CN113501867B

CN113501867B - Corn drought-resistant gene ZmMYBR38 and application thereof

Info

Publication number: CN113501867B
Application number: CN202110795898.6A
Authority: CN
Inventors: 代明球; 孙霄鹏; 向艳丽; 豆楠楠
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-07-14
Filing date: 2021-07-14
Publication date: 2022-06-14
Anticipated expiration: 2041-07-14
Also published as: CN113501867A

Abstract

The invention discloses a corn drought-resistant gene ZmMYBR38 and application thereof, wherein the nucleotide sequence is shown as SEQ ID No. 1. The total 758 amino acids of the protein coded by the gene are 758, and the amino acid sequence is shown as SEQ ID No. 2. The invention verifies the function of the corn drought resistance gene ZmMYBR38 in corn drought resistance, and provides valuable resources for cultivating corn drought resistance varieties in a molecular breeding mode.

Description

Corn drought-resistant gene ZmMYBR38 and application thereof

Technical Field

The invention relates to the field of plant genetic engineering, in particular to a corn drought-resistant gene ZmMYBR38 and application thereof.

Background

Corn is one of the main food crops in China, and the planting area of the corn is mostly overlapped with arid and semiarid areas in China. The increasingly frequent extreme climate causes large-area drought, seriously threatens the corn yield in China, restricts the agricultural development in China, and threatens the benefits of farmers and the national food safety. The cultivation and improvement of corn varieties are carried out by utilizing molecular biology and genetics, so that the tolerance capability of the corn under drought is hopefully enhanced, and the purposes of keeping stable yield and high yield of the corn under drought stress are achieved.

To better adapt to the environment, plants form a complex drought response network. The plants can regulate the expression level of in vivo drought response transcription factors by sensing signal molecules such as abscisic acid and the like, and further regulate and control downstream genes to participate in drought response. MYB transcription factor family is a huge gene family in plants, and many reports show that MYB transcription factor family members participate in abiotic stress responses such as drought of the plants, for example, the heat resistance and the drought resistance of the plants can be improved by over-expressing rice MYB gene OsMYB55 in corn, and the drought resistance of the plants can be improved by over-expressing wheat MYB gene TaMYB31 in arabidopsis. Therefore, the family member has potential application value in the genetic improvement and breeding of crop stress resistance.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a corn drought-resistant gene ZmMYBR38 and application thereof.

In order to achieve the purpose, the invention relates to a corn drought-resistant transcription factor ZmMYBR38 protein, which has the amino acid sequence as follows:

(1) a protein consisting of an amino acid sequence shown in SEQ ID No. 2; or

(2) And the amino acid sequence homology with the amino acid sequence defined by the sequence SEQ ID No.2 is 95-100 percent and encodes the same functional protein.

(3) And (2) the protein which is derived from the protein (1) and has the same activity and is added with, deleted from or substituted for one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.

The invention combines a reference sequence in a database with a reverse transcription PCR technology to obtain a CDS sequence with the full length of 2277bp of the corn drought-resistant gene ZmMYBR38, and the nucleotide sequence is shown as SEQ ID No. 1. The protein coded by the gene has 758 amino acids in total, and the amino acid sequence of the protein is shown as SEQ ID No. 2.

It should be clear that one skilled in the art can substitute, delete and/or add one or more amino acids to the disclosed amino acid sequences without affecting the biological activity of the proteins, and the homology of the protein sequences is more than 90%, so as to obtain mutant sequences of the proteins. Therefore, the invention also comprises a derivative protein which is obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID No.2, has high homology and biological activity.

The invention includes nucleotide sequences encoding the proteins described above.

Furthermore, it will be appreciated that, given the degeneracy of codons and the nature of the codon usage of a species, one skilled in the art can use codons suitable for expression in a particular species as desired.

The nucleotide sequence of the ZmMYBR38 gene for coding the corn drought-resistant transcription factor ZmMYBR38 is shown in SEQ ID No. 1. The protein coded by the gene has 758 amino acids in total, and the amino acid sequence of the protein is shown as SEQ ID No. 2.

