CN117756900A - Application of corn HSF21 protein in improving cold tolerance of plants - Google Patents
Application of corn HSF21 protein in improving cold tolerance of plants Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and particularly discloses application of corn HSF21 protein in improving cold tolerance of plants. The invention discovers that the overexpression of the HSF21 gene in corn can enhance the low temperature resistance of plants, and further provides the application of the corn HSF21 protein or the coding gene thereof or the biological material containing the coding gene thereof in any one of the following aspects: (1) altering the cold tolerance of plants; (2) improving survival rate of plants in low temperature environment; (3) breeding transgenic plants with improved cold tolerance; (4) improving cold tolerant germplasm resources of plants. The invention provides a new gene resource for cultivating new varieties of low-temperature resistant plants.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of corn HSF21 protein in improving cold tolerance of plants.
Background
Corn (Zea mays l.) is an economic crop originating in tropical low latitude regions, and although it gradually enters high latitude and high altitude temperate regions during human domestication and planting, corn is still very sensitive to low temperature cold damage, so that cultivation of cold-resistant and cold-resistant corn is still necessary when expanding corn planting in temperate regions. The sensitivity of corn to low temperatures is mainly due to reduced photosynthesis and metabolic disorders of its own. Short term exposure of maize seedlings to low temperatures can lead to reduced photosynthesis activity, with subsequent involvement of dissipative mechanisms and antioxidant systems, affecting assimilation transport.
The existing transgenic technology can introduce stress resistance genes of plants into corn genetic materials to be improved, and enable the plants to represent stable genetic stress resistance capacity, so that excellent variety resources are provided for agricultural production. The research of new genes for regulating and controlling the cold resistance of the corn has great significance for corn breeding and post-production.
Disclosure of Invention
The invention aims to provide a novel application of HSF21 protein in cold resistance regulation and control of corn.
The invention provides application of a corn cold-resistant gene HSF21 and a coded protein thereof. Corn HSF21 has the highest homology to HSFB1 in arabidopsis, but HSFB1 in arabidopsis has not been found to have a low temperature phenotype. According to the invention, through research on the maize cold related gene HSF21, the transgenic plant over-expressing the gene has an obvious cold-resistant phenotype compared with a wild plant. The invention provides a new gene resource for cultivating a new variety of cold-resistant plants.
The invention provides an application of HSF21 protein and a coding gene thereof in cold resistance and cold resistance of corn. In order to discover the related genes of cold resistance and cold resistance of corn, the invention screens corn libraries of transgenic overexpression lines, observes the phenotypes of the transgenic overexpression lines, discovers that the phenotypes of different overexpression gene lines are different, and further discovers that a plurality of lines of overexpression HSF21 genes all show obvious cold resistance phenotypes.
Specifically, the invention screens the over-expression corn population, performs preliminary screening of low-temperature phenotype by taking the relative injury area of leaf blades as an index, performs re-screening of the over-expression strain with phenotype screened at first, determines the low-temperature related phenotype, discovers that the gene number of the over-expression gene of the strain is GRMZM2G139535 by consulting the over-expression information table, further determines the over-expression gene as corn transcription factor HSF21 according to the gene annotation on the MaizeGDB website, and uniformly compares the over-expression gene to discover that the HSF21 belongs to the class B HSF transcription factor, but the functions of the over-expression gene are not reported at all. The research of the invention confirms that the HSF21 gene is probably a key gene for cold resistance and cold resistance of corn. Furthermore, the invention obtains the cold-resistant and cold-resistant transgenic plant by over-expressing the HSF21 gene in the corn.
The cDNA sequence of the corn HSF21 protein related in the invention is as follows: i) A nucleotide sequence shown as SEQ ID No. 1; or ii) the nucleotide sequence shown as SEQ ID No.1 is substituted, deleted and/or added with one or more nucleotides and expresses the same functional protein; or iii) a nucleotide sequence which is fully complementary to the nucleotide sequence shown in SEQ ID NO. 1.
The corn HSF21 cDNA consists of 1671 bases and has a sequence shown in SEQ ID No. 1. The gene reading frame consists of 2 exons. The amino acid sequence coded by the corn HSF21 gene is shown in SEQ ID No. 2.
