Corn drought-resistant gene ZmYPT3 and application thereof

Abstract

The invention discloses a corn drought-resistant gene ZmYPT3 and application thereof.Whole genome correlation analysis is conducted on 312 parts of natural population genotype and root phenotype data, a target gene ZmYPT3 is obtained, and the gene encodes a Rab protein and is converted between an inactive state and an active state by being combined with GDP or GTP, and the molecular switch effect is achieved. After the gene is over-expressed, the drought resistance of the corn can be improved.

Description

Technical Field

[0001] The invention relates to the field of plant genetic engineering technology, and in particular to a corn drought resistance gene ZmYPT3 and an application thereof. Background Art

[0002] Globally, corn (Zea mays L.) is one of the important food crops. Corn has a high demand for water during its growth period. However, the main corn planting areas are mostly located in arid and semi-arid areas. The natural precipitation in these areas often cannot meet the needs of corn growth and development, resulting in water becoming a key factor limiting corn yield. Therefore, in-depth research on the drought resistance mechanism of corn, the discovery of new drought-resistant genes and excellent natural variations, and the cultivation and promotion of new drought-resistant varieties are important needs to ensure food security. In recent years, the discovery of new drought-resistant genes through GWAS analysis, the study of their functions, the analysis of their drought-resistant genetic and molecular mechanisms, the development of drought-resistant molecular markers, the creation of germplasm combined with gene editing technology, and the application of them to drought-resistant breeding and improvement have become hot spots and focuses in plant drought resistance research. Identifying and obtaining drought-resistant genes that play a decisive role in crops is of great significance for the genetic improvement of crop drought resistance and molecular breeding practice.

[0003] Plant Rab proteins are a class of small GTP-binding proteins, belonging to the largest family in the small G protein superfamily. These proteins regulate their activity by binding to GDP (inactive state) or GTP (active state), and play a key role as molecular switches in the formation, transport, anchoring and fusion of intracellular vesicles with the plasma membrane. When plants are subjected to abiotic stresses such as drought and high temperature, Rab proteins can maintain intracellular water balance, ion homeostasis and redox balance by finely regulating vesicle transport and organelle dynamics, especially by regulating the vesicle transport of key molecules such as aquaporins, ion transporters and antioxidant enzymes, thereby helping plants to effectively cope with stress and enhance their stress tolerance. Current studies have found that Rab proteins play an important role in plant response to abiotic stress. Overexpression of some Rab protein encoding genes in plants can enhance plant tolerance to abiotic stresses such as salt and drought, and reduce yield losses under stress.

[0004] Therefore, Rab protein encoding genes have potential application value in crop stress resistance genetic improvement and breeding. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a corn drought resistance gene ZmYPT3 and its application. The present invention conducts a genome-wide association analysis (GWAS) on 312 natural population genotypes combined with corn root optical phenotype data to obtain the target gene ZmYPT3, which encodes a Rab protein that is involved in corn drought stress response. Overexpression of the gene can effectively improve the drought resistance of corn.

[0006] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0007] The present invention provides a Rab protein, the amino acid sequence of which is:

[0008] (1) a protein consisting of the amino acid sequence shown in SEQ ID No. 2;

[0009] Alternatively, (2) an amino acid sequence having 95-100% homology with the amino acid sequence defined in SEQ ID No. 2 and encoding an amino acid sequence having the same functional protein.

[0010] Alternatively, (3) a protein derived from (1) having the same activity as that of the amino acid sequence shown in SEQ ID No. 2, with one or more amino acids added, deleted or substituted.

[0011] The nucleotide sequence of the maize drought resistance gene ZmYPT3 encoding the maize Rab protein is shown in SEQ ID No.1.

[0012] The present invention also provides a primer pair for obtaining the above-mentioned maize drought resistance gene ZmYPT3, the primer pair being:

[0013] ZmYPT3-F: 5'-ATGTCGTACGCCTACCTCTTCAAG-3',

[0014] ZmYPT3-R: 5'-ACTGCAGCAGCCACCAGC-3'.

