The ZmbHLH30 gene in maize and its application in regulating low-temperature tolerance during maize germination.

Abstract

This invention discloses the maize ZmbHLH30 gene and its application in regulating low-temperature tolerance during maize germination, belonging to the fields of plant genetic engineering and breeding technology. This invention discovers a gene containing the bHLH domain, namely ZmbHLH30. Using overexpressing lines of this gene and the recipient line B104 as materials, the low-temperature tolerance of different materials under low-temperature stress was investigated. The results showed that maize with this overexpressed gene exhibited better low-temperature tolerance than the recipient control, indicating that ZmbHLH30 can effectively improve the low-temperature tolerance of maize. Simultaneously, this invention also proposes a molecular marker related to a maize low-temperature tolerance gene, named DNCAP380. The molecular marker provided by this invention can be used for the cultivation of low-temperature tolerant transgenic plants, providing a new technical means and methodological basis for screening and creating new low-temperature tolerant maize materials, and has significant implications in the field of low-temperature tolerant maize variety breeding.

Description

Technical Field

[0001] This invention relates to a gene containing a bHLH domain and its application in regulating the cold tolerance of maize. It also relates to molecular markers associated with this gene and their applications. This invention belongs to the field of plant genetic engineering technology. Background Technology

[0002] Maize, a C4 crop, originated in subtropical and tropical regions and is a temperature-sensitive crop, requiring relatively high temperatures throughout its growth cycle. Low temperatures can inhibit maize growth, affect morphology, and even cause plant death. Low-temperature stress affects maize growth and development during germination, budding, and seedling stages, resulting in reduced seed germination rates, decreased seedling vigor, and hindered root development. Therefore, the fundamental way to improve maize's low-temperature tolerance is to discover and clone maize low-temperature tolerance genes, clarify their biological functions and analyze their molecular mechanisms, improve the low-temperature tolerance of germplasm resources, and then breed new low-temperature tolerant varieties to enhance maize's adaptability to climate change and provide genetic resources and technological reserves for ensuring food security.

[0003] The bHLH transcription factor family is the second largest transcription factor family in plants after MYB. bHLH proteins are a class of transcription factors possessing bHLH domains. bHLH transcription factors regulate the expression of cold-resistance-related genes by binding to the MYC cis-elements (CANNTG). The bHLH transcription factor family is found in Arabidopsis thaliana, rice, wheat, sweet potato, and pepper. These transcription factors can specifically bind to the MYC element of the CBF / DREB1 gene promoter, activating the CBF gene, and bind to the COR promoter, thereby enhancing the plant's cold resistance.

[0004] Therefore, this invention provides a gene containing the bHLH domain, namely ZmbHLH30, and clarifies the function of this gene and its molecular mechanism in response to low temperature tolerance during maize germination, which is of great value for breeding new low temperature tolerant maize germplasm. Summary of the Invention

[0005] The purpose of this invention is to provide a gene containing the bHLH domain, namely the maize ZmbHLH30 gene, and its application in regulating the low-temperature tolerance of maize during germination.

[0006] To achieve the above objectives, the present invention employs the following technical means:

[0007] This invention first proposes the application of the maize ZmbHLH30 gene or its encoded protein in regulating maize's low-temperature tolerance.

[0008] Furthermore, this invention also proposes the application of biomaterials containing the maize ZmbHLH30 gene or its encoded protein in regulating the low-temperature tolerance of maize.

[0009] Preferably, the sequence of the maize ZmbHLH30 gene is shown in SEQ ID NO.1.

[0010] Preferably, the biomaterial is any one of the following:

[0011] A: Expression cassettes that enable overexpression of the ZmbHLH30 gene;

[0012] B: A recombinant vector containing the expression cassette described in A;

[0013] C: Recombinant microorganisms containing the expression cassette described in A or the recombinant vector described in B.

