CN110184253B

CN110184253B - Application of CiCPK32 gene of caragana intermedia in regulation and control of plant stress resistance

Info

Publication number: CN110184253B
Application number: CN201910491540.7A
Authority: CN
Inventors: 李国婧; 于秀敏; 王瑞刚; 杨飞芸; 丛靖宇; 红格日其其格; 杨天瑞
Original assignee: Inner Mongolia Agricultural University
Current assignee: Inner Mongolia Agricultural University
Priority date: 2019-06-06
Filing date: 2019-06-06
Publication date: 2022-06-07
Anticipated expiration: 2039-06-06
Also published as: CN110184253A

Abstract

The invention relates to the field of biological genetic engineering, in particular to a CiCPK32 gene of an intermediate caragana, and also relates to a functional research of the gene and an application of the gene in regulation and control of plant stress resistance. The invention utilizes the transcriptome database of middle caragana to clone the CiCPK32 gene related to abiotic stress from the middle caragana, after the gene is over-expressed in arabidopsis thaliana, the stress treatment such as drought, high salt and ABA is carried out, the functions of the gene are revealed, and theoretical basis and support are provided for the application of the gene in improving the stress tolerance of agricultural and forestry grass plants in the future.

Description

Application of CiCPK32 gene of caragana intermedia in regulation and control of plant stress resistance

Technical Field

The invention relates to the field of biological genetic engineering, in particular to a CiCPK32 gene of an intermediate caragana, and also relates to a functional research of the gene and an application of the gene in regulation and control of plant stress resistance.

Background

Environmental conditions such as low temperature, drought, land salinization and the like are main adversity stress factors influencing the growth and development of plants, and the research on physiological and biochemical changes of the plants under the adversity stress and the adaptability to the adversity is a research hotspot in recent years. The key problem to be solved urgently is to explore how to improve the stress resistance of plants. Plants can make timely regulation on the levels of molecules, cells, organs, physiology, biochemistry and the like in order to adapt to the stress environment. Along with the evolution of organisms, a complex signal transmission system is generated in plants, so that the stress resistance of the plants is improved. Calcium ion is a second messenger in the cell, is an important signal transduction factor in plants, is involved in plant growth and development, immune defense and hormone regulation, and is responsive to light and various stress signals. Currently, there are three calcium receptor proteins in plants, calmodulin, calcineurin B-like protein (CDPK), and calcineurin, of which calcineurin is the focus of current research. The calcium-dependent protein kinase is one of the main primary calcium ion receptors and is also Ca²⁺The response protein is widely distributed in plants, plays an important role in the plant calcium signal transduction process and plays an important role in the plant response stress signal transmission process.

CDPK is a family of serine/threonine-type protein kinases specific to plants and protists that have not been found in animals, all of which are referred to as: calcium-dependent and Calmodulin-independent protein kinases (calcum-dependent and Calmodulin-independent protein kinases) or Calmodulin-like domain protein kinases (calponin-like protein kinases) are special Calcium ion binding proteins. The protein was first reported in pea (Pisum sativum) and was first purified and characterized in soybean (Glycine max). With the continuous and intensive research on CDPK, CDPK is found to be a large gene family, and CDPK proteins are identified in various plants such as rice (Oryza sativa), wheat (Triticum aestivum), Arabidopsis (Arabidopsis thaliana), poplar (populus trichocarpa), and grape (Vittis viifera) at present, for example, 34 CDPK family members have been identified in the Arabidopsis genome of a model plant, each of which is located on five chromosomes, wherein CPK2/17/20/34 has been shown to be involved in pollen tube elongation; furthermore, early studies showed that the two genes PiCPK1 and PiCPK2 of petunia also may be involved in the regulation of pollen tube growth, the arabidopsis CPK28 gene is considered to be a regulator of Gibberellin (GA) and Jasmonic acid (Jasmonic acid, JA), CPK4 and CPK11 play an important role in ethylene biosynthesis, CPK21 and CPK23 play a negative regulatory role in the salt and drought tolerance pathways of plants, the gene deletion enhances tolerance to corresponding abiotic stresses, whereas over-expressed lines show opposite phenotypes. In rice, 31 CDPK family members have been identified so far, 50 CDPK gene members are found in soybean, 40 CDPK genes are cloned in maize (Zea mays), 20 CDPK genes are found in common wheat and poplar respectively, 25 CDPK genes are identified in the tribulus alfalfa (Medicago truncatula) genome, 29 CDPK genes are found in millet (Setaria italica), 21 CDPK genes are identified in potato (Solanum turberosum), and CDPK genes are identified in melon (cucumis melo), apple (Malus pulima Millapple), grape and peanut (arachi hypogoae). With the continuous and intensive research, CDPK is found to be involved in the regulation of flowering phase, the development of plant root systems, the synthesis of hormones and the regulation of signal pathways, and simultaneously play an important role in regulating and controlling cell differentiation and programmed death, for example, StCDPK5 transgenic potatoes have enhanced resistance to late blight pathogens.

