CN113005107B

CN113005107B - ZmCIPK10.2 protein and application of encoding gene thereof in regulation of low temperature stress tolerance of corn

Info

Publication number: CN113005107B
Application number: CN201911325482.7A
Authority: CN
Inventors: 施怡婷; 杨淑华; 张晓燕
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2023-05-23
Anticipated expiration: 2039-12-20
Also published as: CN113005107A

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to ZmCIPK10.2 protein and application of a coding gene thereof in regulating and controlling low-temperature stress tolerance of corn. The invention discovers that the corn ZmCIPK10.2 gene can positively regulate and control the cold resistance of plants, and can effectively improve the cold resistance of plants by improving the expression quantity of the ZmCIPK10.2 gene. The discovery of the cold resistance function of the ZmCIPK10.2 gene provides a new gene target and resource for cultivating cold-resistant plant varieties, has important significance for researching cold-resistant molecular mechanisms of corn, and lays a certain theoretical foundation for researching the mechanism of plants responding to low-temperature stress and resisting the molecular mechanism of adverse environments.

Description

ZmCIPK10.2 protein and application of encoding gene thereof in regulation of low temperature stress tolerance of corn

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to ZmCIPK10.2 protein and application of a coding gene thereof in regulating and controlling low-temperature stress tolerance of corn.

Background

Corn (Zea mays l.) belongs to the genus Zea of the family poaceae, is an important food crop and feed crop, and is also the crop with the highest total yield worldwide. Corn is very sensitive to cold, especially during the early autotrophic long stage. The activities of C4 and enzymes in the Calvin cycle in corn photosynthesis at low temperature are inhibited, which promotes the dissipation mechanism and affects the antioxidant defenses of corn leaves. Cold stress can affect chloroplast and meristem development, causing irreparable damage to later production. The physiological mechanism of corn cold tolerance remains largely unknown; but now the cold tolerance QTL of maize seedlings has been found. The use of transgenic techniques to introduce endogenous or exogenous stress-resistant genes into the corn genetic material to be improved and to render them subsequently resistant to stable inheritance may help to ultimately elucidate the mechanisms of cold tolerance. Although some studies on maize cold resistance genes have been reported at present, it is important to develop genes having excellent cold resistance functions in order to obtain maize varieties having excellent cold resistance phenotypes.

Ca ²⁺ As a ubiquitous and functionally abundant ion in plant cells, it plays an important role in the growth and development of plants and in signal transduction processes within cells. When the plant cells are subjected to specific external stimulus, ca in the plant body is caused ²⁺ The concentration changes temporally and spatially, thereby producing a calcium signal. Research shows that Ca ²⁺ The phosphorylation state and activity of ion transporters can be regulated by mediating the formation of CBL-CIPK complexes, maintaining intracellular ion homeostasis and plant response to some abiotic stress. Activation of CIPK (CBL-Interacting Protein Kinase) kinase activity is dependent on the interaction of CIPK with CBL and the interaction of both is dependent on Ca ²⁺ . EF hand unit of CBL binds Ca ²⁺ The induced conformational change promotes the interaction of CBL with the NAF domain of CIPK, thereby releasing the inhibition of CIPK kinase activity by the NAF domain and activating the CIPK kinase activity to function. The function of the CBL-CIPK complex is affected by a number of factors, firstly the effect of the acylation modification of CBL on the function of the CBL-CIPK complex.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide ZmCIPK10.2 protein and application of a coding gene thereof in regulating and controlling low-temperature stress tolerance of corn.

The invention carries out preliminary screening of low-temperature phenotype by taking leaf relative injury area as index in polygene over-expression corn population, carries out re-screening on the over-expression strain with low-temperature resistant phenotype obtained by preliminary screening, determines the low-temperature related phenotype, and determines the over-expression gene of the strain with obvious low-temperature resistant phenotype obtained by screening. Through the screening, the targeting gene GRMZM2G409658 (ZmCIPK 10.2) is probably related to low temperature stress tolerance of corn, and the ZmCIPK10.2 belongs to protein kinase of CIPK family through interaction with calcineurin B-like protein CBL. There are also 3 other overexpressed strains of ZmCIPK family members in the overexpressed maize population used for screening, but these have not been found to have a distinct low temperature-related phenotype. Experiments further prove that the ZmCIPK10.2 positively regulates the cold resistance of plants, improves the expression quantity of the ZmCIPK10.2 protein in the plants, and can obviously improve the cold resistance of the plants.