[0014] Furthermore, this invention also proposes a method to improve the low-temperature tolerance of maize, characterized by upregulating the expression level of the ZmbHLH30 gene in maize through any one or more methods such as transgenic, gene editing, hybridization, backcrossing, self-pollination or asexual reproduction, thereby improving the low-temperature tolerance of maize.

[0015] Furthermore, this invention also proposes a dCAPS molecular marker related to low-temperature tolerance during maize germination, the molecular marker being DNdCAPS380, located on maize chromosome 1, the molecular marker containing an SNP site located in the CDS region of the ZmbHLH30 gene, with a mutated base of G / T, corresponding to nucleotide 380 of the sequence shown in SEQ ID NO.12.

[0016] Primer pairs used to amplify the dCAPS molecular marker are also within the scope of protection of this invention. Preferably, the primer pairs include nucleotide sequences as shown in SEQ ID NO.13 and SEQ ID NO.14.

[0017] Furthermore, the present invention also proposes the use of the dCAPS molecular marker or the primer pair in any of the following aspects:

[0018] (1) To assess the low-temperature resistance of corn materials;

[0019] (2) Breeding low-temperature resistant transgenic maize materials through molecular marker-assisted breeding;

[0020] (3) Improve maize germplasm resources.

[0021] Finally, this invention also proposes a method for identifying or breeding low-temperature resistant maize materials, comprising the following steps:

[0022] Genomic DNA was extracted from the maize material samples to be tested, and PCR amplification was performed using the primer pair. The amplification products were digested with NheI restriction endonuclease. Low-temperature resistant materials could not be digested by NheI restriction endonuclease, resulting in one fragment, while low-temperature sensitive materials could be digested by NheI restriction endonuclease, resulting in two fragments.

[0023] Preferably, the cryogenic material cannot be cleaved by the NheI restriction endonuclease to obtain a 246 bp fragment, the sequence of which is shown in SEQ ID NO.15. The cryogenic sensitive material can be cleaved by the NheI restriction endonuclease to obtain two fragments of 221 bp and 25 bp, the sequences of which are shown in SEQ ID NO.16 and SEQ ID NO.17, respectively.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. This invention identifies the low-temperature tolerance of ZmbHLH30 overexpression lines during the germination, bud, and seedling stages and analyzes the low-temperature induced expression pattern. It finds that this gene is an important factor involved in maize's low-temperature tolerance, and compared with the receptor control, the low-temperature tolerance of the overexpression lines is significantly improved.

[0026] 2. This invention found that overexpression of the ZmbHLH30 gene can significantly improve the low-temperature resistance of maize, which is of great significance for the study of the molecular biological mechanism of maize's low-temperature resistance function.

[0027] 3. The ZmbHLH30 and its encoded protein in this invention can be used to improve maize germplasm resources, providing new possibilities for breeding new low-temperature tolerant maize varieties and laying the foundation for improving maize yield and quality. This invention provides a theoretical basis and data support for the low-temperature regulation mechanism of maize and for low-temperature tolerant breeding.

[0028] 4. This invention is based on maize low-temperature tolerance-related genes. By analyzing the sequence variations of the CDS region of low-temperature tolerance-related genes in different maize inbred lines, and performing correlation analysis with the low-temperature tolerance phenotypic indicators of the inbred lines, the associated sites are identified. The restriction enzyme sites of the sites are analyzed and restriction endonucleases are selected to develop an SNP molecular marker associated with plant low-temperature tolerance genes. By detecting the mutation type of this SNP molecular marker in plants, the plant's low-temperature tolerance can be detected. Attached Figure Description

[0029] Figure 1 The DNA, RNA and protein levels of the T1 and T2 generation ZmbHLH30 overexpression lines in Example 2 were detected.