The gene and the protein of the invention can be obtained by cloning or separating from a maize inbred line B73 or by a sequence chemical synthesis method.

The invention also provides a primer pair for obtaining the ZmMYBR38 gene, wherein the primer pair comprises the following components:

and (3) primer F: 5'-ATGGCAGCTGCGGCGCCGG-3' the flow of the air in the air conditioner,

and (3) primer R: 5'-CTATTGCCTGGCTGCTGCTGCTGGCAG-3' are provided.

The invention also provides a corn ZmMYBR38 overexpression vector, wherein the corn ZmMYBR38 overexpression vector is an overexpression vector containing the ZmMYBR38 gene, namely named ZZ0153-UBip-ZmMYBR38-3HA, and the overexpression vector is as follows: ZZ0153-UBIP-3 HA.

The invention also provides an arabidopsis ZmMYBR38 overexpression vector, wherein the arabidopsis ZmMYBR38 overexpression vector is an overexpression vector containing the ZmMYBR38 gene, namely named as pCAMBIA1305-ZmMYBR38-3HA, and the overexpression vector is: pCAMBIA1305-3 HA.

The invention also provides a host cell containing the maize ZmMYBR38 overexpression vector or the Arabidopsis thaliana ZmMYBR38 overexpression vector, and the host cell is Escherichia coli DH5 alpha.

The application of one of the following items in improving the drought resistance of crops,

(1) the aforementioned ZmMYBR38 gene;

(2) the maize ZmMYBR38 overexpression vector described above;

(3) the Arabidopsis ZmMYBR38 overexpression vector;

(4) the host cell described above.

The invention has the beneficial effects that:

the invention verifies the function of the corn drought resistance gene ZmMYBR38 in corn drought resistance, and provides valuable resources for cultivating corn drought resistance varieties in a molecular breeding mode.

According to the invention, the drought-resistant gene ZmMYBR38 of the corn is introduced into arabidopsis and corn, so that the drought-resistant phenotype of a transgenic plant is remarkably improved, and the ZmMYBR38 gene can be used for improving the drought resistance of crops.

Drawings

FIG. 1 is a schematic diagram of construction of maize over-expression vector ZZ0153-UBip-ZmMYBR38-3HA (general vector map, without Chinese annotation);

FIG. 2 is a schematic diagram of the construction of Arabidopsis thaliana overexpression vector pCAMBIA1305-ZmMYBR38-3HA (general vector map, no Chinese annotation);

FIG. 3 shows the expression level identification of ZmMYBR38 in transgenic maize and Arabidopsis plants under drought.

FIG. 4 is an analysis of the drought resistant phenotype of Arabidopsis transgenic plants;

FIG. 5 is the drought resistant phenotype analysis of maize transgenic plants.

Detailed Description

The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.

Example 1ZmMYBR38 gene acquisition

Through prediction analysis, ZmMYBR38 is found to be possibly involved in drought resistance response of corn, and then a primer pair is designed through a reference genome sequence of the corn:

and (3) primer F: 5'-ATGGCAGCTGCGGCGCCGG-3', and the adhesive tape is used for adhering the film to a substrate,

and (3) primer R: 5'-CTATTGCCTGGCTGCTGCTGCTGGCAG-3', respectively;

performing PCR amplification by using a corn genome as a template; obtaining a PCR product, sequencing to obtain a ZmMYBR38 gene (the full-length CDS sequence of ZmMYBR 38), wherein the nucleotide sequence of the gene is shown as SEQ ID No. 1.