[0015] The present invention also provides a method for obtaining the above-mentioned maize drought resistance gene ZmYPT3.

[0016] The maize genomic DNA with gene ZmYPT3 was used as template and the following primer pairs were used:

[0017] ZmYPT3-F: 5'-ATGTCGTACGCCTACCTCTTCAAG-3',

[0018] ZmYPT3-R: 5'-ACTGCAGCAGCCACCAGC-3';

[0019] PCR amplification was performed to obtain the maize drought resistance gene ZmYPT3.

[0020] The present invention also provides the above-mentioned overexpression vector WMV014-ZmYPT3-Flag, which contains the expression vector of the maize drought resistance gene ZmYPT3 described in claim 2.

[0021] Furthermore, the expression vector is a WMV014-Flag vector.

[0022] Use of any one of the following for improving plant drought resistance, wherein:

[0023] (1) the above-mentioned maize drought resistance gene ZmYPT3;

[0024] (2) The above-mentioned overexpression vector WMV014-ZmYPT3-Flag.

[0025] Furthermore, the plant is corn.

[0026] Use of any of the following in breeding new corn varieties, wherein:

[0027] (1) the above-mentioned maize drought resistance gene ZmYPT3;

[0028] (2) The above-mentioned overexpression vector WMV014-ZmYPT3-Flag. Beneficial effects of the present invention:

[0029] Beneficial effects of the present invention:

[0030] The present invention utilizes genome-wide association analysis (GWAS) combined with a method of high-throughput optical phenotyping of maize roots to identify a drought-related gene ZmYPT3, which is involved in drought stress response. Overexpression of the gene can effectively improve the drought resistance of maize. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Manhattan plot and Quantile-Quantile plot of the new drought resistance gene ZmYPT3 located by whole genome association analysis in maize;

[0032] In the figure, A is the Manhattan graph; B is the Quantile-Quantile graph.

[0033] Figure 2 Schematic diagram of the construction of WMV014-ZmYPT3-Flag vector (universal vector map);

[0034] In the figure, A is the WMV014-Flag empty image; B is the WMV014-ZmYPT3-Flag carrier image.

[0035] Figure 3 This is the expression level detection and drought resistance phenotype analysis of ZmYPT3 transgenic plants;

[0036] In the figure, A is the expression level detection diagram of ZmYPT3 overexpression and wild-type plants;

[0037] B is a comparison of water loss rates of detached leaves of ZmYPT3 overexpressing and wild-type plants;

[0038] C is a comparison of the relative water content of leaves of ZmYPT3 overexpressing and wild-type plants under drought;

[0039] D is a comparison of the survival rates of ZmYPT3 overexpressing and wild-type plants under drought;

[0040] The t test was used to determine whether the differences were statistically significant: *P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0042] Example 1 Location of the maize drought resistance gene ZmYPT3

[0043] A genome-wide association analysis was performed using 312 natural maize population genotype data combined with root optical phenotype data under drought stress and normal watering conditions. In-depth analysis found that the root RGB phenotype D39_length_DS (indicates the total root length of the plant under drought conditions 39 days after sowing) was significantly associated with the gene, and the target gene ZmYPT3 was located, which is a new drought resistance gene ( Figure 1 ), which is named ZmYPT3, and its nucleotide sequence is shown in SEQ ID No.1:

