CN113372422B

CN113372422B - ZmGLK44 gene for regulating and controlling moisture utilization efficiency of corn under drought and application thereof

Info

Publication number: CN113372422B
Application number: CN202110646958.8A
Authority: CN
Inventors: 代明球; 张斐; 吴锦峰
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-06-10
Filing date: 2021-06-10
Publication date: 2022-06-14
Anticipated expiration: 2041-06-10
Also published as: CN113372422A

Abstract

The invention discloses a ZmGLK44 gene for regulating and controlling water utilization efficiency under corn drought and application thereof, wherein a ZmGLK44 gene nucleotide sequence is shown as SEQ ID No.1, the invention introduces a corn drought-resistant gene ZmGLK44 into a corn strain C01, the drought-resistant phenotype and the drought-resistant physiological index of a transgenic plant are both remarkably improved, and the ZmGLK44 gene is indicated to be used for improving the drought resistance of crops.

Description

ZmGLK44 gene for regulating and controlling moisture utilization efficiency of corn under drought and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a ZmGLK44 gene for regulating and controlling moisture utilization efficiency under corn drought and application thereof.

Background

Corn (Zea may L) is a cross-pollinated annual grass plant, an important food crop, and also an important feed and industrial raw material. In China, the planting area of corn is larger than that of rice, and the corn is the first large grain crop in China. The production of corn is severely threatened by drought,

with the change of natural environment, the threat of drought to the production of corn will become more serious. The drought-resistant inheritance and molecular mechanism of corn are deeply researched, important drought-resistant genes are identified and cloned, and the breeding speed of varieties with excellent drought resistance can be accelerated by combining plant genetic engineering and traditional breeding. Has great significance for guaranteeing national food safety.

The Golden 2-like (glk) family of transcription factors belongs to the GARP superfamily, a plant-specific family of transcription factors, which was first identified in maize. GLK transcription factors can regulate chloroplast development in plants. In Arabidopsis, the double mutant glk1glk2 appears pale green and cannot synthesize photosynthetic organs. And the over-expression of GLK1 and GLK2 can enhance the expression of chlorophyll biosynthesis genes and enhance light absorption. GLK transduction factors can participate in stress response processes. The existing research shows that GLK can participate in the regulation and control of important stress response hormones abscisic acid (ABA), Salicylic Acid (SA) and Jasmonic Acid (JA) by plants. GLK transcription factors have potential yield increasing effect, and the GLK genes ZmGLK1 and ZmG2 from corn are overexpressed in rice, so that the yield of the rice can be increased by 30-40%.

In view of the dual functions of the GLK transcription factor on yield and stress resistance, the identification and cloning of the GLK transcription factor in the corn has important significance for theoretical research and actual stress resistance of crops.

Disclosure of Invention

The first purpose of the invention is to overcome the defects of the prior art and provide a ZmGLK44 gene for regulating and controlling the water utilization efficiency under corn drought, wherein the gene belongs to a typical GLK transcription factor family; the identification and cloning of the GLK transcription factor (ZmGLK44 gene) in the corn have important significance for theoretical research and actual stress resistance of crops.

The second purpose of the invention is to provide the application of the ZmGLK44 gene in the regulation of the moisture utilization efficiency of corn under drought in improving the drought resistance of crops.

In order to achieve the purpose, the invention obtains ZmGLK44 protein (338 amino acids) for regulating and controlling moisture utilization efficiency under corn drought, and the amino acid sequence of the ZmGLK44 protein is characterized in that:

(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.

It should be clear that one skilled in the art can substitute, actually and/or add one or more amino acids to the disclosed amino acid sequences without affecting the protein activity, and the protein sequence alignment homology is more than 95%, so as to obtain the mutant sequences of the protein. Therefore, the invention also comprises a derivative protein which is obtained by substituting, actually and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID No.2 and has high homology and activity.

The nucleotide sequence of the ZmGLK44 gene for coding the ZmGLK44 protein for regulating the moisture utilization efficiency under the drought of the corn is shown as SEQ ID No. 1.

The ZmGLK44 gene and protein can be obtained by cloning or separating from corn variety B73 or by a sequence chemical synthesis method.

