR amplification was performed using primers Ubip-seq-F (TTTTAGCCCTGCCTTCATACGC SEQ ID No. 5) and NosR-seq-R (AGACCGGCAACAGGATTCAATC SEQ ID No. 6). Colonies with the target size band were identified by electrophoresis as positive clones. The corresponding bacterial cultures were sent to Beijing Sanbo Yuanzhi Biotechnology Co., Ltd. for sequencing to obtain the correct pBCXUN-HA-Flag-MYB28 vector ( Figure 3 (Part A) pBCXUN-HA-Flag-MYB28 was transferred into Agrobacterium EHA105; (3) transgenic plants were obtained by embryo infection; (4) the expression level of the MYB28 gene in the plants was detected. Figure 3 (Partially), sequencing confirmed transgenic positive plants. Ultimately, two MYB28 candidate gene overexpression materials (named MYB28) were obtained. W22 / OE -1 and MYB28 W22 / OE -2).

[0050] Phenotypic analysis of these materials showed that, under control conditions, the aboveground biomass and Na+ of wild-type, myb28 mutant, and overexpression lines were significantly higher than those of other strains. + There was no significant difference in content; under salt stress, the myb28 mutant had a higher biomass and Na content compared to the wild type. + Reduced content, making it more salt-tolerant; MYB28 W22 / OE Biomass decreased, Na + Elevated levels increase sensitivity to salt stress (e.g.) Figure 4The AD portion indicates that MYB28 negatively regulates salt tolerance in maize.

[0051] Example 2: MYB28 Regulation of Na + Transport from roots to above-ground parts

[0052] To further investigate the regulation of Na by ZmNC4 + The molecular mechanism of Na transport was investigated, and the levels of Na in the root and xylem sap of maize seedlings were measured. + Content. Results showed that under normal conditions, there was no significant difference in phenotype between the MYB28 transgenic lines and wild-type plants ( Figure 4 , Figure 5 Under salt stress, the myb28 knockout mutant showed lower Na⁺ content in the aboveground and xylem sap, and higher Na⁺ accumulation in the roots, indicating greater salt tolerance; while the MYB28... OE Overexpression lines showed the opposite trend. Figure 5 These results indicate that MYB28 negatively regulates maize salt tolerance by promoting long-distance Na⁺ translocation from roots to shoots. It is a functional gene regulating the ZmNC4 locus and a key genetic factor leading to the difference in salt tolerance between modern maize (W22) and fodder grass (T8759).

[0053] Example 3: The non-synonymous SNP (SNP1409) is the cause of MYB28 functional variation.

[0054] Conserved domain analysis of the MYB protein showed that MYB28 belongs to the R2R3-MYB family and contains the conserved repressive motif C1 of transcriptional repressors. Figure 6A ), MYB28 W22 and MYB28 T8759 Subcellular localization analysis showed that MYB28 was located in the cell nucleus ( Figure 6B This suggests that it is likely a transcriptional repressor.

[0055] Furthermore, by comparing the near-isogenic line MYB28-NIL W22 and MYB28-NIL T8759 The transcriptional levels of MYB28 in the two groups showed no significant difference. Figure 6C ). Meanwhile, luciferase reporter gene assays showed that, under comparable protein levels, MYB28 W22 and MYB28 T8759 Both act as transcriptional repressors, but MYB28 T8759 Its inhibitory activity was significantly stronger than that of MYB28. W22 ( Figure 6D -E). Furthermore, the applicant compared MYB28. W22 and MYB28 T8759The coding sequences (SEQ ID No. 1 and SEQ ID No. 7) identified 14 non-synonymous SNPs (SEQ ID No. 2 and SEQ ID No. 8) that cause amino acid changes. These non-synonymous SNPs were then analyzed in MYB28. W22 In the background, a point mutation vector was constructed, and luciferase reporter gene experiments showed that MYB28... W22 A single amino acid substitution (W341G, corresponding to position 1021 on the nucleotide sequence, referred to as SNP1409 after calculating the non-coding region) significantly enhanced its transcriptional repression activity, reaching the level of MYB28. T8759 A comparable level ( Figure 6F These results indicate that SNP1409 is the cause of MYB28. T8759 Variations with enhanced transcriptional repression activity are the cause of differences in Na⁺ accumulation and salt tolerance in the aboveground parts of maize (W22) and fodder grass (T8759).

