CN113024644A

CN113024644A - Application of ZmICE1 protein and coding gene thereof in regulation and control of low-temperature stress tolerance of corn

Info

Publication number: CN113024644A
Application number: CN201911359272.XA
Authority: CN
Inventors: 杨淑华; 蒋海芳; 施怡婷
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2021-06-25

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to ZmICE1 protein and application of a coding gene thereof in regulation and control of low-temperature stress tolerance of corn. The invention discovers that the over-expression of ZmICE1 gene in corn can enhance the low temperature resistance of transgenic plants. The invention relates to a cDNA sequence of ZmICE1 gene as shown in SEQ ID NO.1, and the protein coded by the gene has an amino acid sequence as shown in SEQ ID NO. 2. The invention clones ZmICE1 gene, constructs transgenic plant of over-expression ZmICE1 gene, and analyzes the cold resistance of the obtained transgenic plant, and finds that the over-expression ZmICE1 can promote the expression of cold related gene, so that the corn can obtain stronger low temperature tolerance capability. The invention provides a new gene resource for cultivating new varieties of low-temperature-resistant plants, and lays a certain theoretical basis for researching the mechanism of the response of the corn to low-temperature stress.

Description

Application of ZmICE1 protein and coding gene thereof in regulation and control of low-temperature stress tolerance of corn

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to ZmICE1 protein and application of a coding gene thereof in regulation and control of low-temperature stress tolerance of corn.

Background

Plants growing in a fixed manner in nature cannot be influenced by various adversity stresses, and low-temperature stress is one of main external environmental factors, so that the growth, development and geographical distribution of the plants are influenced, and the yield of crops is also seriously influenced. Corn (Zea mays, L.) is an extremely important source of food and energy worldwide. Due to its agricultural and economic value, improving the nutrition or stress resistance of corn has always been the focus of the breeding direction. Maize originates in tropical and subtropical regions and has a certain sensitivity to cold stress. Therefore, the understanding of the molecular and biochemical mechanisms of cold resistance of corn is of great significance to the realization of agricultural sustainable development. However, low temperature tolerance is a complex physiological property and is difficult to measure accurately. Although some potential quantitative trait loci QTL and resistance genes have been discovered, the genetic basis for cold resistance in maize remains unclear.

The cold damage at low temperature has different influences on different development stages of the corn, and has obvious influences on the growth, development and functions of the corn seedlings. The smooth germination and emergence of corn seeds is the first step for obtaining high yield of corn, and has important significance for corn production. In the production of corn, the corn seeds are subjected to low-temperature freezing damage in soil, so that the germination rate of the corn seeds is seriously reduced, and the production is seriously influenced. The corn seedlings are frozen at low temperature in the growth and development process, and the yield and quality of the corn are seriously affected. The low temperature reduces the survival rate of the seedlings, causes the corns in the adult plant stage to be short and small, influences fructification, and further influences reproductive capacity, and is the main reason for unstable yield and low quality of the corns in the northeast of China.

Disclosure of Invention

The invention aims to provide a corn low temperature resistant gene ZmICE1 and application of a coding protein thereof. According to the invention, through the research on the corn cold-related gene ZmICE1, the transgenic plant over expressing the gene is found to have an obvious low temperature resistance phenotype compared with a control plant. The invention has the beneficial effect that the ZmICE1 gene provides gene resources for cultivating new varieties of low-temperature resistant plants.

The invention aims to provide an application of ZmICE1 protein and a coding gene thereof in cold resistance of corn. In order to discover the cold-resistant related genes of corn, the invention screens out the cold-resistant plants of over-expressed GRMZM2G173534 genes from an over-expressed strain library of more than 800 genes in corn. This gene belongs to the bHLH class of transcription factors. . Over-expressing ZmICE1 gene in corn and Arabidopsis thaliana to obtain low temperature resistant transgenic plant.

The sequence of the ZmICE1 gene related by the invention is as follows: i) a nucleotide sequence shown as Seq ID No. 1; or ii) a nucleotide sequence in which one or more nucleotides are substituted, deleted and/or added to the nucleotide sequence shown in Seq ID No.1 and which expresses the same functional protein; or iii) a nucleotide sequence which hybridizes under stringent conditions to the sequence shown in Seq ID No. 1;

the cDNA of corn ZmICE1 consists of 1348 bases, and the sequence is shown in Seq ID No. 1. The reading frame of the gene is cDNA from 93 th site to 1348 th site of 5' end. The reading frame of the gene consists of only 3 exons. The amino acid sequence encoded by the maize ZmICE1 gene is shown in Seq ID No. 2.