The corn HSF21 protein provided by the invention has any one of the following amino acid sequences:
1) An amino acid sequence shown in SEQ ID NO. 2;
2) The amino acid sequence shown in SEQ ID NO.2 is obtained by replacing, deleting or inserting one or more amino acid residues to obtain the amino acid sequence of the protein with the same function.
It will be appreciated that one skilled in the art can, based on the amino acid sequences disclosed herein, substitute, delete and/or add one or more amino acids to obtain mutant sequences of the protein without affecting its activity.
The invention provides the use of a maize HSF21 protein or gene encoding the same, or a biological material containing the same, in any of the following aspects:
(1) Changing the cold resistance of the plants;
(2) The survival rate of the plants in a low-temperature environment is improved;
(3) Selecting transgenic plants with improved cold tolerance;
(4) Improving cold-resistant germplasm resources of plants.
Preferably, the plant cold tolerance is modified to increase plant cold tolerance.
The biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.
The invention also provides cloning vectors or various expression vectors containing the plant low temperature resistant HSF21 gene sequence or fragments thereof, host cells containing the vectors, transformed plant cells and transgenic plants containing the gene sequence or specific fragments thereof. Wherein, the overexpression vector containing the HSF21 gene is a pBCXUN vector containing a Ubi promoter.
The invention also provides a preparation method of the transgenic plant, and the expression quantity of the HSF21 gene is improved by the transgenic method to obtain the plant with improved cold resistance.
The preparation method of the transgenic plant comprises the following steps:
(1) Amplifying the full-length gene cDNA sequence of the HSF21 gene (shown as SEQ ID NO. 1);
(2) Constructing an overexpression vector of the HSF21 gene;
(3) Constructing recombinant agrobacterium containing an overexpression vector of the HSF21 gene;
(4) And constructing a transgenic plant with the HSF21 gene over-expressed by adopting an agrobacterium infection method.
The plant of the present invention is a monocotyledonous or dicotyledonous plant, preferably rice, wheat, soybean, sorghum, millet, cotton, barley or maize, more preferably maize.
The invention also provides a method for changing the low temperature resistance of plants, which controls the expression of the maize HSF21 gene by the plants through transgenic, crossing, backcrossing, selfing or asexual propagation methods.
The transgenic plant line is obtained by silencing expression or reducing expression quantity of the corn HSF21 gene by utilizing a DNA homologous recombination technology, a Cre/Loxp technology and a Crispr/Gas9 technology.
The transgenic method also comprises the steps of introducing a recombinant expression vector containing the corn HSF21 gene into corn by utilizing Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, conductance and agrobacterium-mediated method to obtain a transgenic corn strain.
As one 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 an HSF21 gene by using the cDNA as a template through a primer, constructing an amplified product on an expression vector pBCXUN, and obtaining a recombinant expression vector named pBCXUN-HSF21;
2) Agrobacterium EHA105 was transformed with pBCXUN-HSF21, and then maize callus was infected with the transformed agrobacterium to obtain low temperature resistant transgenic maize seedlings.
Wherein the nucleotide sequence of the primer in the step 1) is shown as SEQ ID No.3 and 4.
The maize being infested is preferably a maize plant of the LH244 homozygous genotype. After overexpression of the HSF21 gene of the present invention, corn exhibits a phenotype of low temperature resistance.
The expression vector is a pBCXUN vector which is modified from a plasmid pCAMBIA1300 to be obtained by connecting a hygromycin resistance gene into the pCAMBIA 1300.
The invention clones HSF21 genes, constructs an over-expression transgenic plant of the HSF21 and Crispr/Cas9 mutant materials, verifies that the HSF21 participates in regulating and controlling the cold resistance of corn, and the over-expression of the HSF21 can lead the corn to obtain stronger low-temperature tolerance. The invention provides new gene resources for cultivating new varieties of low-temperature-resistant plants, and lays a certain theoretical foundation for researching the mechanism of responding to low-temperature stress of corns.
Drawings
FIG. 1 is a graph showing the results of an HSF21 gene overexpression test of the WT group and maize overexpression line in example 2 of the present invention; in the figure, P <0.001 is represented.
FIG. 2 is a photograph showing plant growth after recovery from low temperature treatment of WT group and maize overexpressing strain in example 3 of the present invention;
FIG. 3 is a graph showing the ion leakage rate statistics of WT and maize over-expression lines in example 3 of the present invention.