Specifically, the technical scheme of the invention is as follows:

in a first aspect, the invention provides the use of a zmcipk10.2 protein, a coding gene thereof or a biological material comprising a coding gene thereof for modulating cold resistance in a plant.

In the present invention, the cold resistance includes a property of resisting/tolerating a low temperature above zero or a low temperature below zero.

In particular, the cold resistance may be expressed as ion leakage rate.

In a second aspect, the invention provides the use of a zmcipk10.2 protein, a coding gene thereof or a biological material comprising a coding gene thereof for modulating the survival and/or growth of a plant under low temperature conditions.

In a third aspect, the invention provides the use of a zmcipk10.2 protein, a coding gene thereof or a biological material containing the coding gene thereof for breeding transgenic plants with increased cold resistance and/or yield.

In a fourth aspect, the invention provides the use of zmcipk10.2 protein, its encoding gene or biological material containing its encoding gene in plant cold-resistant germplasm resource improvement.

Preferably, in the above application, the cold resistance and/or yield of the plant is increased or the survival and/or growth of the plant under low temperature conditions is increased by increasing the expression and/or activity of the zmcipk10.2 protein in the plant.

In the invention, the ZmCIPK10.2 protein has any one of the following amino acid sequences:

(1) An amino acid sequence as shown in SEQ ID NO. 1;

(2) The amino acid sequence shown as SEQ ID NO.1 is obtained by replacing, inserting or deleting one or more amino acids to obtain the amino acid sequence of the protein with the same function.

The amino acid sequence shown as SEQ ID NO.1 is the amino acid sequence of ZmCIPK10.2 protein of corn, and the skilled in the art can obtain the mutant of ZmCIPK10.2 protein with the same function as the amino acid sequence shown as SEQ ID NO.1 by substituting, deleting and/or adding one or more amino acids according to the conventional technical means in the art such as the amino acid sequence shown as SEQ ID NO.1, conservative substitution of amino acids and the like on the premise of not affecting the activity of the mutant.

In the invention, the cDNA of the ZmCIPK10.2 protein has any one of the following nucleotide sequences:

(1) A nucleotide sequence as shown in SEQ ID NO. 2;

(2) A nucleotide sequence shown as SEQ ID NO.2 is subjected to substitution, deletion or insertion of one or more nucleotides to obtain a coding nucleotide sequence of the protein with the same function;

(3) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence shown as SEQ ID NO. 2.

The nucleotide sequence shown in SEQ ID NO.2 is a cDNA sequence of ZmCIPK10.2 protein in corn, and consists of 1290 bases, and the reading frame of the gene is from 149 th to 1290 th bases at the 5' end. The gene reading frame consists of only 1 exon. Given the degeneracy of the codons, all nucleotide sequences encoding the zmcipk10.2 protein are within the scope of the invention.

In the invention, the biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

The invention provides a cloning vector or various expression vectors containing the coding gene of the ZmCIPK10.2 protein. The invention also provides a host cell containing the vector, a transformed plant cell or a transgenic plant containing the coding gene of the ZmCIPK10.2 protein.

Specifically, the expression vector may be a pCUN vector (the vector is obtained by ligating a hygromycin resistance gene into pCAMBIA 1300).

In a fifth aspect, the present invention provides a method for improving cold resistance of a plant by improving the expression level of the zmcipk10.2 protein in the plant.

Preferably, the increasing the expression level of the zmcipk10.2 protein in the plant is achieved by introducing an over-expression vector comprising a gene encoding the zmcipk10.2 protein into the plant.

In a sixth aspect, the invention provides a construction method of cold-resistant transgenic corn, which is a method for improving the expression quantity and/or activity of ZmCIPK10.2 protein in corn through transgenic, hybridization, backcross, selfing or asexual propagation; the ZmCIPK10.2 protein has any one of the following amino acid sequences:

(1) An amino acid sequence as shown in SEQ ID NO. 1;

Preferably, the transgene comprises introducing an over-expression vector comprising the gene encoding the zmcipk10.2 protein into maize using Ti plasmid, plant viral vector, direct DNA transformation, microinjection, gene gun, conductance, or agrobacterium-mediated method, resulting in a transgenic maize line.