Caragana intermedia (Caragana inermedia) belongs to the family leguminosae, Caragana, perennial shrubs. The root system is developed, the underground root is nearly 5 meters, the stem is upright or obliquely upward, and the plant height is 1-2 meters; feathery compound leaves, 12-16 small leaves, inverted oval or elliptical, and most of the feather is fur; the middle caragana begins to grow in 4 middle of the month, the flowering phase is 5 middle of the month, the corolla is butterfly-shaped and yellow, the fruit phase is 6 months, the seeds are kidney-shaped, and the caragana carinata has two colors of light green brown and yellow brown. The plant is widely distributed in regions such as inner Mongolia, Shanxi, Ningxia and Shanxi in China, has the characteristics of drought resistance, high temperature resistance, salt and alkali resistance, cold resistance and the like, and is an excellent plant for preventing wind, fixing sand and maintaining water and soil in arid and desert regions; in addition, the middle caragana can also be used as raw materials of feed, fuel, paper making, fertilizer and plates, and also has medicinal value. Other extreme environments such as drought, high salinity and freezing seriously affect the growth and development of plants and the yield of crops. Plants can flexibly and actively avoid various adversity stresses unlike animals, so in order to adapt to various unfavorable growth environments, the plants gradually evolve to form a series of complex stress resistance mechanisms, thereby enhancing the stress resistance of the plants. CDPKs are considered to be important regulators of various plant signal transduction pathways downstream of cytoplasmic calcium ions, and may play a role in enhancing plant stress resistance. The invention clones a CiCPK32 gene related to stress resistance from caragana intermedia, preliminarily studies the functions of the gene and explores the stress resistance mechanism of the gene from various levels such as molecules, physiology and the like.

Disclosure of Invention

The invention utilizes the transcriptome database of middle caragana to clone the CiCPK32 gene related to abiotic stress from the middle caragana, after the gene is over-expressed in arabidopsis thaliana, the stress treatment such as drought, high salt and abscisic acid (ABA) is carried out, the function of the gene is revealed, and theoretical basis and support are provided for the future application of the gene in improving the stress tolerance of agricultural and forestry plants.

The first purpose of the invention is to provide a CiCPK32 gene of an intermediate caragana capable of increasing the stress resistance of plants and a protein coded by the CiCPK32 gene.

The coded amino acid sequence of the middle caragana CiCPK32 gene is shown as SEQ ID NO.1 in a sequence table, and the gene nucleotide sequence is shown as SEQ ID NO.2 in the sequence table.

Another object of the invention is to provide an application of the middle caragana CiCPK32 in improving the stress resistance of plants. In particular, the stress resistance refers to resistance to osmotic stress, drought stress, salt stress and ABA stress.

The recombinant expression vector is inserted with a CiCPK32 gene, wherein the CiCPK32 gene is a nucleotide sequence shown as SEQ ID NO.2, or a nucleotide sequence which is complementary and matched with the SEQ ID NO.2, or a nucleotide sequence which codes SEQ ID NO.1 protein.

The transient and stable expression vector is inserted with CiCPK32 gene, the CiCPK32 gene is the nucleotide sequence shown in SEQ ID NO.2, or the nucleotide sequence complementary and matched with SEQ ID NO.2, or the nucleotide sequence coding SEQ ID NO. 1.

The overexpression and interference expression vector is inserted with a CiCPK32 gene, and the CiCPK32 gene is a nucleotide sequence shown as SEQ ID NO.2, or a nucleotide sequence which is complementary and matched with the SEQ ID NO.2, or a nucleotide sequence which codes the SEQ ID NO. 1.

Another objective of the invention is to provide the application of the CiCPK32 gene in plant breeding.

The CiCPK32 gene, or its recombinant expression vector, or its transient expression and stable expression vector, or its overexpression and interference expression vector is applied in genetic breeding for improving plant resistance, the CiCPK32 gene is the nucleotide sequence shown in SEQ ID NO.2, or the nucleotide sequence complementary and paired with SEQ ID NO.2, or the nucleotide sequence coding SEQ ID NO. 1.

