Promoter of corn disease resistance related gene, molecular marker and application thereof

Abstract

The invention discloses a promoter of a corn disease resistance related gene, a molecular marker and application of the promoter and the molecular marker. The invention provides the promoter of the TPS6 gene for regulating and controlling the resistance of the northern leaf blight, the southern leaf blight, the smut disease and the like of the corn as shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, and the mutant of the promoter, which can regulate and control the expression of the TPS6 gene in the corn, and can be used for improving the resistance of the corn to the northern leaf blight, the southern leaf blight, the smut disease and the like or cultivating a new variety of disease-resistant corn. The invention provides a molecular marker Indel8 as a molecular marker for expression of a TPS6 gene in corn, and application of the molecular marker Indel8 in regulation of expression of TPS6 in corn and improvement of corn disease resistance.

Description

Technical Field

[0001] The present invention belongs to the fields of genetic engineering and molecular biology, and particularly relates to a promoter, a molecular marker and an application thereof of a maize disease resistance-related gene. Background Art

[0002] Maize, as an important food crop, feed and industrial raw material in China, occupies an important position in the national economy. In recent years, with the continuous increase in the maize planting area and the frequent occurrence of extreme weather, the occurrence of maize diseases has become increasingly serious.

[0003] Maize smut, also known as maize head smut, covered smut, etc., is a locally invasive disease caused by Ustilago maydis, which can occur during the entire growth period of maize, and is particularly obvious at the heading stage. Ustilago maydis mainly infects young tissues such as the stems, leaves, tassels, ears, and aerial roots of maize, causing them to become diseased. In the maize production areas of China, maize smut is relatively common, with an incidence rate of about 5-10%, seriously affecting the yield and quality of maize (Liu Fen, Functional study of the znf9 gene during the development of Ustilago maydis, 2014).

[0004] Northern corn leaf blight, also known as maize leaf blight, maize stripe blight, is an important maize disease caused by Exserohilum turcicum, which is widely distributed in maize cultivation areas. It mainly damages maize leaves. In years of severe occurrence, it generally causes a 15%-20% reduction in maize yield, and in severe cases, the reduction can exceed 50%.

[0005] Southern corn leaf blight, also known as maize leaf spot, is a maize disease mainly characterized by small leaf lesions caused by Cochliobolus heterostrophus (asexual state: Bipolaris maydis). It can occur throughout the growth period of maize, but is severe at the tasseling and filling stages. It occurs widely and severely in the warm and humid areas of the Yellow River and Yangtze River basins in China, generally causing a 15-20% reduction in yield, and in severe cases of reduction, it can exceed 50%, or even result in no harvest.

[0006] Breeding and promoting excellent disease-resistant maize varieties is one of the important goals of maize trait improvement and also the most economical and effective means to control maize smut, northern leaf blight, southern leaf blight, etc. Existing studies have shown that the TPS6 gene plays an important role in the disease resistance process of maize. The maize TPS6 gene was first reported by Basse et al. from the Max Planck Institute in Germany. After maize is infected by Ustilago maydis, the TPS6 gene (originally named Umi2) is significantly induced to express (Basse, C.W. (2005) Dissecting defense-related and developmental transcriptional responses of maize during Ustilago maydis infection and subsequent tumor formation. Plant physiology 138, 1774-1784). TPS6 encodes a sesquiterpene cyclase that can catalyze the production of (S)-β-macrocarpene and (S)-β-bisabolene( T.G., Schnee, C., Li, S., Svatos, A., Schneider, B., Gershenzon, J. and Degenhardt, J. (2008) Protonation of a neutral (S)-β-bisabolene intermediate is involved in (S)-β-macrocarpene formation by the maize sesquiterpene synthases TPS6 and TPS11. The Journal of biological chemistry 283, 20779-20788), and finally produces phytoalexin Zealexins under the catalysis of P450 enzyme (Huffaker, A., Kaplan, F., Vaughan, M.M., Dafoe, N.J., Ni, X., Rocca, J.R., Alborn, H.T., Teal, P.E. and Schmelz, E.A. (2011) Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize. Plant physiology 156, 2082-2097; Mao, H., Jiang, L., Fei, R., Peters, R.J. and Qiang, W. (2016) Characterization of CYP71Z18 indicates a role in maize zealexin biosynthesis. Phytochemistry 121, 4-10). Silencing of TPS6 significantly reduces the disease resistance of maize to Ustilago maydis (van der Linde, K., Kastner, C., Kumlehn, J., Kahmann, R. and Doehlemann, G. (2011) Systemic virus-induced gene silencing allows functional characterization of maize genes during biotrophic interaction with Ustilago maydis. New Phytologist 189, 471-483). Maize has experienced a long period of artificial selection and produced a large number of germplasm resources.There are a large number of variations among different varieties. Discovering relevant variations and developing corresponding molecular markers will be of great significance for the breeding of molecular-assisted disease-resistant maize, accelerating the pace of disease-resistant maize breeding. Summary of the Invention