FIG. 4 is a schematic representation of 2 types of HSF21 gene knockouts in example 4 of the present invention;
FIG. 5 is a photograph showing plant growth after recovery from low temperature treatment of WT group and maize HSF21 knockout line in example 4 of the present invention;
FIG. 6 is a graph showing the ion leakage rate statistics of WT group and maize HSF21 knockout strain in example 4 of the present invention.
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 following examples are illustrative of the invention and are not intended to limit the scope of the invention. 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.
The main reagents in the following examples were: various restriction enzymes, taq DNA polymerase, T4 ligase, pyrobest Taq enzyme, KOD from NEB, toyobo and other biological companies; 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; the reagents used for real-time quantitative PCR were purchased from TaKaRa, and the various other chemical reagents used in the examples were all imported or custom analytical pure reagents. The primers used in the examples were synthesized by Hexakuda and subjected to related sequencing.
EXAMPLE 1 construction and detection of HSF21 Gene overexpression vector
Total RNA is extracted from B73 corn (Zea mays L.), cDNA is obtained by reverse transcription, and the cDNA is used as a template, F and R are used as primers, the HSF21 gene is amplified, and the primers are provided with enzyme cutting sites and are connected to an over-expression vector after enzyme cutting. The construction method of the HSF21 gene overexpression vector comprises the following steps:
(1) The total RNA of B73 corn is extracted by using an RNA extraction kit of Magen company, and specific steps are referred to a kit instruction.
(2) The RNA was reverse transcribed into cDNA using a reverse transcription kit from thermo company, and the specific procedure was referred to the kit instructions.
(3) Using corn cDNA as template, F and R as primer, amplifying cDNA of HSF21 (shown as SEQ ID NO.1, its coding amino acid sequence is shown as SEQ ID NO. 2), recovering amplified product by electrophoresis gel cutting, and the recovery method is referred to the instruction book of Tiangen company kit.
The primers used to amplify the HSF21 gene cDNA were:
upstream primer F:5'-ATGGGAGAAGCGGCCGCGGC-3' (SEQ ID No. 3);
the downstream primer R:5'-ATTCTCGCCGCCGCACCGGC-3' (SEQ ID No. 4).
(4) The recovered HSF21 gene cDNA and pBCXUN vector are digested with Xba I and Cla I, and the digested products are subjected to electrophoresis and gel cutting for recovery. The recovered product was ligated with T4 ligase. The HSF21 gene was ligated into the pBCXUN vector, and expression of the HSF21 gene was driven with the Ubi promoter.
The pBCXUN vector is obtained by ligating a hygromycin resistance gene into pCAMBIA1300 by using a commercialized vector pCAMBIA1300 as a skeleton (Guo et al, 2018Stepwis cis-regulatory changes in ZCN8 contribute to maize flowering-time adaptation.Current Bio.28, 3005-3015); meanwhile, the promoter of the corn ubiquitin gene Ubi is cloned to a vector in an enzyme digestion connection mode to drive transcription of a downstream over-expressed gene.
(5) Taking 5 mu L of the product of the enzyme digestion-connection system, and converting the E.coli competence. Screening was performed on LB plates containing 50. Mu.g/mL kanamycin. Colony PCR identifies single clone, and positive clone is selected for sequencing. The obtained recombinant expression vector with correct sequencing was named pBCXUN-HSF21. And (3) carrying out electrophoresis detection after enzyme digestion of the plasmid obtained in the last step, wherein the specific method comprises the following steps: pBCXUN-HSF21 was digested with Xba I and Cla I, electrophoresed on a 1% agarose gel at 120V,50mA, and scanned and imaged by a UVP Gel Documentation gel analysis system.
EXAMPLE 2 construction and detection of HSF21 Gene overexpressing plants
The pBCXUN vector containing the HSF21 gene described in example 1 was transformed into Agrobacterium EHA105 strain (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) and maize LH244 callus was again infested to give transgenic seedlings. The specific method comprises the following steps: inoculating agrobacterium containing target 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, centrifuging at room temperature for 15min at 50g until OD600 value is 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. And then screening, and carrying out a low-temperature stress treatment experiment on the obtained seeds.