The other purpose of the invention is to apply the middle caragana CiCPK32 gene to plant genetic engineering to obtain a transgenic plant with overexpression and interference of CiCPK32, wherein the transgenic plant is used for changing the stress resistance of plants, and particularly the stress resistance refers to osmotic stress resistance, drought stress resistance, salt stress resistance and ABA stress resistance.

Another object of the present invention is to provide a biological agent for improving the stress resistance of plants, particularly, the stress resistance refers to osmotic stress resistance, drought stress resistance, salt stress resistance and ABA stress resistance, and preferably refers to drought stress resistance, salt stress resistance and ABA stress resistance.

A biological agent for improving the stress resistance of plants is characterized in that the active ingredients of the biological agent are derived from a recombinant expression vector or an overexpression and interference expression vector of CiCPK32, or the active ingredients of the biological agent contain a biological product for regulating the expression of CiCPK32 gene, and the CiCPK32 gene is a nucleotide sequence shown as SEQ ID No.2, or a nucleotide sequence which is complementary and matched with the SEQ ID No.2, or a nucleotide sequence which codes the SEQ ID No. 1.

Another object of the present invention is to provide a method for regulating the stress resistance of plants, particularly, the stress resistance refers to osmotic stress resistance, drought stress resistance, salt stress resistance and ABA stress resistance.

A method for regulating plant stress resistance comprises regulating CiCPK32 gene expression, wherein the CiCPK32 gene is a nucleotide sequence shown as SEQ ID NO.2, or a nucleotide sequence complementary and matched with SEQ ID NO.2, or a nucleotide sequence coding SEQ ID NO. 1.

The invention constructs an overexpression and interference expression vector of CiCPK32, transforms the overexpression and interference expression vector to a normal plant variety by a genetic transformation method of an agrobacterium-mediated method, and finally obtains a transgenic plant of the overexpression and interference expression of CiCPK 32.

In particular, the plant is preferably a terrestrial plant. The terrestrial plant may be a dicot and/or a monocot. The dicotyledonous plant can be a cruciferous plant; the cruciferous plant may be arabidopsis thaliana. In a particular embodiment, the plant is preferably a leguminous caragana plant, which may be an intermediate caragana.

In the invention, a middle caragana CiCPK32 obtained by us and applied exploration illustrate the following application modes of the gene:

(1) the middle caragana CiCPK32 gene is obtained by cloning by using RACE technology.

(2) The CiCPK32 gene is induced by drought, NaCl and ABA.

(3) Seed germination rate of CiCPK32 over-expression line was higher than control under treatment with 100mM NaCl, 400mM mannitol, or 0.4 μm ABA.

(4) Under dehydration treatment, the water loss rate of each strain of over-expression CiCPK32 gene is obviously lower than that of a control; after drought treatment, the survival rate of the over-expression strain is obviously higher than that of the wild type.

(5) Under ABA treatment, the expression quantity of stress-related genes in strains over-expressing CiCPK32 genes is obviously higher than that of wild type.

The invention has the following beneficial effects: the CiCPK32 gene related to abiotic stress is cloned from the caragana intermedia, and the stress resistance function of the CiCPK32 gene is disclosed, so that a direction is provided for the application of the CiCPK32 gene in improving the stress tolerance capability of agricultural and forestry plants in the future.

Drawings

FIG. 1: technical route diagrams of the present invention.

FIG. 2 is a schematic diagram: electrophoresis of full-length cDNA clones of CiCPK32 gene (A: CiCPK 323 'RACE B: CiCPK 325' RACE C: CiCPK32 ORF).

FIG. 3: the evolutionary relationship of CiCPK32 to CDPK of other species (amino acid accession numbers in parentheses; numbers on branches indicate the percentage of confidence in Bootstrap validation based on 1000 repeats of the node; scale represents evolutionary distance).

FIG. 4 is a schematic view of: phylogenetic analysis of CiCPK32 and Arabidopsis CDPKs (different colored branches represent members of different subfamilies, blue: subfamily I; purple: subfamily II; green: subfamily III; red: subfamily IV).

FIG. 5: analysis of the expression pattern of CiCPK32 under different treatments.

FIG. 6: screening of transgenic homozygote seedlings of CiCPK 32.

FIG. 7: and (3) identifying the expression level of the CiCPK32 transgenic line.

FIG. 8: detection of germination rates of control and CiCPK32 overexpression strains under mannitol treatment; a: counting the germination rate of each strain on a normal 1/2MS culture medium; b: statistics of germination rates of individual lines on 1/2MS medium containing 400mM Mannitol.