[0007] In response to the needs in the above fields, the present invention provides a promoter of a maize disease-resistant related gene, which can direct the expression of a maize disease-resistant related gene TPS6, enabling maize to exhibit disease resistance; at the same time, it also provides a mutant of this promoter.

[0008] The present invention also provides a molecular marker, which can be used to detect and identify the resistance of maize varieties to northern leaf blight, southern leaf blight, and head smut.

[0009] At the same time, the applications of this promoter and the molecular marker are provided.

[0010] A promoter of a maize disease-resistant related gene TPS6, characterized in that it has a nucleotide sequence shown in (a), (b), (c), or (d):

[0011] (a) The nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.4;

[0012] (b) A nucleotide sequence that can hybridize with the complementary sequence of SEQ ID No.1 or SEQ ID No.4 under stringent hybridization conditions;

[0013] (c) A nucleotide sequence having at least 90% or more similarity with the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.4;

[0014] (d) A nucleotide sequence obtained by deletion, substitution, or insertion of one or more bases based on the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.4, and this nucleotide sequence still has promoter function or activity.

[0015] The mutant of the above-mentioned promoter of the maize disease-resistant related gene TPS6 is characterized in that it has the nucleotide sequences shown in SEQ ID No.2 and SEQ ID No.3. This mutant loses promoter activity, resulting in the non-expression of the TPS6 gene in maize, manifested as a decrease in maize disease resistance.

[0016] A pair of specific primers for amplifying a molecular marker Indel8 that regulates the expression of the disease-resistant related gene TPS6 in maize, and its nucleotide sequence is:

[0017] TPS6P-F: 5'-TATGGTGTTCAACACATGGATGC-3';

[0018] TPS6P-R: 5'-CACAGAGTTGGATGGAAG-3'.

[0019] The molecular marker Indel8 that regulates the expression of the disease-resistant related gene TPS6 gene in maize is characterized in that: using maize genomic DNA as a template, it is obtained by PCR amplification with the following specific primer pair:

[0020] TPS6P-F: 5'-TATGGTGTTCAACACATGGATGC-3';

[0021] TPS6P-R: 5'-CACAGAGTTGGATGGAAG-3',

[0022] Among them, in the materials with normal expression of the TPS6 gene, the amplification product is 441 bp, and in the materials with abnormal expression of the TPS6 gene, the amplification product is 436 bp, or 557 bp.

[0023] Among them, at positions 297-304 of the amplification product in the materials with normal expression of the TPS6 gene is CATGCATC. In the amplification product of 436 bp in the materials with abnormal expression of the TPS6 gene, CATGCATC is deleted starting from the 305th position, a total of 8 bases. Or in the amplification product of 557 bp in the materials with abnormal expression, CATGCATC is deleted starting from the 424th position, a total of 8 bases.

[0024] In the maize inbred line materials where Indel8 in the promoter region of the TPS6 gene lacks the above 8 bases, the TPS6 gene is not expressed, and the risks of northern leaf blight, southern leaf blight, head smut, etc. of maize increase; on the contrary, in the maize inbred line materials where Indel8 in the promoter region of the TPS6 gene inserts the above 8 bases, the TPS6 gene can be normally induced to express, and the risks of northern leaf blight, southern leaf blight, head smut, etc. of maize decrease.

[0025] The molecular marker Indel8 that regulates the expression of the disease-resistant related gene TPS6 gene in maize is applied in screening and identifying maize disease-resistant varieties.