[0044] ATGTCGTACGCCTACCTCTTCAAGTACATCATCATCGGCGACACAGGCGTGGGGAAGTCATGCCTGCTGCTGCAGTTCACGGATAAGCGCTTCCAGCCCGTGCACGATCTCACCATCGGCGTTGAATTTGGCGCCCGCATGATCACCATCGACAACAAGCCCATCAAACTCCAGATTTGGGACACGGCTGGCCAAGAATCATTCAGATCTATTACTAGGTCATACTACAGAGGAGCTGCTGGAGCCCTTTTGGTTTATGATATCACTAGGAGGGAGACCTTCAATCATCTCGCAAGTTGGCTAGAAGATGCCAGGCAACATGCAAATGCTAACATGACAGTGATGCTGATTGGAAACAAATGTGACCTTTCTCATAGGCGTGCTGTCAGCTATGAGGAAGGCGAACAGTTTGCCAAGGAGCATGGTCTGGTTTTTATGGAGGCATCTGCTAAAACTGCACAAAACGTCGAGGAGGCATTCATTAAGACCGCTGGTACAATATACAAGAAAATCCAAGATGGCATATTTGATGTATCAAATGAGTCCAACGGGATCAAAGTTGGCTATGCTGTTCCCAACTCATCTGGAGGTGGTGCTGGCTCGTCCTCACAGGCTGGTGGCTGCTGCAGTTGA;

[0045] Its amino acid sequence is shown in SEQ ID No. 2:

[0046] MSYAYLFKYIIIGDTGVGKSCLLLQFTDKRFQPVHDLTIGVEFGARMITIDNKPIKLQIWDTAGQESFRSITRSYYRGAAGALLVYDITRRETFNHLASWLEDARQHANANMTVMLIGNKCDLSHRRAVSYEEGEQFAKEHGLVFMEASAKTAQNVEEAFIKTAGTIYKKIQDGIFDVSNESNGIKVGYAVPNSSGGGAGSSSQAGGCCS。

[0047] Example 2 Construction of Maize Overexpression Vector

[0048] Using the WMV014-Flag vector ( Figure 2 A), the cDNA fragment of ZmYPT3 was inserted into the vector by homologous recombination to construct the WMV014-ZmYPT3-Flag overexpression vector ( Figure 2 B), laying the foundation for subsequent transgenic research. The specific experimental steps are as follows:

[0049] 1. ZmYPT3 gene amplification

[0050] A pair of homologous recombination primers were designed based on the cDNA sequence of ZmYPT3 for PCR amplification. The primer sequences are as follows:

[0051] ZmYPT3-F: 5′-ATGTCGTACGCCTACCTCTTCAAG-3′, as shown in SEQ ID No. 3;

[0052] ZmYPT3-R: 5′-ACTGCAGCAGCCACCAGC-3′, as shown in SEQ ID No. 4;

[0053] 2. Use Vazyme Phanta Max Super-Fidelity DNA polymerase for gene amplification, and the reaction system is as follows:

[0054] 2x phanta buffer 10ul

[0055] dNTP 0.4ul

[0056] ZmYPT3-F 0.8ul

[0057] ZmYPT3-R 0.8ul

[0058] Phanta max 0.4ul

[0059] cDNA 100ng

[0060] ddH2O 7.1ul.

[0061] 3. The reaction procedure was: 95°C for 3 min; 95°C for 15 s; 66°C for 30 s; 72°C for 30 s; 72°C for 5 min; 35 cycles.

[0062] 4. Preparation of linearized vector: Use restriction endonucleases BamHI and SmaI to double-digest the WMV014-Flag vector to linearize the circular vector.

[0063] 5. Detection and recovery of gene PCR products and vector enzyme digestion products: Detect PCR products and vector enzyme digestion products by agarose gel electrophoresis. Use Omega Bio-tek Gel Extraction Kit to recover the target fragment.

[0064] 6. Recombination of gel recovery products: Use Vazyme ClonExpress II One Step Cloning Kit for homologous recombination. The reaction system is as follows:

[0065] Linearized vector: 200ng

[0066] Insert: 120ng

[0067] 5x CE II buffer: 4ul

[0068] Exnase II: 2ul

[0069] ddH2O: to 20ul

[0070] Use a pipette to gently mix, collect the reaction solution at the bottom of the centrifuge tube after a brief centrifugation; incubate in a 37°C water bath for 30 min, and place on ice to let it cool to room temperature.

[0071] 7. Refer to the molecular cloning experiment guide to transform the recombinant product into DH5α competent cells.

[0072] 8. Extract the recombinant plasmid according to the molecular cloning experimental guide.