A primer set for the ZmGLK44 gene, comprising: the primer pair is as follows:

the invention also provides a recombinant expression vector, which is an expression vector of the ZmGLK44 gene, wherein the expression vector is ZZ0153-RD101p-3 HA.

The invention also provides a host cell containing the recombinant expression vector, and the host cell is Escherichia coli DH5 alpha.

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

(1) the ZmGLK44 gene described above.

(2) The recombinant expression vector described above.

The application comprises the following steps: the gene can be connected to the downstream of an expression vector promoter to construct a recombinant expression vector capable of expressing the protein, and the expression vector can be introduced into various hosts by a genetic transformation method to obtain a transformant of the ZmGLK44 gene.

The invention has the beneficial effects that:

according to the invention, the corn drought-resistant gene ZmGLK44 is introduced into the corn strain C01, so that the drought-resistant phenotype and the drought-resistant physiological index of a transgenic plant are remarkably improved, and the ZmGLK44 gene can be used for improving the drought resistance of crops.

Drawings

FIG. 1 is a schematic diagram of construction of maize drought-induced vector ZZ0153-RD101P-ZmGLK44-3HA (universal vector map);

FIG. 1a is ZZ0153 empty loading diagram,

FIG. 1b is a diagram of the ZZ0153-RD101P-ZmGLK44-3HA vector;

FIG. 2 is a diagram of identification of ZmGLK44 expression level of transgenic plants under drought;

FIG. 2a is a primer design diagram, and FIG. 2b is an exogenous expression diagram of ZmGLK44 transgenic plants under drought;

FIG. 3 is a diagram of the drought resistant phenotype analysis of transgenic plants;

FIG. 3a is a drought phenotype diagram of ZmGLK44 overexpression plant, and FIG. 3b is a physiological and biochemical index diagram of ZmGLK44 overexpression plant before drought; FIG. 3c is a physiological and biochemical index map of ZmGLK44 overexpression plant in drought, and FIG. 3d is a physiological and biochemical index map of ZmGLK44 overexpression plant after rehydration;

note: a. the_NThe photosynthetic rate; e, transpiration rate; IWUE, A/E, water utilization efficiency; chlorophyl, Chlorophyll content; survival Rate, Survival Rate; ion leakage, Ion conductance.

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 1

Acquisition of ZmGLK44 gene for regulating and controlling moisture utilization efficiency of corn under drought

A pair of homologous recombination primers is designed according to the cDNA sequence of ZmGLK44 for PCR amplification, and the primer sequences are as follows:

ZmGLK44-OE-F：	5’-GCACTAGTATCCCGGGAAGGCGCGCCATGGGGCTGGACGTCGG-3’；
		ZmGLK44-OE-R：	5’-CGTCGTATGGGTACATGGCCACGTATTTCCGGCTGTAGCC-3’。

the PCR amplification was carried out by using Vazyme Phanta Max Super-Fidelity DNA polymerase, and the reaction system was as follows:

2x phanta Buffer	10ul
		dNTP	0.4ul
ZmGLK44-OE-F	0.8ul
		ZmGLK44-OE-R	0.8ul
Phanta max	0.4ul
		cDNA	100ng
ddH₂O	7.1ul

the reaction procedure is as follows: 3min at 95 ℃; 15s at 95 ℃; 15s at 60 ℃; 60s at 72 ℃; 5min at 72 ℃; 35, circulating the mixture for a period of time,

obtaining a PCR product, and obtaining the PCR product with the sequence shown in SEQ ID No.1, namely SEQ ID No.1, and the amino acid sequence shown in SEQ ID No.2 through sequencing detection.

Example 2

Construction of ZZ0153-RD101p-3HA vector

1) The promoter sequence of the maize B73 inbred line drought-induced expression gene ZmRD101 is shown as SEQ ID NO.1 and is used as a template design primer pair as follows: RD 101-F: 5' -CCTGTCAAACACTGATAGTTTTTCACTTTTTTAGTCTGGCAAT-3’，RD101-R：5’-CCGGGATACTAGTGCGTTTAAACGCTCACGGTTGCCTTG-3' (the PMEI cleavage site is underlined);

2) carrying out PCR by using a corn drought-induced expression gene ZmRD101 sequence as a template, and sequencing a gene PCR product to obtain a nucleotide sequence shown as SEQ ID No. 3; the PCR amplification conditions were as follows:

reaction system:

2x phanta Buffer	10ul
		dNTP	0.4ul
forward primer RD101-F	0.8ul
		Reverse primer RD101-R	0.8ul
Phanta max	0.4ul
		cDNA	1.5ul
ddH₂O	6.9ul

The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 sec; annealing at 60 ℃ for 30 sec; extension at 72 ℃ for 75 sec; after circulating for 35 times; extension at 72 ℃ for 5 min.