In this example, over-expression lines OE-1 and OE-2 with high expression levels were isolated. In particular, the gene expression of HSF21 in the obtained expressed strains OE-1 and OE-2 is detected by adopting real-time quantitative PCR. The specific method comprises the following steps:
1) Extracting total RNA of plants, and carrying out reverse transcription to obtain cDNA.
2) After 5-fold dilution of the cDNA obtained by reverse transcription, real-time quantitative PCR was performed using a Takara kit, using a reaction system comprising: 2X SYBR Premix ExTaq buffer, 0.2. Mu.L DyII, 0.4. Mu.L Primer (F1/R1), 2. Mu.L cDNA template, and finally ddH 2 O is filled to 20 mu L, and the mixture is put into an ABIPRISM 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.
Wherein, the sequences of the primers F1 and R1 (qRT-PCR primers) are as follows:
F1：5’-CTGACCAAGACGCACCAGAT-3’(SEQ ID No.5)；
R1：5’-GGAGGAGAAGTTGCAGTGCT-3’(SEQ ID No.6)。
after completion of the PCR reaction according to 2 -Δ(ΔCt) The principle of (a) is to calculate the relative expression quantity between the wild type (WT group) and the over-expression strain (OE) and to carry out the graphic analysis, three biological repetition and three biological repetitionThe trends are similar. Simultaneously with the amplification of the identified genes, each sample was amplified simultaneously with the UBI gene as an internal reference. The test results are shown in FIG. 1, and as can be seen from FIG. 1, the expression level of the over-expressed strain is significantly higher than that of the WT control group.
EXAMPLE 3 detection of the Cold resistance of plants overexpressing the HSF21 Gene
Seeds of the WT group (wild type corn) and OE-1 and OE-2 obtained in example 2 were first sown in small pots containing black soil, imported soil and vermiculite (mass ratio 1:1:1) 10cm long, 10cm wide and 10cm high, 5 grains were placed in each pot, 2cm soil was again placed in a tray, watered until the soil was completely wet, placed in a culture chamber at 23℃for 16 hours of light and 8 hours of darkness. After 14 days of growth, 4 ℃ low temperature treatment is carried out for 4 days until the second leaf is wilted, the second leaf is taken out and put into a 23 ℃ culture room for two days of recovery, and then photographing and taking materials are carried out for statistics of ion leakage rate. Three seedlings were taken for each over-expressed strain (OE) and wild-type (WT) and assayed and three biological replicates were performed.
Representative photographs of plant growth after recovery of the WT and maize overexpressing lines from low temperature treatment are shown in FIG. 2 (left panel is control group without low temperature treatment, right panel is experimental group after recovery from low temperature treatment). The results show that wild type WT leaves severely wilt, dry up and even fail to stand, whereas the over-expressed strains OE-1 and OE-2 had only slightly injured leaf tips and remained straight, exhibiting a low temperature resistant phenotype.
The statistics of ion leakage rate in this example were performed by measuring the relative conductivity l= (S1-S0)/(S2-S0) ×100% 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 Table 1, and the ion leakage rate of the over-expressed strain OE-1 and OE-2 is reduced by 41.6% and 41.9%, respectively, compared to the wild-type WT plants (average value of the three times of ion leakage rate differences between the wild-type WT plants and the over-expressed strain), and a significant difference, P <0.05, is achieved, indicating that over-expression of the HSF21 gene can result in enhanced cold resistance of maize.
TABLE 1 ion leakage Rate values (%)
| WT | OE-1 | OE-2 |
| 72.59425 | 31.93767 | 33.469953 |
| 79.60829 | 37.30582 | 37.022639 |
| 67.67797 | 25.72814 | 23.825957 |
Example 4 detection of the Cold resistance of HSF21 Gene knockout plants
To further explore the regulatory effect of HSF21 on maize cold tolerance, gene knockout was performed on the HSF21 gene of wild maize LH244 using CRISPR/Cas9 technology. 2 types of mutant strains of mutant form-hsf 21-1 strain and hsf21-2 strain-were obtained in total. The schematic of knockout is shown in fig. 4. Wherein, the HSF21-1 strain lacks 43bp from 79 th to 121 th of the first exon of the HSF21 gene, and the HSF21-2 strain lacks 22bp from 2313 rd to 2334 th of the second exon of the HSF21 gene.