The ZmICE1 protein has any one of the following amino acid sequences:

1) an amino acid sequence shown as SEQ ID NO. 2; or

2) The amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2. It is understood that one skilled in the art can substitute, delete and/or add one or several amino acids based on the disclosed amino acid sequences without affecting their activity to obtain mutant sequences of the proteins.

The invention provides an application of ZmICE1 protein or a coding gene thereof, or a biological material containing the coding gene thereof in improving the cold resistance of plants.

The invention provides an application of ZmICE1 protein or its coding gene, or biological material containing its coding gene in breeding transgenic plants with improved cold resistance.

The invention provides an application of ZmICE1 protein or a coding gene thereof, or a biological material containing the coding gene thereof in improvement of cold-resistant germplasm resources of plants.

The invention provides an application of ZmICE1 protein or a coding gene thereof, or a biological material containing the coding gene thereof in improving the survival rate of plants in a low-temperature environment.

The biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

The invention also provides a cloning vector or various expression vectors containing the plant low temperature resistant ZmICE1 gene sequence or the fragment thereof, a host cell containing the vector, a transformed plant cell and a transgenic plant containing the gene sequence or the specific fragment thereof. Wherein, the over-expression vector containing ZmICE1 gene is pSuper1300 vector containing Super promoter or pCUN vector containing Ubi promoter. The invention also provides a preparation method of the transgenic plant, which improves the expression quantity of the ZmICE1 gene by a transgenic method to obtain the plant with high cold resistance.

In the present invention, the plant is a dicotyledonous plant or a monocotyledonous plant; preferably rice, wheat, soybean, sorghum, millet, cotton, barley or corn.

The invention also provides a method for constructing cold-resistant transgenic corn, which enables the corn to express or over-express the ZmICE1 gene by a transgenic, crossing, backcrossing, selfing or asexual propagation method.

The transgene comprises the step of introducing a recombinant expression vector containing ZmICE1 gene into corn by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, conductance and agrobacterium-mediated methods to obtain a transgenic corn strain.

In some embodiments, the transgene specifically comprises the steps of:

(1) amplifying a full-length gene cDNA sequence (shown as SEQ ID NO. 1) of the ZmICE1 gene;

(2) constructing an over-expression vector of the ZmICE1 gene;

(3) constructing a recombinant agrobacterium of an overexpression vector containing ZmICE1 gene;

(4) constructing a transgenic plant with over-expressed ZmICE1 gene by adopting an agrobacterium infection method.

In the embodiment of the invention, the specific method for constructing the low-temperature-resistant transgenic plant is as follows:

1) extracting total RNA of corn, carrying out reverse transcription to obtain cDNA, amplifying ZmICE1 gene by taking the cDNA as a template and F and R as primers, constructing an amplification product onto an expression vector pCUN, and naming the obtained recombinant expression vector as pCUN-ZmICE 1;

2) the agrobacterium EHA105 is transformed by pCUN-ZmICE1, and then the transformed agrobacterium is used for infecting corn callus to obtain low temperature resistant transgenic corn seedlings.

Wherein, the nucleotide sequences of the primers F and R in the step 1) are shown as Seq ID No.3 and 4. The maize is preferably a maize plant of the LH244 homozygous genotype. After overexpression of the ZmICE1 gene of the invention, maize shows a low temperature resistant phenotype.

The invention clones ZmICE1 gene, constructs transgenic plant of over-expression ZmICE1 gene, and analyzes the cold resistance of the obtained transgenic corn, and finds that the over-expression ZmICE1 can improve cold response gene expression, promote the expression of cold related gene and enable the corn to obtain stronger low temperature tolerance capability. The invention provides a new gene resource for cultivating new varieties of low-temperature-resistant plants, and lays a certain theoretical basis for researching the mechanism of the response of the corn to low-temperature stress.

Drawings

FIG. 1 shows the expression of ZmICE1 protein in Arabidopsis thaliana overexpression strain of example 3 of the present invention; wherein Col represents a wild type Arabidopsis plant and OE represents a transgenic overexpression line.