[0026] For the application described above, extract maize genomic DNA and perform PCR amplification with the following primer pair,

[0027] TPS6P-F: 5'-TATGGTGTTCAACACATGGATGC-3';

[0028] TPS6P-R: 5'-CACAGAGTTGGATGGAAG-3',

[0029] Among them, the amplified product is 441 bp, indicating the normal expression of the TPS6 gene and the strong disease resistance of this maize variety; the amplified product is 436 bp or 557 bp, indicating the abnormal expression of the TPS6 gene and the weak disease resistance of this maize variety.

[0030] Among them, at positions 297-304 of the amplified product in the material with normal expression of the TPS6 gene is CATGCATC. In the material with abnormal expression of the TPS6 gene, 8 bases of CATGCATC are deleted starting from position 305 of the 436-bp amplified product, or 8 bases of CATGCATC are deleted starting from position 424 of the 557-bp amplified product in the material with abnormal expression.

[0031] A method for cultivating a new disease-resistant maize variety, characterized in that by gene editing of the TPS6 gene promoter, the expression of the TPS6 gene in maize is restored or enhanced, thereby improving the disease resistance of maize.

[0032] The disease resistance mentioned above is resistance to northern leaf blight, southern leaf blight and head smut of maize.

[0033] In the present invention, a promoter of the maize disease-resistant related gene TPS6 was discovered upstream of the ATG of the TPS6 gene. The sequence from 400 bp to 1500 bp upstream of the ATG has promoter function, indicating that the promoter function exists about 400 bp upstream of the ATG. This promoter can regulate the expression of the disease-resistant related gene TPS6. Among 111 randomly selected maize inbred lines, the expression of the TPS6 gene in different maize inbred lines was detected by qPCR. It was found that there were two types of maize inbred lines. One type consisted of 60 maize inbred lines in which the TPS6 gene could be induced to express after ABA treatment; the other type consisted of 51 maize inbred lines in which the TPS6 gene did not express before and after ABA treatment. Referring to the maize B73RefGen_v3 genome sequence, the following primers were designed to amplify the TPS6 promoter sequence in 111 maize inbred lines (the amplified region contains about 400 bp sequence upstream of the ATG). TPS6P-F: 5'-TATGGTGTTCAACACATGGATGC-3'; TPS6P-R: 5'-CACAGAGTTGGATGGAAG-3'; The amplified products were subjected to Sanger sequencing. The sequencing results showed that the TPS6 promoters of the 111 maize inbred lines could be divided into three categories. The first category contained 60 maize inbred lines (such as B73), and the amplified product was 441 bp, which were maize inbred lines in which the TPS6 gene could be induced to express. The second category contained 9 maize inbred lines (such as CML229), and the amplified product was 436 bp, which were maize inbred lines in which the TPS6 gene did not express. The third category contained 42 maize inbred lines (such as B104), and the amplified product was 557 bp, which were maize inbred lines in which the TPS6 gene did not express. One inbred line was selected as a representative from each category, namely B73, CML229, and B104. The alignment results of the promoter sequence about 400 bp upstream of the ATG of the TPS6 gene showed that there were 4 site mutations in CML229 compared with B73, and there were greater differences between B104 and B73. However, compared with B73, CML229 and B104 had 3 identical site mutations, namely Indel4_AACTA / A, Indel1_CA / C, and Indel8_TCATGCATC / T( Figure 7)。Indel4_AACTA / A is located at positions 233 of SEQ ID No.1, positions 238 - 242 of SEQ ID No.2, and positions 354 - 358 of SEQ ID No.3 respectively; Indel1_CA / C is located at positions 280 - 281 of SEQ ID No.1, position 289 of SEQ ID No.2, and position 408 of SEQ ID No.3 respectively; Indel8_TCATGCATC / T is located at positions 297 - 304 of SEQ ID No.1, position 304 of SEQ ID No.2, and position 423 of SEQ ID No.3 respectively. The transient expression system of maize root protoplasts was used to analyze the effects of Indel4, Indel1, and Indel8 on the activity of the TPS6 promoter. The results showed that the promoter activity of 6PF11 - Indel8_T decreased significantly compared with that of 6PF11, while there was no significant difference between 6PF11 - Indel4_A, 6PF11 - Indel1_C and 6PF11. This indicates that Indel8 affects the activity of the TPS6 promoter, and when it mutates, it will lead to very low or no expression of the TPS6 gene.