FIG. 9: detecting germination rates of control and CiCPK32 overexpression strains under NaCl treatment; a and C: statistics of germination and germination rate of each strain on normal 1/2MS medium (photograph of growth 2 d); b and D: statistics of germination and germination rate (photograph of growth 2 d) of each strain on 1/2MS medium containing 100mM NaCl.

FIG. 10: drought resistance and water loss rate analysis of control and CiCPK32 overexpression strains; a, seedlings 2 weeks old before drought stress treatment (upper panel), seedlings 10 days after drought stress treatment (middle panel) and seedlings 3 days after rehydration (lower panel); b, counting the survival rate of each strain; c, counting the water loss rate of the overground part of each strain; denotes P < 0.01.

FIG. 11: detecting germination rates of wild type and CiCPK32 overexpression strains under ABA treatment; a and C: statistics of germination and germination rate of each strain on normal 1/2MS medium (photograph of growth 7 d); b and D: statistics of germination and germination rate (photograph of 7d grown) for each line on 1/2MS medium containing 0.4. mu.M ABA.

FIG. 12: comparing the length of the main root of an Arabidopsis seedling with a control seedling by overexpression of CiCPK32 under ABA treatment; a and C: root growth status and statistics of each strain on normal 1/2MS medium (photograph of growth 10 d); b and D: root length status and statistics (10 d photos of growth) of each line on 1/2MS medium containing 12. mu.M ABA; p <0.05, P <0.01, P < 0.001.

FIG. 13: and (3) detecting the expression quantity of ABA signal pathway genes and stress response genes of different lines of Arabidopsis.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. A specific technical scheme is shown in fig. 1.

Example 1 cloning of the Gene encoding CiCPK32 of the intermediate Caragana

1.1 RACE method for amplifying CiCPK32 gene 3' end sequence

An EST sequence of a CiCPK32 gene which is homologous with Arabidopsis thaliana CPK32 is obtained by screening from a middle caragana drought transcriptome database, the length of the segment is 566bp, and the 3 'end and the 5' end of the gene sequence are both incomplete when NCBI finds through sequence comparison. Designing primers of Outer and Inner according to the requirements of a 3' RACE kit: CiCDPK32-3 '-out and CiCDPK 32-3' -in, and the specific primer sequences are as follows:

1.2 RACE method for amplifying CiCPK32 gene 5' end sequence

According to the requirements of a Smarter RACE cDNA amplification Kit, a 5' RACE primer is designed, and the sequence of the primer is as follows:

the kit is provided with a 5 'UPM primer, and the step of amplifying the 5' terminal sequence is carried out according to the kit instruction.

1.3 cloning of the coding region of the CiCPK32 Gene

Sequencing the RACE amplification results of 1.1 and 1.2 by Shanghai bio-engineering company, splicing by Vector NTI10 software to obtain a full-length cDNA sequence of the CiCPK32 gene, and analyzing to obtain a complete open reading coding frame (ORF) sequence. According to the ORF sequence, primers were designed: HA-CiCPK32-sense and HA-CiCPK 32-anti:

taking cDNA of caragana intermedia as a template and utilizing high fidelity enzyme PrimeSTAR^HSThe DNA Polymerase amplifies the ORF of the gene of interest. The annealing temperature was 63 ℃, the extension time was 1.5min, and the number of reaction cycles was 30.

As can be seen, the sequence of 1196bp 5 'end was obtained by 5' RACE amplification, and the sequence of 854bp 3 'end was obtained by 3' RACE amplification (FIG. 2). After sequencing, the sequence obtained by amplification of RACE technology is spliced with the EST sequence obtained by screening through Vector NTI10 to obtain a cDNA full-length sequence. The middle caragana cDNA is taken as a template, a specific primer is designed, ORF of the gene is cloned, and sequencing verification shows that the splicing is correct.

1.4 sequence analysis of the CiCPK32 Gene

The cDNA amplified from 1.3 is analyzed by using software, and the cDNA total length of the amplified CiCPK32 gene is 2074bp (SEQ ID NO.2), and consists of an Open Reading Frame (ORF)1566bp (SEQ ID NO.3), a non-coding region at the 3 'end and a non-coding region at the 5' end. The gene totally encodes 522 amino acids (SEQ ID NO.1), the initiation codon is ATG, and the termination codon is TAA. It was observed that having a gly (g) residue at the second position of CiCPK32, possibly a myristoylation site, and that the fourth and fifth positions are occupied by cys (c) residues, possibly a palmitoylation site, protein myristoylation is known to promote protein-to-membrane or protein-to-protein interactions, a structure that plays an important role in the membrane localization of CiCPK 32.