[0030] Where M stands for DNA Marker (Trans 2k), W for ddH2O, P for positive control, N for negative control, 1-7 for T0, T1 and T2 generation overexpression positive lines, and WT for negative control;

[0031] Figure 2 The phenotype of the control line of the ZmbHLH30 overexpressing strain in Example 3 under low temperature stress during germination;

[0032] Figure 3 The phenotype of the control line of the ZmbHLH30 overexpressing strain in Example 3 under low temperature stress during the bud stage;

[0033] Figure 4 The phenotypes of ZmbHLH30 overexpression and receptor control lines under low temperature stress during the seedling stage in Example 3;

[0034] Figure 5 This represents the relative expression level of ZmbHLH30 in germinating embryos in Example 4.

[0035] Figure 6 The relative expression levels of the ZmbHLH30 gene in buds and roots during the bud stage in Example 4;

[0036] Where, a: relative expression level of ZmbHLH30 gene in bud, b: relative expression level of ZmbHLH30 gene in root;

[0037] Figure 7 The relative expression levels of ZmbHLH30 in the leaves, stems, and roots during the seedling stage in Example 4;

[0038] Where, a: relative expression level of ZmbHLH30 in leaves, b: relative expression level of ZmbHLH30 in stems, c: relative expression level of the gene in roots;

[0039] Figure 8 The results are the genotype detection results of the molecular marker DNdCAPS380 provided in Example 6;

[0040] In this context, A:M represents the DNA Marker, and 1-6 are the PCR products of Qi319, Ye478, 888-9, Zheng30, Moqun17, and 8415, respectively; B:M represents the DNA Marker, and 1-6 are the enzyme digestion products of Qi319, Ye478, 888-9, Zheng30, Moqun17, and 8415, respectively.

[0041] Figure 9 The results are the verification results of the molecular marker DNdCAPS380 provided in Example 6 in some self-crossing lines;

[0042] Where M is the DNA Marker, and lanes 1-24 are the enzyme digestion products of 888-9, 8415, Moqun17, B84, B104, H10, Jing7, PI42, PI36, L237, 434, Zheng30, M502, K10, Han23, 77, SH15, CA112, Ta5, PI10, Dian11, Zhong451, Ji419, and Shuang105, respectively. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0044] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the test methods involved are all conventional methods unless otherwise specified.

[0045] Example 1: Construction and transformation of ZmbHLH30 gene overexpression vector

[0046] Based on the CDS region of the ZmbHLH30 gene (also known as bhlh170, bHLH-transcription factor 170, whose Gene ID in the Maize GDB database is Zm00001d031818, and whose gene sequence is shown in SEQ ID NO.1), homologous recombination primers with BamHI restriction sites were designed. The homologous recombination PCR primer sequences are shown in SEQ ID NO.2 (5'-GTAAAACGACGGCCAGTGCATGGTGGCGA-3') and SEQ ID NO.3 (5'-TCCGGCTCGTATGTTGTGTGCTGAACCCTGG-3'). Using the maize B73 genome as a template, PCR amplification was performed. The amplified fragment containing the ZmbHLH30 gene was inserted into the pCUB-eGFP-3×FLAG vector (the pCUB-eGFP-3×FLAG vector is based on the pCUB vector (Song Pan, 2015, Construction and genetic transformation of maize ZmPOT gene TALEN knockout and overexpression vector) and uses the BamHI restriction site to insert the GFP protein and 3×FLAG tag between the promoter and terminator of the pCUB vector. The plasmid was constructed using the pCUB-ZmbHLH30-eGFP-3×FLAG plasmid. This plasmid has been disclosed in Chinese patent application No. 202210259256.9, entitled "Application of ZmHIR3 protein or its encoding gene in regulating maize's resistance to maize rough dwarf disease". The plasmid was transformed into Agrobacterium EHA105 using the freeze-thaw method and verified by colony PCR. The gene overexpression line was obtained by transforming maize immature embryos with Agrobacterium into the low-temperature highly sensitive maize inbred line B104.