[0073] 9. The WMV014-ZmYPT3-Flag overexpression vector was obtained through colony PCR, plasmid PCR and sequencing analysis.

[0074] Example 3 Obtaining ZmYPT3 transgenic corn lines

[0075] The vector obtained in Example 2 was sent to Jiangsu Weimi Biotechnology Co., Ltd. for transformation, and the transformation background was maize inbred line KN5585. The T0 transgenic seeds obtained from the company were planted to obtain the T1 generation, and DNA was extracted for PCR detection and herbicide application to screen T1 positive plants. Subsequently, RNA was sampled from the screened T1 plants and extracted. The RNA was digested with DNaseI and then reverse transcribed using Promega MLV reverse transcriptase. The maize actin gene was used as a control, and the expression level was detected using Vazyme SYBR Green Mix. Finally, two overexpression families ( Figure 3A). The T1 overexpression family was self-pollinated to obtain the T2 generation, and the T2 generation plants were tested again by PCR to obtain a homozygous transgenic maize family containing ZmYPT3 stably inherited. The qPCR and PCR detection primers are as follows:

[0076] qZmYPT3-F:5'-AGCTATGAGGAAGGCGAACAG-3'

[0077] qZmYPT3-R: 5'-GGTCTTAATGAATGCCTCCTCGAC-3'

[0078] qActin-F: 5'-GCCGAGCGAGAAATTGTAAG-3'

[0079] qActin-R: 5'-TGGTGATTACTTGGCCATCA-3'

[0080] ZmYPT3-OE-F:5'-CGTACGCCTACCTCTTCAAGT-3'

[0081] 3×Flag-R: 5'-CGTGATCTTTGTAATCTCCATCGTG-3'

[0082] Example 4 Investigation of drought phenotypes of ZmYPT3 overexpressing transgenic materials

[0083] 1. Investigation of water loss rate of detached leaves of ZmYPT3 overexpression and wild-type materials ( Figure 3 B)

[0084] Overexpression and wild-type materials were sown and raised in plug trays, and transplanted into pots (length × width × height = 34 cm × 16 cm × 12 cm) at the 1-leaf stage. The same volume of nutrient soil was placed in each pot, and the overexpression and wild-type plants were planted in half, with 6 replicates. At the 4-leaf stage of corn, overexpression and wild-type plants with good growth were selected, and 5 plants of each material were selected. 5 leaves from the same part were cut and weighed with a 1 / 10,000 balance, and the initial weight was recorded as W0. Then all leaves were placed indoors and allowed to dry naturally. The leaves were weighed once every 1 hour, and the weights were recorded as W1, W2...Wn in turn. The leaves were stopped weighing after 12 hours of in vitro drought, and the water loss rate of the leaves at each time point was calculated as (W0-Wn) / W0.

[0085] Through analysis, it was found that ZmYPT3 overexpressing plants had a lower leaf water loss rate than wild-type plants. In comparison, the overexpressing plants were more drought-resistant, indicating that ZmYPT3 overexpressing plants have a stronger ability to resist drought stress.

[0086] 2. Investigation of leaf water content in ZmYPT3 overexpression and wild-type materials under drought ( Figure 3 C)

[0087] Overexpression and wild-type materials were sown and raised in plug trays, and transplanted into pots (length × width × height = 34 cm × 16 cm × 12 cm) at the 1-leaf stage. The same volume of nutrient soil was placed in each pot, and the overexpression and wild-type materials were planted in half, with 6 replicates. Watering was stopped at the 4-leaf stage of corn, and drought treatment was started. When the leaves were severely curled, the top fully expanded leaves of the corresponding materials were cut, and they were immediately weighed with a 1 / 10,000 balance and recorded as fresh weight (Fresh weight, FW). After weighing, the leaves were placed in a beaker filled with distilled water and placed in a dark environment for 24 hours, and then the leaves were weighed again to obtain the saturated weight (Saturated weight, SW). The weighed leaves were then placed in a 65°C oven for drying for 5 days, and the dry weight (DW) of the leaves was obtained by weighing. Finally, the relative water content of the leaves RWC (%) was calculated as (FW-DW) / (SW-DW)×100.

