Corn drought-resistant gene ZmRVE6 as well as drought-resistant molecular marker and application thereof

Abstract

The invention discloses a corn drought-resistant gene ZmRVE6 as well as a drought-resistant molecular marker and application. The nucleotide sequence of the drought-resistant gene ZmRVE6 is shown as SEQ ID NO: 1. The nucleotide sequence of the corn drought-resistant molecular marker ZmRVE6Type 3 is as shown in SEQ ID NO: 4. According to the invention, a drought-resistant related gene ZmRVE6 is identified. The gene participates in regulation of expression of biological clock related genes and participates in regulation of plant growth and development. Overexpression of the ZmRVE6 can improve the drought resistance of the corn. The structural variation of the promoter region can be used as a corn drought-resistant molecular marker, and amplification of the molecular marker is used for identifying corn varieties with excellent drought resistance. The practical value and the economic value are realized.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and particularly relates to a maize drought-resistant gene ZmRVE6, a drought-resistant molecular marker and its application. Background Art

[0002] Drought is one of the main abiotic stresses affecting crop yields, which can lead to crop yield reduction or crop failure. The main maize-producing areas in China highly overlap with the arid and semi-arid regions in China. Under the background of global warming, drought is extremely likely to occur, posing a great threat to maize production.

[0003] In recent years, the drought-resistant mechanism of plants has been fully studied, and a variety of drought-resistant genes have been identified. Transcription factors are important hubs for plant drought responses. After receiving upstream stress signals, transcription factors interact with their cis-elements to regulate a series of stress-responsive genes, and thus have a greater functional effect compared with single functional genes. RVE belongs to the CCA1-like proteins in the MYB-related family of transcription factors. The MYB-related family is a heterogeneous collection of R1 / R2 and R3-MYB type proteins, usually containing a MYB repeat sequence, and can be divided into 5 subfamilies: circadian clock-related CCA1-like proteins, CAPRICE-like, telomeric DNA-binding-like proteins, I-box-binding-like factors, and R-R type proteins. Among them, CCA1-like proteins usually contain the consensus sequence SHAQK(Y / F)F within a single MYB repeat. CCA1-like proteins play a circadian clock regulatory function by binding to the AAAATATCT sequence. More and more evidence shows that CCA1-like proteins are involved in various biological processes in plants in addition to participating in circadian rhythm regulation, including seed germination, hypocotyl elongation, plant flowering, anthocyanin biosynthesis, plant hormone signal transduction pathways, and stress responses. However, the existing research on the CCA1-like subfamily is limited to a few plant species such as Arabidopsis thaliana and rice, and there are almost no relevant reports in maize.

[0004] In addition to molecular biological breeding, using genetic means to cultivate and improve drought-resistant maize varieties is also of great significance. Screening molecular markers related to maize drought resistance can effectively improve the efficiency of breeding drought-resistant maize varieties. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a maize drought-resistant gene ZmRVE6, a drought-resistant molecular marker and its application. ZmRVE6 regulates the expression of circadian clock-related genes under normal temperature conditions and participates in the regulation of plant growth and development. Maize drought-resistant molecular marker ZmRVE6 Type3, including an insertion of approximately 7318 bp identified by this marker. The presence of this marker significantly improves the drought resistance of maize, and its mechanism of action is to promote the expression of ZmRVE6.

[0006] To achieve the above object, the technical solutions designed by the present invention are as follows:

[0007] The present invention provides a maize drought-resistant gene ZmRVE6, and the nucleotide sequence of the drought-resistant gene ZmRVE6 is shown in SEQ ID NO: 1.

[0008] The present invention also provides a transcript of the above maize drought-resistant gene ZmRVE6, and the CDS nucleotide sequence of the transcript is shown in SEQ ID NO: 3.

[0009] The present invention also provides a drought-resistant protein ZmRVE6 encoded by the above transcript ZmRVE6, and the amino acid sequence of the protein is shown in SEQ ID NO: 2.

[0010] The present invention also provides an application of the above ZmRVE6 or the above drought-resistant protein ZmRVE6 in improving the drought resistance of maize.

[0011] The present invention also provides an application of the above drought-resistant transcript ZmRVE6 or the above drought-resistant protein ZmRVE6 in cultivating new varieties of drought-resistant maize.