[0047] Example 2 Molecular detection of ZmbHLH30 overexpression lines

[0048] DNA-level molecular detection: DNA was extracted from the leaves of overexpressing plants. Primers were designed based on the Ubi promoter and target gene on the vector. The primers for this gene detection are shown in SEQ ID NO. 4-5 (5'-GCCCTGCCTTCATACG-3'; 5'-GTCGGTCAGCAACGCGT-3'), and the primers for the Bar gene detection are shown in SEQ ID NO. 6-7 (5'-GTTGAGCAGATCTCGGTGAC-3'; 5'-GCACCATCGTCAACCACTAC-3'). The amplified products were detected by 1.5% agarose gel electrophoresis. RNA-level molecular detection: Total RNA was extracted from maize leaves using the Trizol method, and cDNA was synthesized using a two-step reverse transcription kit. Test strip detection: The Bar protein test strip (Beijing Aochuang Gold Standard) was used for detection; see the instruction manual for specific operation.

[0049] Agarose gel electrophoresis of the PCR products showed that both the overexpression lines and the recombinant plasmid amplified a 562 bp band of the target Bar gene and a 493 bp band of the target Bar gene, while the negative control and water control showed no bands. Complete RNA was extracted and reverse transcribed into cDNA for RT-PCR detection using primers SEQ ID NO. 8-9 (5'-ATGGTTGTCCAGGAGTTCCAGT-3'; 5'-TACAGCTCGTCCATGCCGAGA-3'). The results showed that the bands of the target Bar gene were clearly visible in the seven overexpression lines of the T1 and T2 generations, indicating normal transcription, while the negative control did not show any target bands of the target Bar gene. Protein expression in the T1 and T2 generations of overexpression lines was detected using test strips. The results showed that all test strips had normal control lines and clearly visible test lines, indicating that all seven overexpression lines could stably translate the Bar protein. Figure 1 ).

[0050] Example 3: Identification of low-temperature tolerance of ZmbHLH30 overexpression lines during germination, budding, and seedling stages.

[0051] The experimental materials were seven stable T2 generation overexpression lines OE-1 to OE-7 obtained in Example 2 and the recipient control B104. The low-temperature tolerance of the overexpression lines of this domain gene was identified during the germination, budding, and seedling stages.

[0052] (1) The specific steps for identifying the low-temperature tolerance during germination were as follows: Each line was set up in 3 replicates, and 50 seeds with plump shape and uniform size were selected as experimental materials for each replicate. First, the seeds were disinfected with 75% ethanol for 10 min, then thoroughly rinsed with sterile water, and after 6 hours of imbibition treatment at a constant temperature of 25℃, the seeds were taken out and placed on germination paper. The seeds in the treatment group were cultured in a low-temperature environment of 10℃ for 7 days, and then transferred to a 25℃ environment for 3 days of recovery; the seeds in the control group were directly cultured in a 25℃ environment for 7 days.

[0053] (2) The specific steps for identifying the low-temperature tolerance of germination are as follows: Seed disinfection treatment is the same as that for germination period. After the seeds have fully absorbed the nutrients, they are placed on special germination paper and germinated in a seed incubator at 25 ℃. When the buds reach about 1 cm in length, buds of similar length are selected and transferred to an incubator at 6 ℃ for low-temperature treatment for 7 days. The control germinates at room temperature of 25 ℃ and grows at a constant temperature of 25 ℃ for 7 days.

[0054] (3) Specific steps for identifying seedling low-temperature tolerance: Seed disinfection treatment was the same as the germination period treatment. Seeds were placed in a 25 ℃ incubator for 2 days. After germination, seeds with uniform sprout length were transferred to sand trays. Each treatment was repeated three times, with 30 seedlings per repeat. All seedlings were placed in a 25 ℃ incubator with a light cycle of 16 hours light / 8 hours darkness. When the seedlings reached the two-leaf-one-heart stage, the treatment groups were transferred to a 4 ℃ incubator for 2-4 days and then a recovery treatment for 2 days. The control group continued to be incubated at 25 ℃. Germination rate, sprout length, root length, root surface area, root volume, and average root diameter of each group were measured and recorded at the germination, sprout, and seedling stages. Germination index, simple vigor index, and vigor index were calculated. The membership function method was used to comprehensively evaluate the low-temperature tolerance of each line.