Example 2 maize ZmMYBR38 overexpression vector construction

Based on an expression vector ZZ0153-UBip-3HA, inserting a cDNA fragment of ZmMYBR38 into the downstream of a UBI promoter and between a 3HA tag through homologous recombination, and expressing the cDNA fragment by the UBI promoter (figure 1); electrophoresis detection and sequencing analysis show that the over-expression vector of the corn ZmMYBR38 is successfully obtained, namely the name ZZ0153-UBip-ZmMYBR38-3HA is obtained, and the specific experimental steps are as follows:

ZmMYBR38 gene amplification:

a pair of homologous recombination primers is designed according to the cDNA sequence of ZmMYBR38 for PCR amplification. The primer sequences are as follows:

ZmMYBR38-F0：5’-CGACAAACGCACTAGTATCCCGGGATGGCAGCTGCGGCGCCGG-3’，

ZmMYBR38-R0：5’-CATTTGGCGCGCCTTCCCCTATTGCCTGGCTGCTGCTGCTGGCAG-3’；

the PCR product was obtained by gene amplification using Vazyme Phanta Max Super-Fidelity DNA polymerase, and the reaction system was as follows (total volume 20 ul):

2x phanta Buffer	10ul
		dNTP	0.4ul
ZmMYBR38-F0	0.8ul
		ZmMYBR38-R0	0.8ul
Phanta max	0.4ul
		cDNA (corn)	1.5ul
ddH₂O	6.9ul

The reaction procedure is as follows: 3min at 95 ℃; 15s at 95 ℃; 30s at 60 ℃; 60s at 72 ℃; 5min at 72 ℃; 35, circulating;

2. preparation of a linearized vector: using restriction enzyme XmaI to singly cut ZZ0153-UBip-3HA vector to linearize the annular vector;

and 3, detecting and recovering the PCR product and the vector enzyme digestion product: and detecting the PCR product and the carrier enzyme digestion product by agarose gel electrophoresis. Recovering the target fragment by using an Omega Bio-tek Gel Extraction Kit;

4. glue recovery product recombination: homologous recombination was performed using the Vazyme Clonexpress II One Step Cloning Kit; the reaction system is as follows:

linearized vector	200ng
		Insert fragment	120ng
5x CE II buffer	4ul
		Exnase II	2ul
ddH₂O	to 20ul

Gently sucking and beating the mixture by using a pipettor, and uniformly mixing the mixture, and collecting the reaction solution to the bottom of a centrifugal tube after short-time centrifugation; water bath at 37 deg.C for 30min, standing on ice to room temperature;

5. and (3) transformation of a recombinant product: the recombinant product was transformed into DH5 α competent cells, according to the molecular cloning protocols.

6. Sequencing and identifying: sequencing analysis by colony PCR and plasmid PCR showed that: successfully obtains a maize ZmMYBR38 overexpression vector ZZ0153-UBip-ZmMYBR38-3 HA; the PCR and sequencing primers were as follows:

0153-F：5’-GCAGGTCGACAAACGCACTA-3’

3HA-R：5’-TACTGAGCAGCGTAGTCTGG-3’。

example 3 Arabidopsis MYBR38 overexpression vector construction

Based on the pCAMBIA1305-3HA vector, the cDNA fragment of ZmMYBR38 was inserted downstream of the 35S promoter and between the 3HA tags by homologous recombination and expressed from the 35S promoter (FIG. 2). Electrophoresis detection and sequencing analysis show that: an Arabidopsis MYBR38 overexpression vector is successfully obtained, namely the vector is named as pCAMBIA1305-ZmMYBR38-3HA, and the specific experimental steps are as follows:

ZmMYBR38 gene amplification

A pair of homologous recombination primers is designed according to the cDNA sequence of ZmMYBR38 for PCR amplification, and the primer sequences are as follows:

ZmMYBR38-F1：5’-CTGCAGGCATGCAAGCTTATGGCAGCTGCGGCGCCGG-3’，ZmMYBR38-R1：5’-AACATCGTATGGGTAAAGCTATTGCCTGGCTGCTGCTGCTGGCAG-3’；

gene amplification was performed using Vazyme Phanta Max Super-Fidelity DNA polymerase to obtain a gene PCR product, which was performed in the following reaction system (total volume 20 ul):

2. preparation of a linearized vector: the pCAMBIA-1305-3HA vector was digested singly with the restriction enzyme HindIII to linearize the circular vector.