[0056] SEQ ID NO.7, MYB28 T8759 :

[0057] G GGGTCCTTGACAGAGTAAACTCAGAGCTGACAGCACAGTCGCCTTCCTTGGTCTAA

[0058] SEQ ID NO.8, MYB28 T8759 :

[0059] MGRSPCCEKIGLKKGPWAPEEDEKLLAFVEEHGHGSWRALPAKAGLQRCGKSCRLRWTNYLRPDIKRGKLSLQEEQTIIQLHALLGNRWSAIATHLPNRTDNEIKNHWNTHLKKRLAKIGIDPVTHKSTCGILTGTTNDRSAKAAASLRHMAQWESARLEAEARLARESK TRTATPTPSALHAQPMDVPASAASPWLDVLHAWQGAKIDLESPTSTVTFTGSNGGMLPTPRTNGLEVSESNSAMSHYQMNDELEGEEAYWQIFSKHQVPEVDSKESEDDFIGCEEPWFSGTAGVGAGFTGMLLDVSNEHELSECWGESSSGQTVEHSKQASDKEDKDYWN G VLDRVNSELTAQSPSLV-

[0060] Example 4: SNP1409T or SNP1419G in the maize salt tolerance gene MYB28 are used as molecular markers.

[0061] Since MYB28-W22-SNP1409T and MYB28-T8759-SNP1409G exist in modern maize inbred lines, SNP1409T or SNP1419G in MYB28 can be used as molecular markers to determine whether an individual is salt-tolerant.

[0062] Primers qNaC3-F1 (forward) and qNaC3-R1 (reverse) were designed based on the flanking sequence of SNP1409, forming primer pair I (qNaC3-F1 / qNaC3-R1). Using primer pair I as primers, genomic DNA from maize inbred lines Zheng 58 and M1016 was used as templates for PCR amplification. The PCR system (30 μl) consisted of: 15 μl of 2×SuperMultiplex PCR Mix, 1.5 μl of 10 μM Primer qNaC3-F1, 1.5 μl of 10 μM Primer qNaC3-R1, 1 μl of DNA, and 18 μl of diH2O. PCR program: pre-denaturation 94 ℃ for 2 min, denaturation 94 ℃ for 30 s, annealing 56 ℃ for 30 s, extension 72 ℃ for 30 s, 35 cycles from denaturation to extension, and a final extension at 72 ℃ for 5 min. Results showed that PCR amplification using W22 and T8759 as templates yielded a 442 bp sequence with primer pair I (detailed sequence see SEQ ID No. 9); PCR amplification using total DNA from the maize inbred line M1016 as a template also yielded a 442 bp sequence with primer pair I (detailed sequence see SEQ ID No. 9). 10), and simultaneously amplified 6 inbred lines using primer pair I. Peak comparison at SNP1409 revealed that NC268, PHP60, 680, and *Eriocaulon buergerianum* T8759 were identical, belonging to the SN1409G haplotype, with Na ion contents of 9.708089159, 11.46660974, and 13.49978801, respectively; 4722, PHN82, WIL900, and maize W22 were identical, belonging to the SNP1409T haplotype, with Na ion contents of 1.624510188, 2.34369536, and 2.460245902, respectively, showing greater salt tolerance. Figure 7 ).

[0063] SEQ ID NO.9:

[0064] ACCTACCTCCACACTGACGTTTACAGGGAGCAATGGTGGCATGCTGCCAACCCCCAGGACCAACGGACCAGAGGTATCAGAAAGCAACTCCGTGATGTCGCATTATCAGATGAGCGATGAGTTGGAGGGTGAAGAAACCTATTGGCAGATCTTCAGCAAGCACCAAGTGCCGGAAGTGGACAGCAAGGAGAGTGAAGATGACTTCATTGGCTGTGAGGAGCCGTGGTTCTCAGGGATGGCTGGGGTTGGAGCTGGCTTCACTGGCATGCTGCTTGATGTATCCTATGAGCATGAGCTATCAGAATGCTGGGGTGAGTCCAGCAGTGGCCAAACTGTTGAGCACAGCAAGCAAGCATCCGATAAGGAGGACAAGGATTATTGGAATTGGGTCCTTGACAGAGTAAACTCAGAGATGACAGCACAGTCGCCTTCCTTGGTCTAA