1.5 Gene bioinformatics analysis

To further understand the evolutionary relationship between CiCPK32 and CDPK of other species, the CDPK protein in soybean, chickpea, medicago truncatula, plum blossom (plum blossom), apple, grape, cotton, cacao (Theobroma cacao), arabidopsis thaliana, sweet orange (Citrus sinensis l. osb.), tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum), potato was selected by aligning the amino acid sequences encoded by the CiCDPK32 gene AT NCBI, and constructing an evolutionary tree (fig. 3) together with the CiCPK32 protein sequence by MAGE7.0, which was found to be 77% identical to arabidopsis thaliana AtCPK32(AT3G 57530). Through sequence analysis, the relation between the middle caragana CiCPK32 and the chickpea, the medicago truncatula and the soybean is close, the two are gathered on a large branch, and the credibility reaches 99%.

In order to further understand the functions of the CiCPK32 gene, the gene and protein sequences coded by 34 CPK genes found in Arabidopsis thaliana are subjected to phylogenetic analysis, a phylogenetic tree is constructed by applying MAGE7.0 (figure 4), and the fact that the CiCPK32 is close to the genetic relationship of the third subfamily of Arabidopsis thaliana and is clustered together is found, so that the gene belongs to the third subfamily of Arabidopsis thaliana. According to research reports, AtCDPK8 and AtCDPK10 of Arabidopsis thaliana subfamily III can enhance the resistance of plants to drought stress, and AtCPK32 controls the polar growth of pollen tubes, thereby providing a direction for the subsequent research of target genes.

Example 2 analysis of the expression characteristics of the CiCPK32 Gene of intermediate Caragana

In order to further determine whether the CiCDPK32 gene is induced by abiotic stress, caragana intermedia is treated under the conditions of dehydration, salt and ABA respectively, and qRT-PCR technology is applied to detect the change condition of the expression quantity of a target gene under different stress treatments.

Selecting seeds with full seeds and no worm damage from the caragana intermedia seeds, sowing the seeds in a plant culture pot containing mixed soil (nutrient soil: vermiculite: 1:3), and culturing under the following conditions: 22 ℃, 16h light/8 h dark, and 7000 and 8000lux light intensity. Selecting the caragana intermedia seedlings with consistent growth states and good development to carry out abscisic acid (ABA), salt and dehydration stress treatment, and taking the caragana intermedia seedlings as raw materials for researching different stress response conditions of target genes.

The detection result shows that the expression of CiCPK32 is rapidly induced after dehydration treatment, the expression level of the target gene reaches the highest level at 3h, which is 2.2 times of that of the target gene when the target gene is not treated, the expression level is basically kept unchanged at 6h, and then the expression level slightly decreases, but is still higher than that of the target gene when the target gene is not treated; after salt treatment, the expression level of the target gene is obviously reduced, the expression level is rapidly reduced to half of that of the untreated gene after 3 hours of treatment, the minimum expression level is reached after 6 hours of treatment, and the expression level is still obviously lower than that of the untreated gene after 12 hours of treatment; after ABA treatment, the expression level of the target gene is increased to 4 times that of the untreated gene at 6 h. The above test results indicate that the CiCPK32 gene may be involved in the response to dehydration, salt and ABA stress (FIG. 5).

Example 3 obtaining of transgenic Arabidopsis thaliana with CiCPK32 Gene and identification of stress resistance

3.1 construction of CiCPK32 Gene expression vector of caragana intermedia and transformation of Arabidopsis thaliana

(1) When cloning ORF of CiCPK32 gene, the restriction enzyme cutting sites contained in the primer are Sal I and Sac I, the target gene fragment is cut from pEASY-Blunt Simple recombinant vector by using the two restriction enzymes, the restriction enzyme cutting sites are proved to be correct, and the restriction enzyme cutting sites are connected with the linear expression vector pCang-HA after double enzyme cutting for construction of over-expression strain.

(2) The ligation product obtained in (1) was transformed into E.coli DH 5. alpha. competent cells containing 20 mg. multidot.mL^-1LB culture of kanamycinScreening positive clone bacteria on the culture medium, extracting plasmids, and performing double enzyme digestion verification by using Sal I and Sac I.