Specifically, as one embodiment of the invention, the construction method of the cold-resistant transgenic corn comprises the following steps:

(1) Extracting total RNA of corn, carrying out reverse transcription to obtain cDNA, using the cDNA as a template and a sequence shown as SEQ ID NO.3-4 as a primer, amplifying CDS sequence of ZmCIPK10.2 gene, and connecting an amplified product to a plant expression vector pCUN to obtain a recombinant expression vector;

(2) Transforming agrobacterium with the recombinant expression vector obtained in the step (1) to obtain recombinant agrobacterium;

(3) Infecting maize callus by adopting the recombinant agrobacterium obtained in the step (2), and screening positive transgenic plants to obtain cold-resistant transgenic maize;

by utilizing the method, the ZmCIPK10.2 over-expression transgenic corn plant is obtained, and compared with wild corn, the cold resistance of the plant is obviously improved.

In the present invention, the plant is a monocotyledonous plant or a dicotyledonous plant. Such plants include, but are not limited to, corn, rice, wheat, cotton, or soybean.

Preferably, the monocotyledonous plant is a plant of the Gramineae family. More preferably corn.

The invention has the beneficial effects that: the invention discovers that the corn ZmCIPK10.2 gene can positively regulate and control the cold resistance of plants, and can effectively improve the cold resistance of plants by improving the expression quantity of the ZmCIPK10.2 gene. The ZmCIPK10.2 over-expression transgenic corn plant is constructed, and compared with wild corn, the cold resistance of the plant is obviously improved. The discovery of the cold resistance function of the ZmCIPK10.2 gene provides a new gene target and resource for cultivating cold-resistant plant varieties, has important significance for researching cold-resistant molecular mechanisms of corn, and lays a certain theoretical foundation for researching the mechanism of plants responding to low-temperature stress and resisting the molecular mechanism of adverse environments.

Drawings

FIG. 1 is an expression level test of ZmCIPK10.2 gene in over-expressed strains OE-5800 and OE-5900 in example 2 of the invention, wherein CK represents wild-type maize plants and #5800 and #5900 represent over-expressed strains OE-5800 and OE-5900, respectively.

FIG. 2 shows the plant growth of the overexpressing strains OE-5800 and OE-5900 of example 3 of the invention after recovery from low temperature treatment; wherein CK represents wild maize plants, 5800 and 5900 represent over-expressed strains OE-5800 and OE-5900, respectively.

FIG. 3 is a statistical plot of the ion leakage rate results for the over-expressed strains OE-5800 and OE-5900 of example 3 of the invention; wherein CK represents wild-type maize plants, #5800 and #5900 represent over-expressed strains OE-5800 and OE-5900, respectively.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 21), or the conditions recommended by the manufacturer's instructions.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The pBSK vector in the examples below is a commonly used cloning vector and is commercially available; the pCUN vector is obtained by ligating a hygromycin resistance gene between cleavage sites of pCAMBIA1300 (Guo et al, 2018stepwis cis-regulatory changes in ZCN, contribute to maize fowering-time adaptation.current bio.28, 3005-3015); the Agrobacterium EHA105 strain is offered by the crop functional genome platform of the university of agricultural college of China and is commercially available (Ma et al 2009,Enhanced tolerance to chilling stress in OsMYB3R-2transgenic rice is mediated by alteration in cell cycle and ectopic expression of stress genes.Plant Physiol.150,244-256). In the following examples, various restriction enzymes, taq DNA polymerase, T4 ligase, pyrobest Taq enzyme, KOD were purchased from biological companies such as NEB, toyobo, etc.; dNTPs are available from Genestar; the plasmid miniprep kit and the agarose gel recovery kit are purchased from Shanghai Jierui bioengineering company; antibiotics such as agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), and rifampicin (Rif), and the like, and companies such as Glucose, BSA, and LB Medium are available from Sigma, bio-Rad, and the like; reagents for real-time quantitative PCR were purchased from TaKaRa; various other chemical reagents used in the examples below were either imported or home-made analytically pure reagents. Primers used in the following examples were synthesized by Hexakuda and subjected to related sequencing.