The wild WT group and the gene knockout lines hsf21-1 and hsf21-2 are subjected to low temperature treatment in the following specific experimental mode: seeds of WT group (wild type corn) and gene knockout lines hsf21-1 and hsf21-2 were sown in small pots containing black soil, imported soil and vermiculite (mass ratio 1:1:1) 10cm long, 10cm wide and 10cm high, 5 grains were placed in each pot, 2cm soil was covered again, placed in a tray, watered until the soil was completely wet, placed in a 23℃incubator, illuminated for 16 hours, darkened for 8 hours, subjected to 4℃low temperature treatment for 3 days after 14 days of growth, then transferred to a 23℃incubator for two days of recovery, photographed and the materials were taken for statistics of ion leakage rate. Three seedlings were taken for each knockout line and WT and assayed and three biological replicates were performed.
Representative photographs of plant growth after recovery of the WT and hsf21 knockout lines from low temperature treatment are shown in FIG. 5 (left panel is a control group without low temperature treatment, right panel is an experimental group after recovery from low temperature treatment). The results show that wild type WT had only slightly injured leaf tips and remained straight, while hsf21 knockout line leaves were severely wilted, and were dry rolled up and even unable to stand, exhibiting a cold-sensitive phenotype.
The statistics of ion leakage rate in this example were performed by measuring the relative conductivity l= (S1-S0)/(S2-S0) ×100% 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 ion leakage rate reflects the integrity of the cell membrane, the test results are shown in fig. 6 and table 2, compared with the WT group, the ion leakage rate of the knockdown lines HSF21-1 and HSF21-2 is respectively increased by 46.7% and 47.8% (the average value of the ion leakage rate difference between the three wild WT plants and the knockdown lines) to reach a significant difference, P <0.05, which indicates that the damage degree of the cells is obviously higher than that of the WT group, and the knockdown of the HSF21 gene can reduce the cold tolerance of corn. Further illustrated are the susceptibility of HSF21 knockout lines (HSF 21-1 and HSF 21-2) to low temperatures.
TABLE 2 ion leakage Rate values (%)
| WT | hsf21-1 | hsf21-2 |
| 23.81285 | 66.25987 | 69.62581 |
| 27.60788 | 83.53274 | 80.20139 |
| 34.2886 | 76.07954 | 79.18962 |
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one 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.
Claims (9)
1. Use of a maize HSF21 protein or gene encoding the same, or a biological material containing the gene encoding the same, in any of the following aspects:
(1) Changing the cold resistance of the plants;
(2) The survival rate of the plants in a low-temperature environment is improved;
(3) Selecting transgenic plants with improved cold tolerance;
(4) Improving cold-resistant germplasm resources of plants.
2. The use of claim 1, wherein altering cold tolerance in a plant comprises improving cold tolerance in a plant.
3. The use according to claim 1 or 2, wherein the maize HSF21 protein has the amino acid sequence shown in SEQ ID No. 2.
4. The use according to claim 1 or 2, wherein the cDNA of the maize HSF21 protein has any one of the following nucleotide sequences:
(1) The nucleotide sequence shown in SEQ ID NO.1, or
(2) A nucleotide sequence fully complementary to the nucleotide sequence shown in SEQ ID NO. 1.
5. The use according to claim 1 or 2, wherein the biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.
6. The use according to claim 1 or 2, wherein the plant is a monocotyledonous plant or a dicotyledonous plant; preferably rice, wheat, soybean, sorghum, millet, cotton, barley or maize.
7. A method for modifying the low temperature resistance of a plant, characterized in that the expression of the maize HSF21 gene in the plant is controlled by means of transgenesis, crossing, backcrossing, selfing or asexual propagation.
8. The method of claim 7, wherein the transgenic plant line is obtained by silencing expression or reducing expression of the maize HSF21 gene using DNA homologous recombination technology, cre/Loxp technology, crispr/Gas9 technology.
9. The method of claim 7, wherein said transgenic comprises introducing a recombinant expression vector comprising said maize HSF21 gene into maize using Ti plasmid, plant viral vector, direct DNA transformation, microinjection, gene gun, conductance, agrobacterium-mediated method, to obtain a transgenic maize line.
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