[0055] result:

[0056] (1) Identification of low temperature tolerance during germination period

[0057] The results are shown in Table 1. Among them, the three overexpression lines OE-4, OE-6, and OE-7 showed the strongest low-temperature tolerance during germination. Compared with the recipient control, the three overexpression lines exhibited longer shoots and roots and stronger low-temperature tolerance after being treated with low-temperature stress during germination. Figure 2 In conclusion, overexpression of the ZmbHLH30 gene can improve the low-temperature tolerance of maize inbred lines during germination.

[0058] Table 1. Comprehensive evaluation of low-temperature tolerance during germination of overexpression and receptor control lines.

[0059]

[0060] (2) Identification of low temperature tolerance during bud stage

[0061] The results are shown in Table 2. The D value of the recipient control was 0.238, with a comprehensive evaluation grade of V. The D values ​​of the overexpression lines ranged from 0.316 to 0.698. OE-2 had the highest comprehensive evaluation grade of 0.698, improving its low-temperature tolerance grade to Grade I. OE-4 improved by three grades, reaching Grade II. OE-7 had a comprehensive evaluation grade of Grade III. Four transgenic lines, including OE-1 and OE-3, improved their low-temperature tolerance grade by one grade, achieving a comprehensive evaluation grade of Grade IV. Figure 3 As shown, after low-temperature stress treatment, the three overexpressing lines OE-2, OE-4, and OE-7 exhibited stronger low-temperature tolerance compared to the recipient control, with increased root and shoot length, indicating improved low-temperature tolerance. Therefore, overexpression of the ZmbHLH30 gene can enhance the low-temperature tolerance of maize inbred lines during the bud stage.

[0062] Table 2. Comprehensive evaluation of low-temperature tolerance during bud stage for overexpression and receptor control lines.

[0063]

[0064] (3) Identification of seedling cold tolerance

[0065] The results are shown in Table 3. Three transgenic lines, OE-4, OE-5, and OE-6, which exhibited improved low-temperature tolerance during the seedling stage, were subjected to low-temperature stress during the seedling stage. Figure 4 As shown, the three overexpression lines exhibited less leaf wilting compared to the receptor control. In conclusion, overexpression of ZmbHLH30 can improve the low-temperature tolerance of maize inbred lines during the seedling stage.

[0066] Table 3. Comprehensive evaluation of low-temperature tolerance during seedling stage for overexpression and receptor control lines.

[0067]

[0068] Example 4: Analysis of the induction expression pattern of ZmbHLH30 overexpression lines

[0069] The selected superior overexpression lines and the control line B104 were subjected to low-temperature treatment during seed germination, budding, and seedling stages. The specific steps are as follows:

[0070] (1) Germination period: Soak seeds in sterile water at 25℃ for 6 h, and treat them with low temperature at 10℃ and control at 25℃ for 2, 4, 6, 8, 10 and 12 h respectively, and then dissect and separate the embryos.

[0071] (2) Germination period: When the seeds are germinated at a constant temperature of 25 ℃ until the buds are about 1 cm long, they are placed in incubators at 6 ℃ and 25 ℃ for 2, 4, 8 and 12 h respectively, and the buds and roots are taken respectively.

[0072] (3) Seedling stage: When the seedlings grow to the two-leaf-one-heart stage, they are treated with low temperature of 25 ℃ and 4 ℃ for 2, 4, 8 and 12 h respectively, and leaves, stems and roots are taken respectively.