[0034] The beneficial effects of the present invention are as follows: The molecular marker Indel8 of the disease - resistant related gene TPS6 gene expressed in maize provided by the present invention is applied in screening and identifying disease - resistant maize varieties. When the disease - resistant related gene TPS6 gene is expressed, it can improve the disease - resistant ability of maize. This molecular marker plays an important role in molecular - assisted disease - resistant maize breeding and selecting disease - resistant and high - quality crop germplasm resources. Brief Description of the Drawings

[0035] Figure 1 is a structural diagram of a plant expression vector,

[0036] where A is a schematic diagram of pCB - Ubi - TPS6, B is the CRISPR target sequence of TPS6, and C is a schematic diagram of the CRISPR editing vector pCB - Ubi - Cas9 - TPS6;

[0037] Figure 2 is the detection of the expression level of the TPS6 gene in the transformant,

[0038] where A is the detection of the expression level of the TPS6 gene in the TPS6 - OX transformant, and B is the detection of the expression level of the TPS6 gene (the mutated form of the target sequence) in the TPS6 - KO transformant,

[0039] Figure 3 is the result after inoculating the leaves of TPS6 - OX, TPS6 - KO and the control material B73 maize with fungi;

[0040] where (A) is Exserohilum turcicum, (B) is Bipolaris maydis, and (C) is Ustilago maydis;

[0041] Figure 4 For the expression of the TPS6 gene in 111 maize inbred lines before and after ABA treatment.

[0042] Figure 5 For the comparison of maize inbred lines B73 and B104,

[0043] where A is the alignment result of the CDS sequence of the TPS6 gene, and B is the three-dimensional structure alignment result;

[0044] Figure 6 For the activity comparison of the TPS6 promoter sequences 6PF1 (1500 bp upstream of ATG) and 6PF11 (400 bp upstream of ATG) of maize inbred line B73,

[0045] where A is the GUS histochemical staining in Arabidopsis thaliana, and B is the GUS enzyme activity analysis;

[0046] Figure 7 For the alignment of the TPS6 promoter sequences in different maize inbred lines,

[0047] where A is the PCR amplification, and B is the alignment result of Sanger sequencing;

[0048] Figure 8 For the activity analysis of 6PF11 and promoters with different mutation forms,

[0049] where A is the schematic diagram of the promoter vectors with different mutation forms, and B is the activity analysis result of the promoters with different mutation forms. Detailed implementation manners

[0050] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further elaborates the present invention in detail with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not impose any limitation on the scope of the present invention.

[0051] The biological materials involved below are all preserved in the laboratory of the applicant and can be distributed externally. Except as otherwise stated, they are all commercially available products.

[0052] Example 1. Verification of the function of the TPS6 gene

[0053] 1. Construction of the overexpression and CRISPR-Cas9 gene editing vectors of TPS6

[0054] Referring to the maize B73 RefGen_v3 genome sequence, the coding sequence of the TPS6 gene was artificially synthesized and constructed into the pCB-Ubi vector by seamless cloning (the pCB-Ubi vector was modified from the commercial vector pCAMBIA3300, and the maize Ubi promoter and NOS terminator were inserted into the multiple cloning sites HindIII and EcoRI). The obtained TPS6 overexpression vector was named pCB-Ubi-TPS6 (for the vector schematic diagram, see Figure 1 Figure A). Using the SnapGene software and the B73 RefGen_v3 gene sequence of TPS6 to search for the target editing sequence of the TPS6 gene, the obtained target sequence was then aligned with other genomic sequences of B73 to remove multi-copy and highly similar sequences to other genomic sequences. The secondary structure was predicted through the RNAFolding Form system, and finally, 1 optimal target sequence (20 bp, see the underlined part in Figure 1 Figure B) was selected for editing the TPS6 gene. The sgRNA expression cassette of the TPS6 target sequence was artificially synthesized (this expression cassette consists of the maize U6 promoter, the TPS6 target sequence, and the gRNA scaffold), and the expression cassette of the TPS6 target sequence was constructed into the pCB-Ubi-Cas9 vector by seamless cloning (the pCB-Ubi-Cas9 vector was constructed based on the pCB-Ubi vector in the previous step, and the Cas9 gene was inserted between the Ubi promoter and the NOS terminator of the pCB-Ubi vector by seamless cloning). The obtained CRISPR editing vector of TPS6 was named pCB-Ubi-Cas9-TPS6 (for the vector schematic diagram, see Figure 1 Figure C).