3) Preparation of a linearized vector: the pZZ01523 vector was digested singly with the restriction enzyme PMEI to linearize the circular vector.

4) And (3) detecting and recovering a gene PCR product and a carrier enzyme digestion product: and detecting the PCR product and the carrier enzyme digestion product by agarose gel electrophoresis. The desired fragment was recovered using the Omega Bio-tek Gel Extraction Kit.

5) 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
5 x 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, and setting on ice to room temperature.

6) Transformation of recombinant products: the recombinant product was transformed into DH5 α competent cells, according to the molecular cloning protocols.

7) Colony PCR of the recombinant product; extracting recombinant plasmids: and (3) obtaining the recombinant expression vector ZZ0153-RD101p-3HA by referring to a molecular cloning experimental manual.

2. Construction of recombinant expression vectors

Based on ZZ0153-RD101p-3HA vector, the cDNA fragment of ZmGLK44 was inserted downstream of RD101 promoter and between 3HA tags by homologous recombination and expressed from RD101 promoter (see FIG. 1). Electrophoresis detection and sequencing analysis show that the recombinant expression vector ZZ0153-RD101p-ZmGLK44-3HA is successfully obtained; the specific experimental steps are as follows:

1) ZmGLK44 Gene amplification (same as example 1)

2) Preparation of a linearized vector: carrying out single enzyme digestion on ZZ0153-RD101p-3HA vector by using a restriction enzyme ASCI (anaerobic digestion-polymerase) 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 a 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:

linearized vector	0.03pmol
		Insert fragment	0.06pmol
5 x CE II buffer	4ul
		Exnase II	2ul
ddH₂O	To 20ul

Gently sucking and beating the mixture by using a pipettor, and mixing the mixture uniformly, and collecting the reaction solution to the bottom of a 0.2ul centrifuge tube after short-time centrifugation; reacting at 37 deg.C for 30min in PCR instrument, and cooling to 4 deg.C;

5) transformation of recombinant products: transforming DH5 alpha competent cells with the recombinant product, referring to molecular cloning experimental guidelines (Huang Petang 2002);

6) extracting recombinant plasmids: reference molecular cloning experimental guidelines (huang petang 2002);

7) sequencing and identifying the recombinant plasmid: the colony PCR, the plasmid PCR and the sequencing analysis show that the ZZ0153-RD101p-ZmGLK44-3HA overexpression vector is obtained. The PCR and sequencing primers are

RD101p-F	5’-TCAAATCTGTCCGAATGCC-3’
		RD101p-R	5’-TCTGGAACGTCGTATGGG-3’

Example 3 transgenic maize line acquisition

1. The vector obtained in the example 2 is sent to Jiangsu Mimi biological science and technology limited company for transformation, the transformation background is a corn inbred line C01, and T1 generations are obtained by cultivating T0 generations of transgenic seeds obtained from the company;

2. screening T1 positive plants by coating herbicide, harvesting seeds, and cultivating to obtain T2 generations;

3. 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 ZmGLK44 stable inheritance and corresponding negative segregation materials; the PCR detection primer is (RD101p-F/ZmGLK 44-OE-R).