[0073] (4) Each of the above samples was treated three times, with five samples mixed each time to extract RNA, reverse transcribe to synthesize cDNA, and perform PCR detection.

[0074] The results are as follows Figure 5-7 As shown. Figure 5 The relative expression level of the ZmbHLH30 gene in germinating embryos is shown. Figure 6 The relative expression levels of the ZmbHLH30 gene in buds and roots during the bud stage are given. Figure 7 The values ​​represent the relative expression levels of the ZmbHLH30 gene in leaves, stems, and roots during the seedling stage. These results indicate that the ZmbHLH30 gene is an important factor involved in maize's cold tolerance, and that overexpression lines of this gene showed significantly improved cold tolerance compared to the receptor control.

[0075] Example 5: Sequence Variation Analysis of ZmbHLH30

[0076] 1. Based on the sequence of the ZmbHLH30 gene, primers as shown in SEQ ID NO.10-11 (5'-CTACGAGCGTTTACGCATTA-3'; 5'-TAAATTCCACTATCGCCAACA-3') were designed to amplify its DNA sequence in 123 maize inbred lines with different low-temperature tolerance. The CDS region of the gene was extracted using Snap Gene software, and sequence variation analysis of the CDS region of the gene in the 123 maize inbred lines was performed using DNASPv 6.0 software. The results showed that there were a large number of mutations in the second exon region of the gene.

[0077] 2. ZmbHLH30 monomer type analysis

[0078] This invention detected 12 SNP sites in the CDS region of the gene of 123 maize inbred lines ZmbHLH30 with different low-temperature tolerance levels, corresponding to nucleotides 124, 339, 354, 360, 372, 380, 508, 528, 576, 585, 587, and 678 of the sequence shown in SEQ ID NO. 12, and two Indel sites located at positions 35-40 and 325-330 of the sequence shown in SEQ ID NO. 12 (Table 4). Using DNA SPv6.0 software, polymorphic sites were classified according to the type and number of nucleotide variations. A total of 12 haplotypes were identified (Table 5), with a polymorphism index of 0.8337. The major haplotypes HAP3, HAP4, HAP5, and HAP7 accounted for 77.24% of the tested samples, while other haplotypes were rare variations. HAP3 consists of 36 inbred lines, mainly including materials with medium-low temperature resistance and high sensitivity to low temperatures; HAP4 consists of 22 materials with low or medium low temperature resistance; HAP5 consists of 18 inbred lines, mainly including materials with medium resistance and high sensitivity to low temperatures; HAP7 consists of 19 inbred lines, including materials with medium resistance and high sensitivity to low temperatures. Based on the above results, it is preliminarily considered that the 36 materials with medium or higher low temperature resistance in HAP3 belong to excellent haplotypes.

[0079] Table 4. SNPs, Indels, and monomer types in the CDS region of ZmbHLH30

[0080]

[0081] Table 5. Inbred lines corresponding to ZmbHLH30 haplotype

[0082]

[0083] 3. Analysis of changes in amino acid levels in ZmbHLH30

[0084] In this invention, four non-synonymous SNP mutations were found in the CDS region of the ZmbHLH30 gene in 123 maize inbred lines. SNP124 and SNP587 were mutated from alanine (A) and asparagine (N) to serine (S), respectively. SNP380 was mutated from serine (S) and SNP508 was mutated from valine (V) to isoleucine (I), as shown in Table 6.

[0085] Table 6. Amino acid changes corresponding to SNPs of nonsynonymous mutations.

[0086]

[0087] 4. Nucleotide diversity analysis of ZmbHLH30

[0088] Based on different levels of low-temperature resistance, the materials were classified, and the nucleotide diversity and haplotype diversity of each level were analyzed. As shown in Table 7, among the 123 inbred lines studied, 3 high-low-temperature-resistant materials had a nucleotide diversity π value of 0.01027 and an Hd value of 1.000; 5 low-temperature-resistant materials had a π value of 0.00700 and an Hd value of 0.900; 87 medium-low-temperature-resistant materials had a π value of 0.00640 and an Hd value of 0.773; and 21 highly sensitive materials had a π value of 0.00384 and an Hd value of 0.834.