[0055] 2. Obtaining of TPS6 transgenic materials and detection of disease resistance

[0056] The pCB-Ubi-TPS6 and pCB-Ubi-Cas9-TPS6 vectors were respectively transformed into the maize inbred line B104 by the Agrobacterium-mediated method and backcrossed to the inbred line B73. The transformants with overexpression and knockout of TPS6 were detected using the primers of the TPS6 gene and the target sequence. The primer sequences are as follows:

[0057] TPS6-qPCR-F: 5'-CAAATGAGAGAAAAGGCTGC-3';

[0058] TPS6-qPCR-R: 5'-AGCCTCAACAAAATTCCCAAG-3';

[0059] TPS6-C-F: 5'-TAAAGGAAGAAGTGAGAAAC-3';

[0060] TPS6-C-R: 5'-ATGAAACATCGTAGCCA-3';

[0061] The TPS6-OX transformants ( Figure 2 A in) and TPS6-KO mutants ( Figure 2 B in) were obtained by detection. Further, inoculation tests for common smut, northern leaf blight, and southern leaf blight were conducted on the TPS6-OX transformants, TPS6-KO mutants, and the control material B73 ( Figure 3 ). The results showed that for the resistance to common smut, northern leaf blight, and southern leaf blight, the TPS6-OX transformants were generally higher than B73 and much higher than the TPS6-KO transformants. These results indicate that the TPS6 gene has a broad-spectrum disease resistance function in maize, and changing the expression level of TPS6 can affect the disease resistance of maize.

[0062] Example 2. Expression analysis of the TPS6 gene in different maize inbred lines

[0063] A total of 111 inbred lines were randomly selected from 527 maize inbred lines (Yang, X., Gao, S., Xu, S., Zhang, Z., Prasanna, B. M., Li, L., Li, J., and Yan, J. (2011). Characterization of a global germplasm collection and its potential utilization for analysis of complex quantitative traits in maize. Molecular Breeding 28, 511 - 526.). The seeds were sown in soil (vermiculite: nutrient soil = 1:1) and cultivated in an artificial climate chamber. The temperature was 25 °C, the photoperiod was 16 h (L) / 8 h (D), and the light intensity was 100 - 150 mE m -2 S -1 , and the humidity was 40 - 60%. Maize inbred line seedlings grown in soil for 2 weeks were treated by irrigating the roots with abscisic acid (ABA) (100 μM) solution. After 6 h, the roots were taken, frozen in liquid nitrogen, ground into powder, and total RNA was extracted and reverse transcribed into cDNA. The roots before ABA treatment were used as the control. The expression of the TPS6 gene in different maize inbred lines was detected by qPCR, with the maize actin1 gene as the internal reference gene. The primer sequences are as follows:

[0064] TPS6-qPCR-F: 5'-CAAATGAGAGAAAAGGCTGC-3';

[0065] TPS6-qPCR-R: 5'-AGCCTCAACAAAATTCCCAAG-3';

[0066] actin-qPCR-F: 5'-CGAATGCCCAGCAATGTA-3';

[0067] actin-qPCR-R: 5'-TTAGGTGGTCGGTGAGGT-3';

[0068] The qPCR results showed that 111 maize inbred lines could be divided into two categories. One category was the maize inbred lines in which the TPS6 gene could be induced to express after ABA treatment (such as B73), with a total of 60 lines; the other category was the maize inbred lines in which the TPS6 gene did not express before and after ABA treatment (such as B104), with a total of 51 lines ( Figure 4 ). The TPS6 gene sequences of B73 and B104 were downloaded from the MaizeGDB database. CDS sequence alignment found that there were 14 single-base mutations at 14 sites between the TPS6 of B104 and the TPS6 gene of B73. Among them, the mutations at 7 sites caused amino acid changes, but these mutations did not occur in metal cations, substrate binding sites, and the DDXXD motif ( Figure 5 in A). Three-dimensional structure model alignment also did not find obvious structural changes between the two ( Figure 5 in B). We speculated that the reason for the non-expression of the TPS6 gene in some maize inbred lines might be due to mutations in the promoter region of TPS6.