FIG. 2 is a photograph showing the growth of plants recovered from the low-temperature treatment of the overexpression lines of Arabidopsis thaliana in example 4 of the present invention; wherein, A is a survival rate statistical chart of 5 strains; in the diagram B, the left 1 is the normal growth phenotype of Arabidopsis before the low-temperature treatment, and the right 1 and the right 2 are the survival phenotype of Arabidopsis seedlings after the low-temperature treatment at-7 ℃ for 1 hour; wherein Col represents a wild type Arabidopsis plant and OE represents a transgenic overexpression line.

FIG. 3 shows the growth of a maize over-expression strain after recovery of the strain by low-temperature treatment in example 4 of the present invention; wherein, the A picture is before cold treatment, and the B picture is after cold treatment; CK represents control maize plants and #1 to #5 represent 5 overexpression lines, respectively.

FIG. 4 shows that ZmICE1 and ion leakage rate in maize over-expression strains are detected by qRT-PCR in examples 4-5 of the invention; wherein, the A picture is the detection of the expression quantity of ZmICE1-OE in the transgenic corn; b, a graph is a measurement result of the ion leakage rate of the corn seedlings after cold treatment; wherein CK represents a control maize plant and #1 to #5 represent 5 overexpression lines, respectively.

FIG. 5 shows the low temperature resistance of a ZmICE1 knock-out plant of example 6, wherein, A is the deletion of amino acids D44 and D19; b, the growth condition of the plant after low-temperature treatment and recovery; the C picture is the ion leakage rate measuring result after cold treatment; where CK represents a control maize plant and D44 and D19 represent knock-out ZmICE1 gene strains, respectively.

FIG. 6 shows germination of maize ZmICE1 over-expressed strain at 12 degrees in example 7 of the present invention; wherein, A picture is the result display of seed germination of control and ZmICE1-OE over-expression in transgenic corn; b, the chart is the germination rate statistical data of the corn seeds at low temperature; where CK represents a control maize plant and OE represents a transgenic overexpression line.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

The pBSK vector in the following examples is a commonly used cloning vector, and is commercially available; pSuper1300-GFP vector and pCUN vector are cited in the literature (Ni et al, 1995; Yang et al, 2010); the agrobacterium EHA105 strain originates from the crop functional genome platform of the university of chinese agriculture institute of biology (Ma et al, 2009).

The main reagents in the following examples are: various restriction enzymes, Taq DNA polymerase, T4 ligase, Pyrobest Taq enzyme, KOD from NEB, Toyobo, etc.; dNTPs were purchased from Genestar; the plasmid miniextraction kit and the agarose gel recovery kit are purchased from Shanghai Czeri bioengineering company; antibiotics such as agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), and rifampicin (Rif), and Glucose, BSA, and LB Medium were purchased from Sigma, Bio-Rad, and the like; reagents for real-time quantitative PCR were purchased from TaKaRa, and various other chemical reagents used in the examples were imported or home-made analytical reagents.

The primers used in the examples were synthesized from Hexa Huada and subjected to related sequencing.

Example 1 construction and detection of ZmICE1 Gene overexpression vector

Through large-scale corn over-expression strain population screening by a reverse genetics means, the GRMZM2G173534 gene is found to be possibly involved in regulating and controlling the cold resistance of corn. A plurality of over-expression strains of the gene have low temperature resistant phenotype (see result figure 2), primers F and R are designed according to coding region sequence analysis, the coding region of the gene is amplified and connected to an over-expression vector pSuper1300 with a Super promoter. The primers used were:

an upstream primer F: 5'-ATGGACGACTCGGCGGAGGT-3' (Seq ID No.3)

A downstream primer R: 5'-CATTGCGTTGTGGAGGCCGG-3' (Seq ID No.4)

The specific method for connecting ZmICE1 gene (the cDNA sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2) to the vector pSuper1300-GFP with Super promoter is as follows: firstly, taking cDNA as a template, amplifying ZmICE1 by using upstream and downstream primers (shown as SEQ ID NO. 3-4), connecting a PCR product with a pBSK vector, and naming a connection product as ZmICE 1-pBSK; the ligated product of ZmICE1 recovered from the correctly sequenced ZmICE1-pBSK by digestion with SalI and KpnI was named pSuper1300-ZmICE1-GFP vector. Carrying out enzyme digestion on the plasmid obtained in the last step, and carrying out electrophoresis detection, wherein the specific method comprises the following steps: pSuper1300-ZmICE1-GFP was digested with SalI and KpnI, and scanned for imaging using a UVP Gel Documentation Gel analysis system after electrophoresis on a 1% agarose Gel, 120V, 50 mA.