3. And (3) detecting and recovering a gene PCR product and a carrier enzyme digestion product: detecting the PCR product and the carrier enzyme digestion product by agarose Gel electrophoresis, and recovering the target fragment by using an Omega Bio-tek Gel Extraction Kit.

4. Glue recovery product recombination: homologous recombination was performed using the Vazyme Clonexpress II One Step cloning kit, as follows:

Gently sucking and beating the mixture by using a pipettor, and uniformly mixing the mixture, and collecting the reaction solution to the bottom of a centrifugal tube after short-time centrifugation; water bath at 37 deg.c for 30min, and setting on ice to room temperature.

5. And (3) transformation of a recombinant product: transforming the recombinant product into agrobacterium-infected cells according to the molecular cloning experimental guidelines;

6. sequencing and identifying: sequencing analysis by colony PCR and plasmid PCR showed that: the Arabidopsis ZmMYBR38 overexpression vector pCAMBIA1305-ZmMYBR38-3HA is successfully obtained; the PCR and sequencing primers were:

1305-F：5’-TCATTTGGAGAGAACACGGG-3’

3HA-R：5’-AGGATGAGACCAAGCGTAAT-3’。

example 4 transgenic Arabidopsis and maize lines

The maize ZmMYBR38 overexpression vector ZZ0153-UBip-ZmMYBR38-3HA obtained in example 2 is sent to Jiangsu Miami Biotech limited to be transformed, and the transformation background is maize inbred line KN 5585. Transgenic seeds of T0 generation obtained from the company were cultivated to obtain T1 generation, T1 positive plants were screened by applying herbicide and seeds were harvested, and cultivated to obtain T2 generation. And continuously carrying out PCR detection on the T2 generation plants, determining that the target gene is not subjected to genetic segregation loss, and finally obtaining two positive homozygous transgenic corn families containing ZmMYBR38 stable inheritance and corresponding negative segregation materials. The PCR detection primers were as in example 2.6.

The Arabidopsis MYBR38 overexpression vector pCAMBIA1305-ZmMYBR38-3HA obtained in example 3 is transferred into Col-0 Arabidopsis thaliana in full-bloom stage by utilizing an Agrobacterium dip-dye transformation method (the flower buds of the Arabidopsis thaliana are completely immersed in the Agrobacterium for 1min and then taken out), the Arabidopsis thaliana treated by the Agrobacterium is covered by a black plastic bag, is grown for 24h in a dark place, and is placed in a light culture room for 3-4 weeks to harvest. Seeds of T0 generation are dibbled on MS culture medium containing hygromycin resistance, the MS culture medium is placed in a 22 ℃ illumination incubator to grow for a week, plants which can normally grow on the resistant MS dish are transplanted into pots filled with nutrient soil to be planted and harvested, and meanwhile, the primers in example 3.6 are used for determining the expression of ZmMYBR 38.

Example 5 expression level detection of ZmMYBR38 transgenic Arabidopsis and maize plants

1. Respectively planting ZmMYBR38 Arabidopsis overexpression material and Col, taking Arabidopsis leaves growing for two weeks, extracting RNA by using a Trizol method, digesting the extracted RNA by DNaseI, performing reverse transcription by using Promega MLV reverse transcriptase, and performing RT-PCR detection by using the extracted cDNA. The arabidopsis Actin7 gene was used as a control.

The detection result shows that: the ZmMYBR38 gene was successfully transferred into Arabidopsis.

2. Respectively planting ZmMYBR38 over-expression corn material and wild type, taking corn leaves growing for three weeks, extracting RNA by using a Trizol method, digesting the extracted RNA by DNaseI, carrying out reverse transcription by using Promega MLV reverse transcriptase, carrying out RT-PCR detection by using the extracted cDNA, and using a corn Actin gene as a control.