[0089] Table 7 Nucleotide diversity analysis of ZmbHLH30

[0090]

[0091] Example 6 Development of dCAPS molecular markers for ZmbHLH30

[0092] 1. Association analysis between ZmbHLH30 sequence variation and maize cold tolerance

[0093] This invention combines the phenotypic identification results of low-temperature stress treatment of maize inbred lines in our laboratory in the previous period, and uses TASSEL 5.0 software to conduct correlation analysis on the SNP sites and InDel sites in the CDS region of the ZmbHLH30 gene of 123 maize inbred lines with the phenotypic indicators of low-temperature tolerance during germination, namely, relative germination rate (RGR), relative shoot length (RGL), relative root length (RRL), relative vigor index (RVI), relative root surface area (RRSA), and relative root volume (RRV). The results are shown in Table 8. The nonsynonymous mutation SNP380 showed a very strong association with RRL, RVI, RSVI, RRSA, and RRV during germination (p<0.01). The contribution rate of SNP380 to these phenotypes ranged from 7.47% to 12.62%. Although the synonymous mutation sites SNP339, SNP528, and SNP576 did not directly change the protein sequence, they still showed a very significant association with eight indicators, including relative germination rate, relative root volume, and relative germination index during germination (p<0.01), with a phenotypic contribution rate of 5.03%-17.45%.

[0094] Table 8. Results of the association analysis between sequence variation of CDS of ZmbHLH30 and maize cold tolerance.

[0095]

[0096]

[0097] 2. Design of dCAPS molecular markers for ZmbHLH30

[0098] SNP380 (G / T), a non-synonymous mutation site with the highest phenotypic contribution, was selected. The optimal restriction enzyme site was screened and the restriction endonuclease was determined using the dCAPs Finder online platform. The marker DNCAP380 was developed, and primer sequences for amplifying this molecular marker were designed as shown in SEQ ID NO.13-14 (5'-CGCTCGATCGTGCAGATGG-3'; 5'-TGACGCGCCGCGGCGGCGGCTAG-3'). Using these primers, PCR amplification was performed on maize materials with cold-resistant and cold-sensitive genotypes. Materials with the cold-resistant genotype could not be specifically cleaved by NheI to obtain a 246 bp fragment, while the amplification product of the cold-sensitive genotype maize material, with a G-to-T mutation forming a restriction enzyme site, could be cleaved by NheI into two fragments: 221 bp and 25 bp. Figure 8 ).

[0099] Example 7: Verification of DNdCAPS380 Functional Markers

[0100] Using 123 amplified inbred lines (Table 9) as materials, the DNCAP380 marker was validated, such as... Figure 8 As shown, the PCR products of the cryotolerant and cryosensitive genotypes, after enzyme digestion verification, yielded bands of 246 bp and 221 bp, respectively. Of the 123 inbred lines, 91 exhibited a cryotolerant phenotype, with 70 of these (including 8415, 7884, and 77) producing enzyme digestion products of 246 bp, indicating a cryotolerant genotype (genotype-phenotype concordance rate of 76.92%). The remaining 32 inbred lines exhibited a cryosensitive phenotype, with 27 of these (including PI42, Dan1324, and JiA-034) producing enzyme digestion products of 221 bp and 25 bp, indicating a cryosensitive genotype (genotype-phenotype concordance rate of 84.38%).

[0101] Table 9. Results of low-temperature tolerance evaluation and enzyme digestion verification of 123 maize inbred lines.