Example 2 construction and detection of ZmICE1 Gene overexpression vector

The specific method for connecting ZmICE1 gene (cDNA sequence is shown as SEQ ID NO.1, amino acid sequence is shown as SEQ ID NO. 2) to vector pCUN with 35S promoter is as follows: firstly, taking cDNA as a template, amplifying ZmICE1 by using upstream and downstream primers (shown as SEQ ID NO. 3-4), connecting a PCR product with a pBSK vector, and naming a connection product as ZmICE 1-pBSK; ZmICE1 was digested from the correctly sequenced ZmICE1-pBSK using SalI and KpnI, and ligated into pCUN vector, and the ligation product was named 35S ZmICE 1. Carrying out enzyme digestion on the plasmid obtained in the last step, and carrying out electrophoresis detection, wherein the specific method comprises the following steps: the 35S, ZmICE1, was digested with SalI and KpnI, and scanned and imaged by a UVP Gel Documentation Gel analysis system after electrophoresis with 1% agarose Gel, 120V, 50 mA.

Example 3 construction and detection of ZmICE1 Gene overexpression Arabidopsis thaliana

(1) Construction:

the pSuper1300 vector containing ZmICE1 gene described in example 1 was transformed into Agrobacterium GV3101 strain (Clough and Bent,1998), the Agrobacterium containing the desired vector was inoculated into 100mL LB three-antibody liquid medium (Kan 50. mu.g/mL, Rif 50. mu.g/mL, Gen 50. mu.g/mL), cultured overnight with shaking at 28 ℃ until OD600 value was 1.0-2.0, centrifuged at 5000g at room temperature for 15min, and the cells were collected; the cells were suspended in 200mL of the transformation solution (1/2MS, 5% sucrose, 40. mu.L Silwet L-77); soaking the arabidopsis inflorescence in the transformation liquid of the agrobacterium for 5min, covering a freshness protection package for moisturizing, placing in a dark place to ensure that the temperature is lower, taking out the plant from the freshness protection package the next day, and placing the plant back on an illumination culture rack for normal growth until the plant is harvested. The screened resistance gene carried by the pSuper1300 vector is hygromycin, the hygromycin resistance is used for screening Arabidopsis transgenic seedlings, the obtained positive seedlings with hygromycin resistance in T1 generations are subjected to single plant seed collection, the hygromycin resistance test is carried out on seeds in T2 generations, strains with 3/4 resistance and the rest 1/4 resistance are selected, and the over-expression vector carrying the target gene in the strains is inserted in a single copy form. And (3) removing the plants with hygromycin resistance from the strains, harvesting the single plants, screening the hygromycin resistance, and if the plants are not separated, indicating that the transgenic strains are homozygotes which can be used for seed reproduction and low-temperature stress treatment experiments.

(2) And (3) detection:

protein expression of ZmICE1 in the over-expressed strains OE-2 and OE-10 was detected by Western blotting.

1) Extraction of plant Total protein

The 14d seedlings grown on the culture dish are wrapped by tinfoil paper and quickly frozen in liquid nitrogen for later use. Grinding the plant material into powder with liquid nitrogen, adding protein extraction buffer solution, vortex shaking, and centrifuging at 13000g for 15min at 4 deg.C.

2) Detection of protein content in OE-2 and OE-5 strains by Western blotting

Adding 5 xSDS protein loading buffer into plant proteins of wild type, OE-2 and OE-5 Arabidopsis strains with equal concentration, boiling at 100 deg.C for 5min, running glue at 90V for 15min, changing to running glue at 120V for 1h, and transferring to membrane at 200mA for 2 h. Protein content in OE-2 and OE-10 strains was determined using Anti-GFP antibody (Abiramate, cat # 294175). Rubisco staining is a quantitative picture as an internal control. The results are shown in FIG. 1, which shows that the protein content of ZmICE1 in both OE-2 and OE-5 transgenic lines is highly expressed.

Example 4 construction and detection of ZmICE1 Gene overexpression maize

(1) Construction:

the pCUN vector containing ZmICE1 gene described in example 2 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 target vector into 100mL LB three-antibody liquid culture medium (Kan 50. mu.g/mL, Rif 50. mu.g/mL, Gen 50. mu.g/mL), shaking and culturing at 28 deg.C overnight until OD600 value is 1.0-2.0, centrifuging at 50g at room temperature for 15min, and collecting thallus; the cells were suspended in 2mL of transformation medium (1/2MS, 5% sucrose, 40. mu.L Silwet L-77); soaking the corn callus in agrobacterium transformation liquid, and sealing. And putting the seeds back to the illumination culture shelf to grow normally until plants grow. And then, carrying out a low-temperature stress treatment experiment on the screened seeds.