[0012] The present invention also provides a maize drought-resistant molecular marker ZmRVE6 Type 3 , the maize drought-resistant molecular marker ZmRVE6 Type 3 has a nucleotide sequence shown in SEQ ID NO: 4.

[0013] The present invention also provides a primer pair for obtaining the above maize drought-resistant molecular marker ZmRVE6 Type 3 , and the primer pair is:

[0014] ZmRVE6 Type 3 -F1: 5'-CAAGCTCTGGCTCTGCAAC-3',

[0015] ZmRVE6 Type 3 -R1: 5'-GGCGGCACAAGACTCCAC-3'.

[0016] The present invention also provides an application of the above primer pair in preparing a kit for identifying maize varieties with excellent drought resistance.

[0017] The present invention also provides a kit for preparing and identifying maize varieties with excellent drought resistance, and the kit includes the primer pairs described above.

[0018] The present invention also provides a method for identifying maize varieties with excellent drought resistance using the above primer pairs or the above kit, comprising the following steps:

[0019] (1) Extract the DNA of the maize variety to be detected;

[0020] (2) Using the above DNA as a template, perform PCR amplification with the primer pairs,

[0021] (3) Electrophoresis: If a target band of 1634 bp appears in the amplification, it indicates that the maize variety to be detected has strong drought resistance, that is, this variety is a maize variety with excellent drought resistance.

[0022] Principle of the present invention:

[0023] The present invention obtains maize ZmRVE6 overexpression materials by using maize transformation technology. The overexpression materials and the isolated negative control materials were subjected to drought treatment at the seedling stage, and it was found that the drought resistance of the overexpression materials was significantly higher than that of the wild type. Further determination at the physiological and biochemical levels found that the content of malondialdehyde in the overexpression materials was lower after drought stress, indicating that the positive materials with ZmRVE6 overexpression had stronger drought tolerance compared to the negative isolates. Using association population amplification, it was found that there were large structural variations in the promoter region of maize ZmRVE6. Taking B73 as the reference sequence, there was an insertion sequence of 7318 bp in the type3 promoter. According to the statistical results and combined with the existing data on expression levels and survival rates before and after drought in the laboratory, the relationship between the structural variations in the upstream sequence of ZmRVE6 and the total expression level and drought survival rate of the gene before and after drought was statistically analyzed, and it was found that the type1 structural variation was significantly correlated with the expression level and survival rate of ZmRVE6.

[0024] Beneficial effects of the present invention:

[0025] The present invention identified a drought-related gene ZmRVE6. This gene is involved in regulating the expression of genes related to the biological clock and participates in regulating plant growth and development. Overexpression of ZmRVE6 can improve the drought resistance of maize. The structural variation in its promoter region can be used as a molecular marker for maize drought resistance; amplifying this molecular marker is used to identify maize varieties with excellent drought resistance; it has practical and economic value. Description of the drawings

[0026] Figure 1 It is a schematic diagram for constructing the overexpression vector pZZ0153-RD101p-ZmRVE6-3HA,

[0027] Figure 2Identification diagram of the protein and expression level of ZmRVE6 in transgenic materials;

[0028] In the figure, A is the identification diagram of protein level before and after drought treatment,

[0029] B is the identification diagram of the expression level of ZmRVE6 before and after drought treatment.

[0030] Figure 3 Schematic diagram for measuring the drought phenotypes and physiological indexes of ZmRVE6 overexpression materials and the control at the seedling stage;

[0031] In the figure, A is the photo of the phenotype taken before and after drought treatment,

[0032] B is the statistical data diagram of the survival rate,

[0033] C is the measurement result diagram of the malondialdehyde content.

[0034] Figure 4 Allelic variation analysis diagram of ZmRVE6;

[0035] In the figure, A is the distribution diagram of the promoter structural variation of ZmRVE6 in the association population and in 378 association populations,

[0036] B is the distribution diagram in 378 association populations, C is the analysis diagram of the expression level and survival rate of the ZmRVE6 promoter structural variation before and after drought.

[0037] Figure 5 Electrophoresis diagram of PCR products:

[0038] In the figure, CIMBL55, CIMBL70, CIMBL90, B73 and TY2 are five different maize inbred lines. Specific implementation manners

[0039] The present invention will be further described in detail below in conjunction with specific embodiments for those skilled in the art to understand.