(3) Preparing competent cells of Agrobacterium GV3101, and transforming Agrobacterium GV3101 with the plasmid verified to be correct in (2) by electric transformation. The constructed over-expression vector pCang-CiCPK32 is transformed into wild type Arabidopsis thaliana (Col-0) by a floral dip method, and mature T is collected₁Generation of seeds, drying seeds in the air, and adding T₁Seeding the seed generation on 1/2MS culture medium (containing 25 mg. L)^- ¹Kan) for transgenic seedling screening. Transferring green seedling (figure 6A) in culture medium into culture bowl filled with mixed soil (vermiculite: nutrient soil: 3:1) for culture, harvesting single plant after seed maturation, and obtaining transgenic T₂And (5) seed generation. Continue to put T₂The seeds were sown on 1/2MS medium (containing 25 mg. L)^-1Kan) and transplanting the green seedlings on the medium according to the mendelian segregation law into a culture pot filled with mixed soil for culture (fig. 6B). After the plants are mature, T is collected by single plant₃Generation of seeds, collecting T₃The seeds were sown on 1/2MS medium (containing 25 mg. L)^-1Kan), observing the growth condition of seedlings on the plate for about 7 days, wherein the whole green seedlings are homozygote strains (figure 6C).

Through screening and identification, 12 CiCPK32 transgenic Arabidopsis homozygote strains are obtained in total. T obtained by screening₃The generation homozygote strain was tested for expression level of CiCPK32 by qRT-PCR (FIG. 7). In subsequent experiments, strains of OE-2, OE-6 and OE-12 homozygotes were selected for phenotypic testing.

3.2 detection of seed Germination Rate under different treatments

To test the tolerance of CiCPK32 to osmotic stress during seed germination, homozygous lines transformed with pCang-HA empty vector (as control) and 3 over-expressing homozygous lines OE-2, OE-6, OE-12 seeds were sown on 1/2MS medium containing 0mM and 400mM mannitol, 55 seeds per line. There was no significant difference in germination rates between control and CiCPK32 overexpressing Arabidopsis seeds in media containing 0mM mannitol (FIG. 8A). Whereas in the medium containing 400mM mannitol, the germination rates of the CiCPK32 overexpression lines were all significantly higher than that of the wild type Arabidopsis thaliana, e.g., at germination day 5, the germination rates of the overexpression lines were all higher than 92.1%, while the germination rate of the wild type Arabidopsis thaliana was only 74.5% (FIG. 8B), indicating that overexpression of CiCPK32 enhanced the tolerance of Arabidopsis thaliana to osmotic stress.

3.3 detection of tolerance of Arabidopsis seed germination to salt stress by overexpression of CiCPK32 Gene

To test the tolerance of control and CiCPK32 overexpression Arabidopsis thaliana to salt stress during seed germination, 55 seeds of each of 3 overexpression lines and control Arabidopsis thaliana were dibbled on 1/2MS medium containing 0mM and 100mM NaCl, respectively. There was no difference in germination rates between control and CiCPK32 overexpressing strains in NaCl-free medium (FIGS. 9A and C); whereas in the medium containing 100mM NaCl the germination rate of 3 CiCPK32 overexpressing strains was significantly higher than wild type (FIGS. 9B and D). For example, when seeds germinate at day 2, the germination rate of the wild type is 60.0%, while the germination rates of 3 overexpression lines are all higher than 87.3%, which indicates that overexpression of CiCPK32 in the germination stage of Arabidopsis seeds can improve the salt stress tolerance of the Arabidopsis seeds.

3.4 drought stress phenotype and Water loss Rate detection of Arabidopsis with overexpression of CiCPK32 Gene

To further explore the function of the CiCPK32 gene in drought stress, the water loss rates of the controls grown for 3 weeks and the aerial parts of CiCPK32 overexpressing Arabidopsis were examined and the water loss rates of the overexpressing lines were both lower than the controls (FIG. 10C). After drought treatment was performed on the control and overexpression lines that grew normally for 2 weeks, it was found that after 10 days of watering stopped, the wild leaves lost water severely, the leaves were in a curled and wilted state, while the wilting phenomenon of the leaves of the transgenic lines was significantly lighter than that of the control (fig. 10A). The survival rate of the control was 56.3% when the drought-treated plants were rehydrated and counted after 3d, whereas the survival rates of the over-expressed strains OE-2, OE-6 and OE-12 of CiCPK32 were 99.3%, 100% and 99.6%, respectively, which were significantly higher than the wild type (FIG. 10B). In conclusion, after the CiCPK32 is over-expressed, the drought resistance of Arabidopsis is improved.