EXAMPLE 1 construction and identification of ZmCIPK10.2 Gene overexpression vector

By screening corn lines with a cold tolerance phenotype in a corn pool of transgenic overexpressing lines, a zmcipk10.2 overexpressing line with a pronounced cold tolerance phenotype was found. In the maize pool of transgenic overexpressing lines, zmcipk10.2 has a total of 6 transformation events. The identification by real-time quantitative PCR shows that 2 strains have the ZmCIPK10.2 gene expression level obviously up-regulated, and the 2 strains have no obvious growth and development phenotype but all have obvious cold-resistant phenotype. The coding region sequence of the corn ZmCIPK10.2 gene is analyzed, primers F and R are designed according to the coding region sequence, the coding region of the gene is amplified, and the amplified coding region is connected to an over-expression vector pCUN with a 35S promoter (the vector is obtained by connecting hygromycin resistance gene between SalI and Kpn enzyme cleavage sites of pCAMBIA 1300). The primer sequences are specifically as follows:

upstream primer F:5'-ATGGGGAAGCTGCTGGGGAA-3' (Seq ID No. 3);

the downstream primer R:5'-CTTCTGCTGCAGATCATCTC-3' (Seq ID No. 4).

The specific method for ligating zmcipk10.2 gene to pCUN vector with 35S promoter is: firstly, using cDNA as a template, amplifying ZmCIPK10.2 by using an upstream primer F and a downstream primer R, connecting a PCR product with a pBSK vector, and naming the connection product as ZmCIPK10.2-pBSK; zmCIPK10.2 was digested with SalI and KpnI and ligated into pCUN vector after cleavage from the properly sequenced ZmCIPK10.2-pBSK, and the ligation product was designated 35S: zmCIPK10.2. The obtained plasmid is subjected to enzyme digestion and then is subjected to electrophoresis detection, and the specific method comprises the following steps: the samples were digested with SalI and KpnI 35S: ZMCIPK10.2, and subjected to electrophoresis at 120V and 50mA using a 1% agarose gel, and then scanned and imaged by a UVP Gel Documentation gel analysis system. The results indicate that the pCUN overexpression vector of zmcipk10.2 was successfully constructed.

Example 2 construction and characterization of ZmCIPK10.2 Gene overexpressing maize

The pCUN vector containing zmcipk10.2 gene constructed in example 1 was transformed into agrobacterium EHA105 strain, and maize callus was infected to obtain transgenic seedlings. The specific method comprises the following steps: inoculating Agrobacterium containing the objective vector into 100mL LB three-antibody liquid culture solution (Kan 50 μg/mL, rif 50 μg/mL, gen 50 μg/mL), shake culturing at 28deg.C overnight, and standing for OD ₆₀₀ Centrifuging at room temperature for 15min at 50 Xg and with a value of 1.0-2.0, and collecting thallus; 2mL of the transformant (1/2 MS,5% sucrose, 40. Mu.L Silwet L-77) was used to suspend the cells; soaking corn callus in agrobacterium transformation liquid, and sealing. And (5) placing the plants back to the illumination culture rack for normal growth until the plants grow out. Then the seeds obtained by screening are subjected to low-temperature stressAnd (5) treating the experiment.

In this example, over-expressed strains OE-5800 and OE-5900 were isolated and the gene expression of ZmCIPK10.2 in the over-expressed strains OE-5800 and OE-5900 was detected by real-time quantitative PCR, as follows:

(1) Total RNA of corn is extracted and cDNA is obtained by reverse transcription.

(2) After 5-fold dilution of the cDNA obtained by reverse transcription, real-time quantitative PCR was performed using Takara kit, using the following reaction system: 2X SYBR Premix ExTaq buffer, 0.2. Mu.L DyII, 0.4. Mu.L LPrimer (F/R), 2. Mu.L cDNA template, and finally ddH ₂ O is filled to 20 mu L, and the mixture is put into an ABI PRISM 75 real-time quantitative PCR instrument for PCR amplification by a two-step method after being fully and evenly mixed, and the reaction conditions are as follows: 95 ℃ for 30s;95 ℃ for 5s; 40s at 60 ℃;40cycles. Simultaneously with the amplification of the identified genes, each sample was amplified simultaneously with UBI as an internal reference. After completion of the PCR reaction according to 2 ^-Δ(ΔCt) The relative expression level between the wild type and the over-expressed strain is calculated and plotted. The results are shown in FIG. 1 and demonstrate that the ZmCIPK10.2 gene in OE-5800 and OE-5900 is up-regulated by 39-fold and 78-fold, respectively.

Example 3 detection of Low temperature resistance of corn overexpressing ZmCIPK10.2 Gene

Seeds of wild type corn (control group CK) and over-expressed strains OE-5800 and OE-5900 were first sown in small pots 10cm long, 10cm wide and 10cm high containing black soil, imported soil and vermiculite (1:1:1), 5 grains were placed in each pot, 2cm of soil was then placed in a tray, watered until the soil was completely wet, placed in a culture chamber at 23℃for 16h of light and 8h of darkness. After 14d growth, 4 ℃ low temperature treatment is carried out until the second leaf is wilted, the second leaf is taken out and put into a 23 ℃ culture room for two days to recover, and then photographing and taking materials are carried out for statistics of ion leakage rate.