[0088] Through analysis, it was found that under drought stress, ZmYPT3 overexpressing plants had higher leaf water content than wild-type plants, indicating that ZmYPT3 overexpression significantly enhanced the tolerance of corn to drought.

[0089] 3. Investigation of the survival rate of ZmYPT3 overexpression and wild-type materials under drought ( Figure 3 D)

[0090] The overexpression and wild-type materials were sown and raised in plug trays, and transplanted to pots (length × width × height = 34 cm × 16 cm × 12 cm) at the 1-leaf stage. The same volume of nutrient soil was placed in each pot, and the overexpression and wild-type materials were planted in half, with a total of 12 plants planted in each pot, of which 6 plants of each material were planted, and 6 replicates were set. Watering was stopped at the 4-leaf stage of corn, and drought treatment was started. When the corn plants showed changes such as lodging, soft stems, and wilting leaves, rehydration was started. After 3 days of rehydration, the survival rate of each replicate was counted. Through analysis, it was found that ZmYPT3 overexpression plants had a higher survival rate than wild-type plants under drought stress, indicating that ZmYPT3 overexpression significantly improved the drought resistance of corn.

[0091] The above results indicate that ZmYPT3 is involved in plant drought stress response, and its overexpressed plants have higher drought resistance. Under drought stress, compared with wild-type plants, ZmYPT3 overexpressed plants have higher survival rate, lower leaf water loss rate and higher leaf water content. The above results indicate that overexpression of this gene improves maize drought resistance. It also indicates that this gene has the potential to achieve stable yield under drought stress.

[0092] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A corn Rab protein, whose amino acid sequence is: (1) a protein consisting of the amino acid sequence shown in SEQ ID No. 2; Alternatively, (2) an amino acid sequence encoding a protein with the same functional identity as the amino acid sequence defined by SEQ ID No. 2 with a homology of 95-100%; Alternatively, (3) a protein derived from (1) having the same activity as that of the amino acid sequence shown in SEQ ID No. 2, with one or more amino acids added, deleted or substituted.

2. The gene encoding the maize Rab protein according to claim 1, ZmYPT3, whose nucleotide sequence is shown in SEQ ID No.

1.

3. A primer pair for obtaining the maize drought resistance gene ZmYPT3 according to claim 2, characterized in that: The primer pairs are: ZmYPT3-F: 5'-ATGTCGTACGCCTACCTCTTCAAG-3', ZmYPT3-R: 5'-ACTGCAGCAGCCACCAGC-3'.

4. A method for obtaining the maize drought resistance gene ZmYPT3 according to claim 2, characterized in that: The maize genomic DNA with gene ZmYPT3 was used as template and the following primer pairs were used: ZmYPT3-F: 5'-ATGTCGTACGCCTACCTCTTCAAG-3', ZmYPT3-R: 5'-ACTGCAGCAGCCACCAGC-3'; PCR amplification was performed to obtain the maize drought resistance gene ZmYPT3.

5. An overexpression vector WMV014-ZmYPT3-Flag, characterized in that: It contains the expression vector of the maize drought resistance gene ZmYPT3 described in claim 2.

6. The overexpression vector WMV014-ZmYPT3-Flag according to claim 1, characterized in that: The expression vector is WMV014-Flag vector.

7. Use of any one of the following for improving drought resistance of plants, characterized in that: (1) The maize drought resistance gene ZmYPT3 according to claim 2; (2) The overexpression vector WMV014-ZmYPT3-Flag according to claim 5.

8. The use according to claim 7, characterized in that: The plant is corn.

9. Use of any one of the following in breeding new corn varieties, characterized in that: (1) The maize drought resistance gene ZmYPT3 according to claim 2; (2) The overexpression vector WMV014-ZmYPT3-Flag according to claim 5.

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