[0065] SEQ ID NO.10:

[0066] ACCTACCTCCACAGTGACGTTTACAGGGAGCAATGGTGGCATGCTGCCAACCCCCAGGACCAACGGACTAGAGGTATCAGAAAGCAACTCCGCGATGTCGCATTATCAGATGAACGATGAGTTGGAGGGTGAAGAAGCCTATTGGCAGATCTTCAGCAAGCACCAAGTGCCGGAAGTGGACAGCAAGGAGAGTGAAGATGACTTCATTGGCTGTGAGGAGCCGTGGTTCTCAGGGACGGCTGGGGTTGGAGCTGGCTTCACTGGCATGCTGCTTGATGTATCCAATGAGCATGAGCTATCAGAATGCTGGGGTGAGTCCAGCAGTGGCCAAACTGTTGAGCACAGCAAGCAAGCATCCGATAAGGAGGACAAGGATTATTGGAATGGGGTCCTTGACAGAGTAAACTCAGAGCTGACAGCACAGTCGCCTTCCTTGGTCTAA

[0067] Therefore, primer pair I can be used for molecularly-assisted breeding of maize with salt tolerance, and the salt tolerance molecular marker based on primer pair II is named qNaC3. The sequences of each primer are as follows:

[0068] Primer qNaC3-F1: GGTCACTTTGATTACTGCTGCT (SEQ ID No. 11);

[0069] Primer qNaC3-R1: TCGAATGGAGGGCTCAGTAC (SEQ ID No. 12).

[0070] Example 5: Detecting salt tolerance in maize using salt-resistance molecular markers

[0071] 115 maize inbred lines (including some core maize inbred lines) were selected for testing. The detection method was as described in Example 4. Using the primer pairs for detecting the salt-resistant molecular markers of the present invention in Example 4, PCR amplification and sequencing were performed using the maize genomic DNA to be tested as a template. The results showed that the sequencing results of SNP1409 at the primer pair I of 96 inbred lines were T, indicating a salt-resistant genotype; the sequencing results of SNP1409 at the primer pair I of the other 19 inbred lines were G, indicating a salt-sensitive genotype. The names of the inbred lines used and the identification results are shown in Table 1 below:

[0072] Table 1. Results of inbred line identification

[0073] Name of inbred line SNP1409(T / G) <![CDATA[Na + Content (mg / g DM) Fang Yin T 0.278387014 PHP85 T 0.327443212 PHJ33 T 0.529492551 Double 741 T 0.606328904 Huang Zao 4 T 0.812642783 Qi 35 T 0.829842799 LH123HT T 0.834382641 196 T 1.057107843 Yu 82 T 1.079114106 433-7 T 1.188189897 7146 T 1.237662572 Jinsui 54 T 1.274947257 XF223 T 1.291177523 D857 T 1.312341772 PHP55 T 1.357950117 D619 T 1.372974768 Radiation 8521 T 1.445494994 LH38 T 1.517828784 78599 T 1.590183616 4722 T 1.624510188 S8324 T 1.76422629 MM402A T 1.778696498 PHR36 T 1.816784038 N138 T 1.958117266 Ji 846 T 2.142331349 LH128 T 2.16569083 Radiation 8701 T 2.227841358 49 AM T 2.269157088 PHN82 T 2.34369536 PHT69 T 2.359294437 Jingnuo 2 T 2.364145265 Ji 444 T 2.369193742 WIL900 T 2.460245902 Big MO T 2.532446809 Strive for 72 T 2.543806991 E28 T 2.602746212 K14 T 2.610175268 Ay420 T 2.658997373 H84 T 2.824264706 030-1 T 2.928760163 L061F T 3.067511013 MBST T 3.415913903 Lu Yuan 133 T 3.448360266 West 502 T 3.483697007 L135 T 3.517357143 PHN29 T 3.544992019 C-Wu 215B T 3.552643824 B47 T 3.564698163 128 T 3.574837574 Jun 971 T 3.615076183 SS99 T 3.62589803 Ms71 T 3.646094927 Miscellaneous C546 T 3.661568444 LH208 T 3.673113208 LH202 T 3.838758846 PHK05 T 3.882018717 IB014 T 3.902218805 785 T 4.1853602 Ji 53 T 4.192783912 PHW79 T 4.228384279 787 T 4.240542606 897 T 4.283907104 Maxa T 4.375227273 794 T 4.375536543 KP3130 T 4.396514745 W182bn T 4.39844086 Cheng 435 T 4.438768397 ML606 T 4.476133019 W8304 T 4.543909915 M131-5 T 4.594180162 H21 T 4.625596184 Qi 410 T 4.714940445 2-1 cattle T 4.745671547 Yu 87-1 T 4.966230769 R150 T 5.057114255 FR14 T 5.145394208 S8326 T 5.21001171 6 T 5.330379483 CR14 T 5.382865169 W499 T 5.416901408 99122 T 5.683455583 85 White 64 T 5.715361446 49 T 5.760483871 17-1 T 5.902098907 Yuan Wu 05 T 5.957965213 H-5 T 5.967662835 LH132 T 6.242923387 ys06 T 6.262489425 LH196 T 6.606548155 1121 T 6.651002406 DF20 T 6.684696157 R136 T 6.772826087 SC24-1 T 6.909269038 D886 T 6.934361093 Shen 977 T 7.057700422 B73 T 7.246436939 XOP2 G 7.253927911 9711 G 12.45476886 Cheng Zi 2142 G 9.003895815 NC268 G 9.708089159 Ning 45 G 8.906382979 OH7 G 8.692309065 67 G 11.15306772 Xing K36 G 4.795636095 Oh07B G 12.375547 PHW52 G 11.1572549 PHG35 G 7.384210526 FAPW G 9.659253247 PHPR5 G 9.62792172 2MA22 G 11.06534539 PHM10 G 13.47733579 PHP60 G 11.46660974 PHV37 G 12.95163425 W23 G 6.012317518 680 G 13.49978801