[0040] Example 1 Mapping of maize drought-resistant gene ZmRVE6

[0041] By analyzing the RNA-seq data of maize leaves under different drought degrees, we found that the expression of some RVE family genes in maize was inhibited by drought when the maize was subjected to drought treatment, while the expression level of ZmRVE6 increased and continued to rise with the deepening of drought stress. It shows that the expression of ZmRVE6 is induced by drought, and ZmRVE6 may be involved in the drought stress response in maize. Its nucleotide sequence is shown in SEQ ID No: 1; the amino acid sequence encoded by this gene is shown in SEQ ID No: 2.

[0042] Example 2 Construction of Maize ZmRVE6 Overexpression Vector

[0043] Based on the expression vector pZZ0153-RD101p-3HA, the cDNA fragment of ZmRVE6 was inserted downstream of the RD101 promoter and in the middle of the 3HA tag through homologous recombination, and was expressed by the RD101 promoter( Figures 1-2 ); Electrophoresis detection and sequencing analysis showed that the maize ZmRVE6 overexpression vector was successfully obtained, namely named pZZ0153-RD101p-ZmRVE6-3HA. The specific experimental steps are as follows:

[0044] 1. Amplification of ZmRVE6:

[0045] According to the cDNA sequence of ZmRVE6, a pair of homologous recombination primers were designed for PCR amplification. The primer sequences are as follows:

[0046] ZmRVE6--F0: 5’-GCACTAGTATCCCGGGAAGGCGCGCCAT GGTGTCGACGAGCTCG-3’,

[0047] ZmRVE6-R0: 5’-CGTCGTATGGGTACATGGCCTTGCTCGTT ATCATGAACG-3’;

[0048] Vazyme Phanta Max Super-Fidelity DNA polymerase was used for gene amplification to obtain the PCR product, and its CDS nucleotide sequence is shown in SEQ ID NO: 3; The reaction system is as follows (total volume 20 μL):

[0049] 2×Phanta Buffer 10 μL dNTP 0.4 μL ZmRVE6-F0 0.8 μL ZmRVE6-R0 0.8 μL Phanta max 0.4 μL cDNA (maize B73) 1.5 μL <![CDATA[ddH2O]]> 6.9 μL

[0050] The reaction program was: 95°C for 3 min; 95°C for 15 s; 58°C for 30 s; 72°C for 60 s; 72°C for 5 min; 35 cycles;

[0051] 2. Preparation of linearized vector:

[0052] The pZZ0153-RD101p-3HA vector was digested with the restriction endonuclease AscΙ alone to linearize the circular vector;

[0053] 3. Detection and recovery of PCR product and vector digestion product:

[0054] The PCR product and the vector digestion product were detected by agarose gel electrophoresis. The target fragment was recovered using the Omega Bio-tek GelExtraction Kit;

[0055] 4. Recombinant of Gel Recovery Products:

[0056] Perform homologous recombination using Vazyme ClonExpress II One Step Cloning Kit; the reaction system is as follows:

[0057] Linearized vector 200 ng Insert fragment 120 ng 5×CE II buffer 4 μL Exnase II 2 μL <![CDATA[ddH2O]]> to 20 μL

[0058] Gently pipette and mix well, and collect the reaction solution to the bottom of the centrifuge tube after brief centrifugation; incubate in a water bath at 37°C for 30 min, and place on ice until reaching room temperature;

[0059] 5. Transformation of Recombinant Products:

[0060] Transform the recombinant products into Escherichia coli DH5α competent cells, referring to the Molecular Cloning Experiment Guide.

[0061] 6. Sequencing and Identification:

[0062] After colony PCR and plasmid PCR, select positive plasmids for sequencing. The correct sequencing results indicate that the overexpression vector pZZ0153-RD101p-ZmRVE6-3HA of maize ZmRVE6 is successfully obtained; the PCR primers are as follows:

[0063] Primer F:

[0064] 5’-GCACTAGTATCCCGGGAAGGCGCGCCATGGTGTCGAC GAGCTCG-3’,

[0065] Primer R: 5’-CGTCGTATGGGTACATGGCCTTGCTCGTTATCA TGAACG-3’.