Example 4 detection of expression levels of ZmGLK44 transgenic maize plants

Simultaneously planting the positive transgenic material and the negative separation material in a square box with the specification of 30x40x15cm, stopping watering when the materials grow to the four-leaf one-heart stage under the normal state, and normally watering a control group; extracting RNA from the uppermost unfolded leaf when the water content of the soil is reduced to 10%, digesting the RNA by DNaseI, performing reverse transcription by using Promega MLV reverse transcriptase, and then detecting the expression quantity by using Vazyme SYBR Green Mix. The maize actin gene was used as a control;

the detection result shows that the ZmGLK44 gene expression quantity is induced by drought stress in the transgenic positive plant (figure 2). The detection primers used were as follows:

example 5 drought resistance phenotype testing of ZmGLK44 transgenic maize plants

0.2L of flowerpots are filled with the same amount of matrix (peat: vermiculite: 7: 3), and two transgenic positive plants or corresponding negative segregating plants are planted in each flowerpot. The plants were grown in a growth chamber at 22-26 ℃ with a circadian rhythm of 16h of sunlight/8 h of darkness. Plant chlorophyll content and photosynthetic rate were measured 16 days after seed germination (fig. 3b), while watering was stopped to start drought treatment. Plants showed significant leaf curl 15 days after drought, at which time the photosynthetic rate during drought was measured (FIG. 3c) and rehydrated. The survival rate, ionic conductivity, photosynthetic rate and chlorophyll content of the plants were measured 6 days after rehydration (fig. 3 d).

Under the normal growth state, the transgenic positive plants and the transgenic negative plants are not obviously different in phenotype. In drought, transgenic positive plants show higher carbon dioxide assimilation and higher water utilization efficiency. The transgenic positive plants after rehydration show higher carbon dioxide assimilation capability and higher water utilization efficiency. Meanwhile, the survival rate, the ion conductivity (reflecting the integrity of cell membranes) and the chlorophyll content of the transgenic positive plants after rehydration are obviously higher than those of negative separated plants.

The results show that the ZmGLK44 transgenic plant has stronger drought stress resistance. And the ZmGLK44 transgenic plant can keep higher water utilization efficiency during and after drought stress, and has the potential of realizing yield preservation and stable yield under drought.

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> ZmGLK44 gene for regulating and controlling moisture utilization efficiency of corn under drought and application thereof

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gagtcctgca tcaggagcct cgaggaggag cgccggaaga tcgaggtgtt caggcgcgag 180

ctcccgctct gcgtgcgtct cctcgccgat gtgatcgacg aattgaagga tgaggccgcc 240

aagagaggcg gagacgcgga ggccaaggcc gacgacggcg ataagcggaa atggatgagc 300

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<213> corn (Zea may L)

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Asp Leu Gly Leu Phe Ala Ala Arg Ser Ala Gly Gly Met Ala Ala Ala

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Glu Glu Arg Arg Lys Ile Glu Val Phe Arg Arg Glu Leu Pro Leu Cys

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Val Arg Leu Leu Ala Asp Val Ile Asp Glu Leu Lys Asp Glu Ala Ala

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Lys Arg Gly Gly Asp Ala Glu Ala Lys Ala Asp Asp Gly Asp Lys Arg

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Asp Glu Ser Asp Lys Glu Gln Leu Ser Glu Ile Thr Ser Pro Glu Pro

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Lys Leu Leu Gly Gly Ala Pro Met Pro Ile Arg Ala Val Ala Ala Val

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Pro Pro Leu Pro Pro Pro Phe Phe Arg Arg Glu Asp Ser Ser Ala Gly

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Ser Gly Leu Ser Leu Val Pro Pro Ala Ala Lys Pro Pro Ile Pro Pro

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aacgcgtgtg aagctgaatc taggccggga gagcaggaga tgccgccgtc gtgttgcgat 720

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ttttcgttta gatatgtgaa atttaggtac agataagcta atattatttt atacataata 1200

tatttatata ttaccgtagt catatatgag atatttatac actacatttt tattatagat 1260

gagtaagttt aaagggtatg tcatgaatta cggagtagag tcatggacta ctgatacatg 1320

aatcaatttc cacctactcg ttcaataaat ttgacatagg cttatatcta aatattgcaa 1380

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aacgattaat attttagaac gggattttag acgacgtata gtgcttactg tataaagcgc 1620

cgacgataaa agttatttac attggaaagg gcaaaaaacc tacaatttga taccgaggaa 1680

gtgggtttta ctcgtagaaa aagatattta tccgctaatt tgaaaagaga cgtttgatac 1740

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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）ZmGLK44 the nucleotide sequence of the gene is shown as SEQ ID No. 1;

(2) a recombinant expression vector; the recombinant expression vector contains the aboveZmGLK44 The gene expression vector is ZZ0153-RD101p-3 HA.