The detection result shows that the ZmMYBR38 gene is successfully transferred into the corn (figure 3).

The primers used in the detection process were as follows:

qMYBR38-F：5’-ACACGCCTCTGTTGGCTCAG-3’，

qMYBR38-R：5’-CAGTGCCGGTGCCAGTGAAG-3’；

qZmActin-F：5’-GCTGGATCTTGCTGGCCGTG-3’，

qZmActin-R：5’-AGGCGCCACGACCTTGATCT-3’；

AtActin-F：5’-GGCCGATGGTGAGGATATTCAGCCACTTG-3’，

AtActin-R：5’-TCGATGGACCTGACTCATCGTACTCACTC-3’。

example 6 drought resistance phenotype testing of ZmMYBR38 transgenic Arabidopsis

Seeds of Arabidopsis thaliana overexpressing plants of ZmMYBR38 and wild type Col-0 plants were both treated at 4 ℃ for three days in the dark and subsequently grown on MS culture dishes in 22 ℃ incubators. After one week the over-expressed and wild type plants were grown in half in square pots and placed in a 22 ℃ culture room with normal watering for two weeks. Watering is then stopped, plants are rehydrated after severe wilting and survival rates for ZmMYBR38 over-expressed material and wild-type material are performed three days after rehydration.

The results show that: the survival rate of the over-expression material of the ZmMYBR38 is obviously higher than that of the wild-type material, which indicates that the ZmMYBR38 transgenic Arabidopsis plant has stronger drought stress resistance (figure 4).

Example 7 drought resistance phenotype testing of ZmMYBR38 transgenic maize plants

The ZmMYBR38 positive transgenic material and the negative separation material are planted in a square box with the specification of 30cm multiplied by 40cm multiplied by 15cm at the same time, and the two materials are planted in half left and right. Plants were grown in a greenhouse at 28 ℃ and were watered off for drought treatment when growing normally to the four-leaf stage. And (4) carrying out rehydration when the plants show obvious wilting states after 10 days of drought treatment, and carrying out statistics on the plant survival rates of the positive transgenic materials and the negative separation materials in each square box 7 days after rehydration.

The results show that: the survival rate of the over-expression material of the ZmMYBR38 is obviously higher than that of the negative separation material, which indicates that the ZmMYBR38 transgenic corn has stronger drought stress resistance (figure 5).

Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> corn drought-resistant gene ZmMYBR38 and application thereof

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ccactgaaac tgactcctgt ggacgggaaa gctaaccctg taactgcggc tgggagaggt 1860

aagcctgtag ctgctggagt ggcatcaacc ggaaagaagg ctacaatccc tatctcacat 1920

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<213> Artificial Sequence (Artificial Sequence)

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Claims

1. The application of one of the following items in improving the drought resistance of corn is characterized in that:

(1) the ZmMYBR38 gene; the nucleotide sequence of the ZmMYBR38 gene is shown in SEQ ID No. 1;

(2) a maize ZmMYBR38 overexpression vector; the over-expression vector of the corn ZmMYBR38 is an over-expression vector containing the ZmMYBR38 gene, namely named ZZ0153-UBip-ZmMYBR38-3HA, wherein the over-expression vector is as follows: ZZ0153-UBip-3 HA;

(3) a host cell; the host cell containing the maize ZmMYBR38 overexpression vector is Escherichia coli DH5 alpha.

2. The application of one of the following items in improving the drought resistance of arabidopsis thaliana is characterized in that:

(2) an Arabidopsis ZmMYBR38 overexpression vector; the Arabidopsis thaliana ZmMYBR38 overexpression vector is an overexpression vector containing the ZmMYBR38 gene, namely named as pCAMBIA1305-ZmMYBR38-3HA, wherein the overexpression vector is as follows: pCAMBIA1305-3 HA;

(3) a host cell; the host cell containing the arabidopsis ZmMYBR38 overexpression vector is escherichia coli DH5 alpha.