[0066] Example 3 Obtaining Transgenic Maize Plant Lines

[0067] Send the overexpression vector pZZ0153-RD101p-ZmRVE6-3HA of maize ZmRVE6 obtained in Example 2 to China National Seed Group Co., Ltd. for transformation, and the transformation background is maize inbred line KN5585. The T0 generation transgenic seeds obtained from the company are cultivated to obtain the T1 generation. Screen T1 positive plants by applying herbicides and harvest the seeds, and then cultivate to obtain the T2 generation. Continue to perform PCR detection on the T2 generation plants to determine that the target gene has not undergone genetic segregation and loss. Finally, two positive homozygous transgenic maize families with stable inheritance of ZmRVE6 and corresponding negative segregating plants are obtained. The PCR detection primers are the same as those in Example 2.6.

[0068] Example 4 Detection of Expression Levels of ZmRVE6 Transgenic Maize Plants

[0069] Separate ZmRVE6 transgenic maize plants and wild types were planted. Maize leaves at the three-week growth stage were taken, and RNA was extracted using the Trizol method. The extracted RNA was digested with DNaseI and then reverse-transcribed using Promega MLV reverse transcriptase. RT-PCR detection was performed using the extracted cDNA, and the maize Actin gene was used as a control.

[0070] The detection results showed that the ZmRVE6 gene was successfully transferred into maize ( Figure 2 ).

[0071] The primers used in the detection process are as follows:

[0072] qZmRVE6-F: 5’-GAAATGGATCCCATTGATGC-3’,

[0073] qZmRVE6-R: 5’-CATCCGAAGAGAGCAACCTC-3’;

[0074] qZmActin-F: 5’-GCTGGATCTTGCTGGCCGTG-3’,

[0075] qZmActin-R: 5’-AGGCGCCACGACCTTGATCT-3’;

[0076] Example 5 Seedling-stage drought phenotype of ZmRVE6 transgenic maize plants

[0077] ZmRVE6 transgenic maize plants and negative segregating materials were planted simultaneously in square boxes with a specification of 30 cm × 40 cm × 15 cm, planted in two equal parts left and right. The plants grew in a greenhouse at a temperature of 28°C. When they grew to the four-leaf stage under normal conditions, watering was stopped for drought treatment. When the plants showed obvious wilting after 10 days of drought treatment, rehydration was carried out. Seven days after rehydration, the plant survival rates of ZmRVE6 transgenic maize plants and negative segregating plants in each square box were counted.

[0078] The results showed that: the survival rate of ZmRVE6 transgenic maize plants was significantly higher than that of negative segregating plants, indicating that ZmRVE6 transgenic maize plants had stronger drought resistance ( Figure 3 ).

[0079] Example 6 Genotype identification of ZmRVE6 in the association population Type 3 and association analysis of expression levels before and after drought

[0080] The ZmRVE6 sequences of the maize materials B73 and SK with published sequencing sequences were analyzed, and identified in 378 associated population materials. It was found that the number of type1 was the least, and the remaining types were evenly distributed in various materials. Using the associated population amplification, it was found that there were large structural variations in the promoter region of maize ZmRVE6. Taking B73 as the reference sequence, there was an insertion sequence of 7318bp in the type3 promoter. According to the structural variation of the 7318bp insertion, all materials were roughly divided into 4 categories. Combining the existing data of expression levels and survival rates before and after drought in the laboratory, the relationship between the structural variation of the upstream sequence of ZmRVE6 and the total expression level and drought survival rate of the gene before and after drought was statistically analyzed. It was found that the type1 structural variation was significantly correlated with the expression level and survival rate of ZmRVE6.

[0081] Primer ZmRVE6 for identifying the type3 promoter Type 3 -F1 / R1 are as follows (amplifying partial sequences):

[0082] ZmRVE6 Type 3 -F1: 5’-CAAGCTCTGGCTCTGCAAC-3’,

[0083] ZmRVE6 Type 3 -R1: 5’-GGCGGCACAAGACTCCAC-3’.

[0084] The PCR amplification system is as follows (total volume 20μL):

[0085] DNA 100 ng 2×Taq mix 10 μL <![CDATA[ZmRVE6 Type3 -F1]]> 0.4 μL <![CDATA[ZmRVE6 Type3 -R1]]> 0.4 μL <![CDATA[ddH2O]]> to 20 μL

[0086] The reaction program is: 95℃ for 3min; 95℃ for 30s; 58℃ for 30s; 72℃ for 45s; 72℃ for 5min; 35 cycles.