The phenotype of the wild type plants and the over-expressed strains OE-5800 and OE-5900 after recovery from low temperature treatment is shown in FIG. 2, and the leaf wilting degree of the over-expressed strains OE-5800 and OE-5900 is obviously reduced compared with the control, which indicates that the over-expressed strains OE-5800 and OE-5900 both show the phenotype of low temperature resistance. In the above experiments, 3-5 seedlings were independently repeated 3 times for each experiment.

The statistics of ion leak rate in this example were performed by detecting the relative conductivity l= (S1-S0)/(S2-S0) of the leaf. All the whole plants of the corn subjected to low-temperature treatment are placed into a 15ml centrifuge tube filled with 10ml of distilled water, a vacuum pump is used for pumping air for 30min, then the whole plants are placed into a shaking table for shaking at room temperature for 1h, then a conductivity meter is used for measuring the initial conductivity value of the whole plants to be S1, then a sample is placed into boiling water for water bath for 15min, the whole plants are taken out and placed into the shaking table for shaking for 2h, and then the conductivity is measured and recorded as S2. S0 is the conductivity of the control distilled water. The results are shown in FIG. 3, and compared with wild type plants (70%), the ion leakage rates of the over-expressed strains OE-5800 and OE-5900 are 47% and 42% respectively, which are significantly lower than that of the wild type plants, indicating that over-expression of the ZmCIPK10.2 gene can enhance the freezing resistance of corn. In the above experiments, 3-5 seedlings were tested each time, and the independent experiments were repeated 3 times.

While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

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Claims

Application of ZmCIPK10.2 protein, coding gene thereof or biological material containing the coding gene thereof in regulation and control of cold resistance of corn;

the application is that the cold resistance of the corn is improved by improving the expression quantity and/or activity of the ZmCIPK10.2 protein in the corn;

the amino acid sequence of the ZmCIPK10.2 protein is shown as SEQ ID NO. 1;

the biological material is an expression cassette, a vector or a host cell.
Application of ZmCIPK10.2 protein, coding gene thereof or biological material containing the coding gene thereof in regulating and controlling survival rate and/or growth of corn under low-temperature conditions;

the application is that the survival rate and/or the growth of the corn under the low-temperature condition are improved by improving the expression quantity and/or the activity of the ZmCIPK10.2 protein in the corn;

the amino acid sequence of the ZmCIPK10.2 protein is shown as SEQ ID NO. 1;

the biological material is an expression cassette, a vector or a host cell.
Application of ZmCIPK10.2 protein, coding gene thereof or biological material containing the coding gene thereof in breeding transgenic corn with improved cold resistance;

the application is that the cold resistance of the corn is improved by improving the expression quantity and/or activity of the ZmCIPK10.2 protein in the corn;

the amino acid sequence of the ZmCIPK10.2 protein is shown as SEQ ID NO. 1;

the biological material is an expression cassette, a vector or a host cell.
Application of ZmCIPK10.2 protein, coding gene thereof or biological material containing the coding gene thereof in cold-resistant germplasm resource improvement of corn;

the application is that the cold resistance of the corn is improved by improving the expression quantity and/or activity of the ZmCIPK10.2 protein in the corn;

the amino acid sequence of the ZmCIPK10.2 protein is shown as SEQ ID NO. 1;

the biological material is an expression cassette, a vector or a host cell.
5. The use according to any one of claims 1 to 4, wherein the nucleotide sequence of the cDNA of the zmcipk10.2 protein is shown in SEQ ID No. 2.
6. A construction method of cold-resistant transgenic corn is characterized in that the expression quantity and/or activity of ZmCIPK10.2 protein in corn is improved by a transgenic, hybridization, backcross or selfing method;

the amino acid sequence of the ZmCIPK10.2 protein is shown as SEQ ID NO. 1.
7. The method of claim 6, wherein the transgenic comprises introducing an over-expression vector comprising a gene encoding the zmcipk10.2 protein into corn using a Ti plasmid, plant viral vector, direct DNA transformation, microinjection, gene gun, conductance, or agrobacterium-mediated method to obtain a transgenic corn line.