3.5 tolerance detection of Arabidopsis seed germination with overexpression of CiCPK32 gene to ABA

To test whether the regulation effect of CiCPK32 in the seed germination stage is related to ABA, control and CiCPK32 overexpression lines were respectively dibbled on 1/2MS culture media containing 0 and 0.4. mu.M ABA, and the germination rate of seeds was counted. There was no significant difference in germination rates between control and over-expression on medium without ABA addition (fig. 11A and C); on the other hand, on the medium containing 0.4. mu.M ABA, the germination rates of the CiCPK32 overexpression lines were all significantly higher than those of the control (FIGS. 11B and D), for example, at the 7 th day of germination, the germination rate of the wild type was 83.0%, and the average value of the germination rates of the overexpression lines was 95.8%, indicating that the overexpression of CiCPK32 improves the ABA tolerance of the Arabidopsis seeds at the germination stage.

3.6 Effect of ABA on the root Length of Arabidopsis seedlings overexpressing the CiCPK32 Gene

To further understand whether the CiCPK32 gene has an effect on the growth and development of the root system of Arabidopsis seedlings, 5d germinated Arabidopsis seedlings were treated with 12. mu.MABA, and the growth of the root system of the seedlings was observed and recorded. Arabidopsis seedlings grown for 5d in 1/2MS medium were transferred to 1/2MS medium containing 0 and 12. mu.M ABA, and root length L was measured and recorded₁Measuring and recording the root length L after vertical cultivation of 10₂When the length of the root is L (L ═ L)₁-L₂) As shown in FIG. 12, the elongation of the root length of the over-expressed line after 10 days of growth on 1/2MS medium is slightly longer than that of the control (FIGS. 12A and C), while the elongation of the root length of the over-expressed line after 10 days of growth on the medium containing 12. mu.M ABA is significantly higher than that of the control (FIGS. 12B and D), and the growth state of the leaves of the over-expressed line is significantly better than that of the over-expression, which indicates that the gene can also improve the tolerance of the plant root system and the overground part to ABA during the seedling period.

3.7 ABA Signal pathway Gene and stress response Gene expression level detection

The expression levels of the following 10 genes in the normal growth state and 100. mu.M ABA treated control and CiCPK32 transgenic water culture seedlings were detected by real-time fluorescent quantitative PCR. The results show (fig. 13) that ABF1, ABF2, ABI1, ABI2 and 5 stress response genes MYC2, MYB2, KIN1, COR15A, RD29A, etc. among the 4 selected ABA signaling pathway genes were not significantly different among the strains under normal growth conditions and expression was low; after ABA treatment, the expression level of the detected gene is obviously improved, which shows that the ABA treatment is effective; and the expression level of each detected gene in the CiCPK32 overexpression strain is higher than that of the wild type. The fact that CiCPK32 has little influence on the genes under the normal growth condition after being over-expressed in Arabidopsis thaliana is shown; when plants are stressed by drought, salt and ABA, the expression of the genes is induced, so that the plants can resist the influence of external adverse environment.

SEQUENCE LISTING

<110> Applicant

<120> application of CiCPK32 gene of caragana intermedia in regulation and control of plant stress resistance

<130> application of CiCPK32 gene of caragana intermedia in regulation and control of plant stress resistance