[0074] Under salt stress, the Na content in the leaves of the salt-tolerant genotype material (SNP1409T) is... + The content was significantly lower than that of the salt-sensitive genotype material (SNP1409G). Figure 8 This is consistent with the results of molecular marker identification.

Claims

1. A maize MYB-type transcription factor salt tolerance gene, characterized in that, The nucleotide sequence of the salt tolerance gene is SEQ ID NO.

1.

2. The maize MYB-type transcription factor encoded by the salt tolerance gene according to claim 1, characterized in that, The amino acid sequence of the maize MYB transcription factor is SEQ ID NO.

2.

3. The application of the maize salt tolerance gene according to claim 1 or the maize MYB transcription factor according to claim 2 in regulating maize salt tolerance.

4. The application according to claim 3, wherein the salt tolerance gene in maize is knocked out to improve the salt tolerance of maize; or the salt tolerance gene is overexpressed in the application to make maize more sensitive to salt stress.

5. The application according to claim 4, wherein the salt tolerance gene is knocked out by the CRISPR-Cas9 system; or the salt tolerance gene is overexpressed by the pBCXUN vector.

6. The application according to claim 5, wherein the target sequences SEQ ID NO.13 and SEQ ID NO.14 are knocked out by the CRISPR-Cas9 system.

7. Maize salt-tolerant molecular marker SNP1409, characterized in that, The maize salt tolerance molecular marker SNP1409 is located at position 1023 of the salt tolerance gene shown in SEQ ID NO.1, and its polymorphism is T or G; when it is T genotype, it is salt resistant, and when it is G genotype, it is salt sensitive.

8. A primer pair for detecting the maize salt-tolerant molecular marker SNP1409 according to claim 7, characterized in that, The primer pairs include qNaC3-F1 and qNaC3-R1, whose nucleotide sequences are shown in SEQ ID NO.11 and SEQ ID NO.

12.

9. A method for detecting whether corn is salt-tolerant, characterized in that, The method includes: using maize genomic DNA as a template, performing PCR amplification using the primer pair according to claim 8; sequencing the amplification product to detect the genotype of the maize salt tolerance molecular marker SNP1409 site according to claim 7; if it is the T genotype, it is determined to be salt resistant; if it is the G genotype, it is determined to be salt sensitive.

10. The application of the maize salt-tolerant molecular marker SNP1409 according to claim 7, the primer pair according to claim 8, or the method according to claim 9 in salt-tolerant maize breeding.

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