[0102]

[0103]

[0104]

[0105]

[0106] Enzyme digestion verification revealed that the DNCAP380 marker can effectively identify the two parental materials, Ye 478 and Qi 319. Therefore, 102 families in the RIL population constructed using Ye 478 and Qi 319 as parents were used for verification. Ye 478 is a low-temperature tolerant inbred line, and Qi 319 is a low-temperature highly sensitive inbred line, yielding two types of bands: 246 bp and 221 bp, and 25 bp, respectively. Figure 9 As shown in Table 10, 70 of the 102 RIL populations exhibited a low-temperature tolerant phenotype. Among them, 57 materials, including ZYQ010, ZYQ054, and ZYQ113, had enzyme digestion products of 246 bp, with sequences shown in SEQ ID NO.15, indicating a low-temperature tolerant genotype. The genotype-phenotype concordance rate was 81.43%. The enzyme digestion products of 13 inbred lines, including ZYQ042, ZYQ155, and ZYQ218, were 221 bp and 25 bp, respectively, with sequences shown in SEQ ID NO.16 and SEQ ID NO.17, indicating a low-temperature sensitive genotype.

[0107] Table 10. Low-temperature tolerance and enzyme digestion verification results of 102 maize RIL population inbred lines.

[0108]

[0109] In summary, this gene ZmbHLH30 containing the bHLH domain enhances the low-temperature tolerance of maize during germination, and overexpressed plants have a stronger ability to resist low-temperature stress. The molecular marker DNdCAPS380 developed based on this gene can be used to identify the low-temperature tolerance of maize for the purpose of improving and breeding low-temperature tolerant maize varieties.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Contains corn ZmbHLH30 Application of biological materials containing genes or their encoded proteins in improving the low-temperature resistance of maize, wherein the maize ZmbHLH30 The gene sequence is shown in SEQ ID NO.1, and the biological material is any one of the following: A: Enables ZmbHLH30 Expression cassettes for gene overexpression; B: A recombinant vector containing the expression cassette described in A; C: Recombinant microorganisms containing the expression cassette described in A or the recombinant vector described in B.

2. A method for improving the low-temperature resistance of corn, characterized in that, Upregulating maize using one or more methods, including transgenic technology, gene editing, hybridization, backcrossing, self-pollination, or asexual reproduction. ZmbHLH30 The gene expression level improves the low-temperature resistance of maize. ZmbHLH30 The gene sequence is shown in SEQ ID NO.

1.

3. The application of a dCAPS molecular marker associated with low-temperature tolerance during maize germination in any of the following aspects: (1) To assess the low-temperature resistance of corn materials; (2) Breeding low-temperature resistant maize materials through molecular marker-assisted breeding; The molecular marker, DNdCAPS380, is located on maize chromosome 1. The dCAPS molecular marker is obtained by amplification using maize genomic DNA as a template with the primer pairs shown in SEQ ID NO. 13 and SEQ ID NO.

14. The dCAPS molecular marker contains one SNP site, which is located at... ZmbHLH30 The mutation in the CDS region of the gene is G / T, corresponding to nucleotide 380 of the sequence shown in SEQ ID NO.

12.

4. A method for identifying or breeding low-temperature resistant maize materials, characterized in that, Includes the following steps: Genomic DNA was extracted from the maize material samples to be tested and PCR amplification was performed using the primer pairs shown in SEQ ID NO.13 and SEQ ID NO.

14. The amplification products were digested with NheI restriction endonuclease. Low-temperature resistant materials could not be digested by NheI restriction endonuclease, resulting in one fragment, while low-temperature sensitive materials could be digested by NheI restriction endonuclease, resulting in two fragments.

5. The method as described in claim 4, characterized in that, The cryogenic material cannot be cleaved by the NheI restriction endonuclease, yielding a 246 bp fragment, the sequence of which is shown in SEQ ID NO.

15. The cryogenic sensitive material can be cleaved by the NheI restriction endonuclease, yielding two fragments of 221 bp and 25 bp in length, the sequences of which are shown in SEQ ID NO.16 and SEQ ID NO.17, respectively.

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