[0069] Example 3. Analysis of the active region of the TPS6 promoter in maize inbred line B73

[0070] Referring to the maize B73 RefGen_v3 genomic sequence, primers were designed to clone the 1500 bp sequence upstream of the ATG of the TPS6 gene as the full-length sequence of the TPS6 promoter (denoted as 6PF1), and the 400 bp sequence upstream of the ATG of the TPS6 gene as the truncated sequence of the TPS6 promoter (denoted as 6PF11, see sequence SEQ ID No. 4). 6PF1 and 6PF11 were respectively linked into the commercial pKGWFS7 plant expression vector by the Gateway method and transformed into Arabidopsis thaliana. GUS was used as a reporter gene to analyze the promoter activity. GUS staining and enzyme activity analysis were performed on the obtained transgenic Arabidopsis thaliana materials. The results showed that both 6PF1 and 6PF11 had promoter functions and there were no significant differences in the expression sites and expression intensities of the reporter genes ( Figure 6 ), indicating that the 400 bp upstream of the ATG of the TPS6 gene in B73 had the function of the full-length promoter. Subsequently, this region of different maize inbred lines could be compared and analyzed to find differences.

[0071] Example 4. Alignment analysis of TPS6 promoter sequences in different maize inbred lines

[0072] According to the results of Example 3 and referring to the maize B73 RefGen_v3 genome sequence, the following primers were designed to amplify the TPS6 promoter sequences in 111 maize inbred lines (the amplified region contains approximately 400 bp of sequence upstream of ATG).

[0073] TPS6P-F: 5'-TATGGTGTTCAACACATGGATGC-3';

[0074] TPS6P-R: 5'-CACAGAGTTGGATGGAAG-3';

[0075] The amplified products were subjected to Sanger sequencing. The sequencing results showed that the TPS6 promoters of the 111 maize inbred lines could be divided into three categories. The first category contained 60 maize inbred lines (such as B73), and the sequence was shown in SEQ ID No.1. The second category contained 9 maize inbred lines (such as CML229), and the sequence was shown in SEQ ID No.2. The third category contained 42 maize inbred lines (such as B104), and the sequence was shown in SEQ ID No.3. To more clearly display the alignment results, one inbred line was selected as a representative from each category, namely B73, CML229, and B104. The alignment results of the promoter sequences approximately 400 bp upstream of the ATG of the TPS6 gene showed that there were 4 site mutations in CML229 compared with B73, and there were relatively large differences between B104 and B73. However, compared with B73, CML229 and B104 had 3 identical site mutations, namely Indel4_AACTA / A, Indel1_CA / C, and Indel8_TCATGCATC / T( Figure 7 ). Indel4_AACTA / A was located at positions 233 of SEQ ID No.1, positions 238-242 of SEQ ID No.2, and positions 354-358 of SEQ ID No.3, respectively; Indel1_CA / C was located at positions 280-281 of SEQ ID No.1, position 289 of SEQ ID No.2, and position 408 of SEQ ID No.3, respectively; Indel8_TCATGCATC / T was located at positions 297-304 of SEQ ID No.1, position 304 of SEQ ID No.2, and position 423 of SEQ ID No.3, respectively. According to the TPS6 gene expression analysis results in Example 2, the TPS6 gene was not expressed in the maize inbred lines of the CML229 and B104 classes. We speculated that Indel4, Indel1, and Indel8 might be related to the expression of TPS6.