The overexpression strain obtained by separation in this example was tested for gene expression of ZmICE1 in the overexpression strain by real-time quantitative PCR.

(2) And (3) detection:

5 different transformation event overexpression strains separated in the embodiment are selected and the gene expression of ZmICE1 in the overexpression strains is detected by real-time quantitative PCR.

1) Extracting total plant RNA and reverse transcribing to obtain cDNA.

2) After the cDNA obtained by reverse transcription was diluted 5 times, real-time quantitative PCR was carried out using a Takara kit, and the reaction system used included: 2 × SYBR Premix ExTaq buffer, 0.2. mu.L DyII, 0.4. mu.L LPrimer (qRT-F/qRT-R), 2. mu.L cDNA template, and finally ddH₂And (3) supplementing the O to 20 mu L, fully and uniformly mixing, and then putting the mixture into an ABI PRISM 75 real-time quantitative PCR instrument to perform two-step PCR amplification, wherein the reaction conditions are as follows: 30s at 95 ℃; 5s at 95 ℃; 40s at 60 ℃; 40 cycles. After completion of PCR reaction according to 2^-Δ(ΔCt)The relative expression between wild type or control group and overexpression was calculated and mapped. While amplifying the identified genes, each sample was simultaneously amplified with the UBI gene as an internal control. The results are shown in FIG. 4A.

Example 5 detection of Low temperature resistance of plants overexpressing ZmICE1 Gene

1) Low temperature resistance detection of transgenic arabidopsis thaliana with over-expression ZmICE1

More than 10 independent overexpression events were obtained by screening arabidopsis positive seedlings. 5 strains are selected and treated at low temperature, and all strains have anti-freezing phenotype. The specific operation method comprises the following steps: wild type Arabidopsis seedlings and the ZmICE1 gene overexpression strain obtained in example 3 were grown on a culture dish for 14 days, transferred to a 4 ℃ low-temperature incubator for 3 days for cold acclimation, and taken out after 3 days for programmed cooling (1 ℃/hour) to-7 ℃ for 1 hour of low-temperature treatment. The treated seedlings were left in the dark for 24 hours for slow thawing. Then the cells were transferred to a 22 ℃ light incubator for 3 days, and photographing and survival rate statistics were performed.

The survival rate test results are shown in fig. 2, wherein fig. 2A is a statistical graph of the survival rate of 5 strains, and all 5 strains showed higher survival rate compared with the control group. In FIG. 2B, FIG. 1 on the left is the normal growth phenotype of Arabidopsis before cryo-treatment; the right 1 and right 2 figures show that after being treated at-7 ℃ for 1 hour, the Arabidopsis seedlings have a survival phenotype, and both OE-2 and OE-5 show an antifreeze phenotype. This indicates that the low temperature resistance of ZmICE1 over-expression plants is obviously improved.

2) Low temperature resistance detection of over-expression ZmICE1 transgenic corn

Firstly, seeds of a CK control group and an overexpression strain are sown in black soil, small pots which are 10cm in length, 10cm in width and 10cm in height and contain imported soil and vermiculite (1:1:1), 5 seeds are placed in each pot, then 2cm of soil is covered, the pots are placed in a tray, water is poured until the soil is completely wet, the pots are placed in a 23-degree culture room, and the pots are illuminated for 16 hours and are dark for 8 hours. After the control group grows for 14 days, the control group is subjected to 4 ℃ low-temperature treatment until the second leaf shrinks and wilts, the control group is taken out and placed in a 23 ℃ culture room to be recovered for two days, then photographing is carried out (the result is shown in figure 3, wherein, a picture A is before cold treatment, and a picture B is after cold treatment), and statistics of ion leakage rate is carried out by taking materials (the result is shown in figure 4B). The ion leakage rate reflects the integrity of the cell membrane. The statistical result shows that the ion leakage rate in the overexpression strain is obviously reduced compared with that in the control group. When the ion leakage rate of the control group is 0.35, the over-expression strain is only 0.1, which indicates that after low-temperature treatment, the over-expression plant has a more perfect cell membrane structure, thereby showing a low-temperature resistant phenotype.