[0087] The results showed that: the nucleotide sequence (7318bp) of the maize drought-resistant molecular marker ZmRVE6 Type 3 amplified is shown in SEQ ID NO: 4. This structural variation (7318bp sequence insertion) was significantly correlated with the expression level and survival rate of ZmRVE6. Among them, the expression level of the type3 promoter before and after drought was the highest, and that of type2 was the lowest; the survival rate of the type3 promoter after drought was the highest ([[ID=3�]] Figure 4 ).

[0088] Example 7 Kit for identifying maize varieties with excellent drought tolerance and instructions for use Kit for identifying maize varieties with excellent drought tolerance, which includes the following primer pairs:

[0089] ZmRVE6 Type 3 -F1: 5’-CAAGCTCTGGCTCTGCAAC-3’,

[0090] ZmRVE6 Type 3 -R1: 5’-GGCGGCACAAGACTCCAC-3’.

[0091] The instructions for using this kit are as follows:

[0092] (1) Extract the DNA of the maize variety to be detected;

[0093] (2) Use the above primer pair and perform PCR amplification with the above DNA as the template;

[0094] (3) Electrophoresis: If a target band of 1634 bp appears in the amplification, it indicates that this variety has excellent drought resistance.

[0095] Example 8 Identification of Drought Resistance of Different Maize Varieties

[0096] In this example, 5 maize varieties (CIMBL55, CIMBL70, CIMBL90, B73, and TY2) were collected and identified using the kit of Example 7.

[0097] As Figure 5 shown: The genomes of maize varieties CIMBL55, CIMBL70, and CIMBL90 all contain the maize drought resistance molecular marker ZmRVE6 Type 3 , indicating that the three maize varieties CIMBL55, CIMBL70, and CIMBL90 have excellent drought resistance, and they are all drought-tolerant varieties, while B73 and TY2 are drought-sensitive varieties.

[0098] Other parts not described in detail are all prior art. Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to this example without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A maize drought-resistant gene ZmRVE6, characterized by: The nucleotide sequence of the drought-resistant gene ZmRVE6 is shown in SEQ ID NO:

1.

2. The transcript of the maize drought-resistant gene ZmRVE6 described in claim 1, characterized in that: The CDS nucleotide sequence of the transcript is shown in SEQ ID NO:

3.

3. A drought-resistant protein ZmRVE6 encoded by the transcript ZmRVE6 according to claim 2, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO:

2.

4. Use of the ZmRVE6 according to claim 2 or the drought-resistant protein ZmRVE6 according to claim 3 in improving the drought resistance of corn.

5. Use of the drought-resistant transcript ZmRVE6 according to claim 2 or the drought-resistant protein ZmRVE6 according to claim 3 in breeding new drought-resistant corn varieties.

6. A molecular marker for drought resistance in maize, ZmRVE6 Type3 , characterized in that: The maize drought-resistant molecular marker ZmRVE6 Type3 has a nucleotide sequence as shown in SEQ ID NO:

4.

7. A primer pair for obtaining the maize drought-resistant molecular marker ZmRVE6 described in claim 5 Type3 , characterized in that: The primer pairs are: ZmRVE6 Type3 -F1: 5’-CAAGCTCTGGCTCTGCAAC-3’, ZmRVE6 Type3 - R1: 5'-GGCGGCACAAGACTCCAC-3'.

8. Use of the primer pair according to claim 7 in preparing a kit for identifying corn varieties with excellent drought resistance.

9. A kit for identifying maize varieties with excellent drought resistance, characterized in that: The kit comprises the primer pair according to claim 7.

10. A method for identifying maize varieties with excellent drought resistance using the primer pair according to claim 7 or the kit according to claim 9, characterized in that: The following steps are involved: (1) Extracting DNA from the corn variety to be tested; (2) using the above DNA as a template and performing PCR amplification using primer pairs; (3) Electrophoresis: The amplified target band of 1634 bp appears, indicating that the corn variety to be tested has strong drought resistance, that is, the variety is a corn variety with excellent drought resistance.

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