[0076] Example 5. Indel8 Affects the Activity of the TPS6 Promoter

[0077] After the Indel4, Indel1, and Indel8 of B73 were mutated into the B104 / CML229 form respectively, the 6PF11, 6PF11-Indel4_A, 6PF11-Indel1_C, and 6PF11-Indel8_T sequences were respectively constructed into the commercial pGreenII-0800 vector ( Figure 8 in A), and the maize root protoplast transient expression system was used to analyze the effects of Indel4, Indel1, and Indel8 on the activity of the TPS6 promoter. The results showed that the promoter activity of 6PF11-Indel8_T was significantly decreased compared with 6PF11, while there was no significant difference between 6PF11-Indel4_A, 6PF11-Indel1_C and 6PF11 ( Figure 8 in B). This indicates that Indel8 affects the activity of the TPS6 promoter, and when it mutates, it will lead to very low or no expression of the TPS6 gene.

[0078] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand. According to all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the protection scope of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. The promoter of a maize disease resistance-related gene TPS6, characterized in that Having a nucleotide sequence shown in (a), (b), (c) or (d): (a) The nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.4; (b) A nucleotide sequence that can hybridize with the complementary sequence of SEQ ID No.1 or SEQ ID No.4 under stringent hybridization conditions; (c) A nucleotide sequence having at least 90% or more similarity with the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.4; (d) A nucleotide sequence obtained by deletion, substitution or insertion of one or more bases on the basis of the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.4, and this nucleotide sequence still has promoter function or activity.

2. Use of the promoter according to claim 1 in regulating the expression of the disease resistance-related gene TPS6 gene in maize.

3. A specific primer pair for amplifying the molecular marker Indel8 that regulates the expression of the disease resistance-related gene TPS6 gene in maize, and its nucleotide sequence is: TPS6P-F: 5'-TATGGTGTTCAACACATGGATGC-3'; TPS6P-R: 5'-CACAGAGTTGGATGGAAG-3'.

4. The molecular marker Indel8 for regulating the expression of the disease-resistant related gene TPS6 gene in maize, characterized in that: Using maize genomic DNA as a template, it is obtained by PCR amplification with the following specific primer pair: TPS6P-F: 5'-TATGGTGTTCAACACATGGATGC-3'; TPS6P-R: 5'-CACAGAGTTGGATGGAAG-3', wherein, in the material with normal expression of the TPS6 gene, the amplification product is 441bp, and in the material with abnormal expression of the TPS6 gene, the amplification product is 436bp, or 557bp.

5. The molecular marker Indel8 according to claim 4, wherein, At positions 297-304 of the 441bp amplification product in the material with normal expression of the TPS6 gene is CATGCATC. In the material with abnormal expression of the TPS6 gene, the 436bp amplification product has a deletion of 8 bases of CATGCATC starting from position 304, or in the material with abnormal expression of the TPS6 gene, the 557bp amplification product has a deletion of 8 bases of CATGCATC starting from position 423.

6. Application of the molecular marker Indel8 that regulates the expression of the disease resistance-related gene TPS6 gene in maize according to claim 4 or 5 in screening and identifying disease-resistant maize varieties.

7. According to the application described in claim 6, extract maize genomic DNA and amplify it with the following primer pair, TPS6P-F: 5'-TATGGTGTTCAACACATGGATGC-3'; TPS6P-R: 5'-CACAGAGTTGGATGGAAG-3', Among them, If the amplification product is 441bp, it indicates that the TPS6 gene is normally expressed and the maize variety has strong disease resistance; if the amplification product is 436bp, or 557bp, it indicates that the TPS6 gene is abnormally expressed and the maize variety has weak disease resistance.

8. The application according to claim 7, wherein, At positions 297 - 304 of the amplification product in materials with normal expression of the TPS6 gene, it is CATGCATC; in materials with abnormal expression of the TPS6 gene, starting from position 304 of the 436bp amplification product, CATGCATC, a total of 8 bases, is deleted, or starting from position 423 of the 557bp amplification product in materials with abnormal expression, CATGCATC, a total of 8 bases, is deleted.

9. A method for cultivating a new variety of disease-resistant corn, characterized in that, By performing gene editing on the promoter of the TPS6 gene, the expression of the TPS6 gene in maize is restored or increased, thereby enhancing the disease resistance of maize.

10. According to the method described in claim 9, insert CATGCATC, a total of 8 bases, at the deletion of the molecular marker Indel8 in the promoter region of the TPS6 gene to enable normal induced expression of the TPS6 gene in maize materials, and the diseases against which the resistance is provided are northern leaf blight, southern leaf blight, and head smut of maize.

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