Example 6 detection of Low temperature resistance of ZmICE1 knock-out Gene plants

The gene is knocked out by using CRISPR/Cas9, and 2 mutant strains in independent mutation forms are obtained and named as D44 and D19 respectively. Both strains were deleted for 44 and 19 amino acids (as shown in FIG. 5A), respectively, resulting in premature termination of ZmICE1 during translation.

Firstly, seeds of CK and CRISPR/Cas9 knockout strains in a control group are sown in small pots, 10cm in length, 10cm in width and 10cm in height, of black soil, imported soil and vermiculite (1:1:1), 5 seeds are placed in each pot, 2cm of soil is covered on each pot, the pots are placed in a tray, water is poured until the soil is completely wet, the pots are placed in a culture room with the temperature of 23 ℃, and the pots are illuminated for 16 hours and are dark for 8 hours. After 14 days of growth, the leaves of the control group are treated at low temperature of 4 ℃ until all the leaves of the control group are damaged and wilted. Taking out, placing in a 23 ℃ culture room, taking pictures after two days of recovery (the result chart is shown in figure 5B), and taking materials for counting the ion leakage rate.

Statistical results (see fig. 5C) show that the ion leakage rate in the knock-out line is significantly higher than that in the control group. When the ion leakage rate of the control group is 20%, the knockout strain approaches to 100%, which indicates that the mutant plant has serious damage to the cell membrane after low-temperature treatment, thereby showing a cold-sensitive phenotype. That is, deletion of the ZmICE1 protein results in reduced cold tolerance in maize.

Example 7 overexpression of ZmICE1 Gene improves maize seed germination rates at Low temperatures

3 ZmICE1 overexpression strains are selected to carry out corn seed germination treatment at low temperature, and the corn seed germination rate can be obviously improved.

The specific operation method comprises the following steps: seeds of the control group and the ZmICE1 overexpression line were first placed on wet filter paper and at 25 ℃ for 48 h. Then the culture medium is transferred into a low-temperature incubator at 12 ℃ for culturing for several days, and the filter paper is kept wet in the period. And counting the germination rate every 24 hours. And photographing and counting the survival rate after the seeds are all germinated.

The results are shown in FIG. 6, where Panel A is a representation of seed germination results for control and ZmICE1-OE overexpression in transgenic maize; b, the chart is the germination rate statistical data of the corn seeds at low temperature; where CK represents a control maize plant and OE represents a transgenic overexpression line. It can be seen that ZmICE1 overexpression can promote germination rate of corn seeds at low temperature.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggacgact cggcggaggt 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cattgcgttg tggaggccgg 20

Claims

The application of ZmICE1 protein or its coding gene, or the biological material containing its coding gene in improving the cold resistance of plant.
Application of ZmICE1 protein or its coding gene, or biological material containing its coding gene in breeding transgenic plant with improved cold resistance.
The application of ZmICE1 protein or its coding gene, or the biological material containing its coding gene in improving cold-resistant germplasm resources of plants.
The application of the ZmICE1 protein or the coding gene thereof, or the biological material containing the coding gene thereof in improving the survival rate of plants in a low-temperature environment.
5. The use of any one of claims 1 to 4, wherein the ZmICE1 protein has an amino acid sequence selected from any one of the following:

1) an amino acid sequence shown as SEQ ID NO. 2; or

2) The amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2.
6. The use of any one of claims 1 to 4, wherein the cDNA of the ZmICE1 protein has a nucleotide sequence of any one of:

(1) a nucleotide sequence shown as SEQ ID NO. 1; or

(2) The coding nucleotide sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 1; or

(3) A nucleotide sequence which can be hybridized with the nucleotide sequence shown in SEQ ID NO.1 under strict conditions.
7. The use according to any one of claims 1 to 4, wherein the biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.
8. The use according to any one of claims 1 to 4, wherein the plant is a dicotyledonous plant or a monocotyledonous plant; preferably rice, wheat, soybean, sorghum, millet, cotton, barley or corn.
9. The method for constructing cold-resistant transgenic corn is characterized in that the ZmICE1 gene is expressed or over-expressed in the corn by a transgenic, crossing, backcross, selfing or asexual propagation method.
10. The method of claim 9, wherein said transgene comprises introducing a recombinant expression vector comprising the ZmICE1 gene into maize using Ti plasmid, plant viral vector, direct DNA transformation, microinjection, gene gun, conductance, agrobacterium-mediated methods to obtain transgenic maize lines.