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 522

<212> PRT

<213> Caragana inermedia

<400> 1

Met Gly Asn Cys Cys Ala Thr Pro Pro Ser Val Ala Gly Glu Glu Thr

1 5 10 15

Lys Lys Lys Lys Asn Lys Lys Gly Lys Lys Glu Asn Pro Phe Ala Ile

20 25 30

Asp Tyr Ala Phe Asn Asn Asn Asn Asn Ile Asn Thr Gly Ser Lys Leu

35 40 45

Thr Val Leu Lys Asn Pro Thr Gly Lys Glu Ile Glu Val Arg Tyr Glu

50 55 60

Leu Gly Arg Glu Leu Gly Arg Gly Glu Phe Gly Ile Thr Tyr Leu Cys

65 70 75 80

Thr Asp Lys Glu Thr Gly Glu Glu Leu Ala Cys Lys Ser Ile Ser Lys

85 90 95

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

100 105 110

Glu Ile Met Arg His Met Pro Lys His Pro Asn Ile Val Thr Leu Lys

115 120 125

Asp Thr Tyr Glu Asp Asp Asn Ala Val His Leu Val Met Glu Leu Cys

130 135 140

Glu Gly Gly Glu Leu Phe Asp Arg Ile Val Ala Arg Gly His Tyr Thr

145 150 155 160

Glu Arg Ala Ala Ala Ala Val Thr Lys Thr Ile Val Glu Val Val Gln

165 170 175

Met Cys His Lys His Gly Val Met His Arg Asp Leu Lys Pro Glu Asn

180 185 190

Phe Leu Phe Ala Asn Lys Lys Glu Thr Ala Pro Leu Lys Ala Ile Asp

195 200 205

Phe Gly Leu Ser Val Phe Phe Lys Pro Gly Glu Arg Phe Asn Glu Ile

210 215 220

Val Gly Ser Pro Tyr Tyr Met Ala Pro Glu Val Leu Lys Arg Asn Tyr

225 230 235 240

Gly Pro Glu Val Asp Ile Trp Ser Ala Gly Val Ile Leu Tyr Ile Leu

245 250 255

Leu Cys Gly Val Pro Pro Phe Trp Ala Glu Thr Glu Gln Gly Val Ala

260 265 270

Gln Ala Ile Ile Arg Ser Val Val Asp Phe Lys Arg Asp Pro Trp Pro

275 280 285

Lys Val Ser Asp Asn Ala Lys Asp Leu Val Lys Lys Met Leu Asp Pro

290 295 300

Asp Pro Arg Arg Arg Leu Thr Ala Gln Glu Val Leu Asp His Pro Trp

305 310 315 320

Leu Gln Asn Ala Lys Lys Ala Pro Asn Val Ser Leu Gly Glu Thr Val

325 330 335

Arg Ala Arg Leu Lys Gln Phe Ser Val Met Asn Lys Leu Lys Lys Arg

340 345 350

Ala Leu Arg Val Ile Ala Glu His Leu Ser Val Glu Glu Ala Ala Gly

355 360 365

Leu Lys Glu Gly Phe Lys Val Met Asp Thr Ser Asn Lys Gly Lys Ile

370 375 380

Asn Ile Asp Glu Leu Arg Val Gly Leu Leu Lys Leu Gly His Gln Ile

385 390 395 400

Pro Asp Ala Asp Val Gln Ile Leu Met Glu Ala Gly Asp Val Asp Arg

405 410 415

Asp Gly Tyr Leu Asp Tyr Gly Glu Tyr Val Ala Ile Ser Val His Leu

420 425 430

Arg Lys Met Gly Asn Asp Glu His Leu His Lys Ala Phe Glu Phe Phe

435 440 445

Asp Glu Asn Gln Ser Gly Tyr Ile Glu Ile Asp Glu Leu Arg Asn Ala

450 455 460

Ile Ser Asp Glu Val Glu Thr Asn Ser Glu Glu Val Ile Asn Ala Ile

465 470 475 480

Met His Asp Val Asp Thr Asp Lys Asp Gly Arg Ile Ser Tyr Glu Glu

485 490 495

Phe Ala Thr Met Met Lys Ala Gly Thr Asp Trp Arg Lys Ala Ser Arg

500 505 510

Gln Tyr Ser Arg Glu Arg Phe Asn Asn Leu

515 520

<210> 2

<211> 2074

<212> DNA

<213> Caragana inermedia

<400> 2

acatggggga gaaagagaga gacgacgatg gacgaagtct ctgcgaaaca agttttgtgc 60

ttttgacctt ctcagctacc accatcgatg caacccatta atcccctttc gtccactctt 120

ccaataacgc gttaattaaa ttcattcatc ctcattcccc aaatcccctt tttcttaaat 180

caccctctca ctctgaaatc ccatttgcgt tattcttcat tttctcaaaa gaaacccctt 240

ttgttttaca acccagatct ggtagaaccc gaaccttagt taaaccatgg gtaactgttg 300

cgcaacccct ccttctgttg cgggagaaga aacgaaaaag aagaaaaaca aaaaggggaa 360

aaaggaaaac cctttcgcaa tcgactacgc cttcaacaac aacaacaaca taaacaccgg 420

gtcaaaactc accgttttga aaaacccaac agggaaagag atcgaggttc ggtacgagct 480

aggtcgggaa ctaggtcggg gtgagttcgg gataacgtac ctgtgtacgg ataaggagac 540

aggggaagag ctagcgtgca agtcgatatc gaagaagaag cttagaactg cgatagatat 600

tgaagatgtg aggagagaag ttgagatcat gagacacatg cctaaacacc ctaatattgt 660

gactttgaag gatacctatg aagatgacaa tgccgttcac cttgttatgg agctttgtga 720

gggtggtgag ctttttgatc gaatcgtggc gcgtggacac tacaccgaac gcgccgctgc 780

tgctgtcacc aaaaccatag ttgaagttgt tcagatgtgc cacaaacatg gtgtgatgca 840

tagggatctt aagcctgaga actttttgtt tgcgaataag aaggaaacag cacctctgaa 900

agctatagat tttggattgt cagtgttctt taaaccaggg gaaagattta atgagatagt 960

tggaagtcca tattacatgg ctcctgaggt attgaagcga aattatggcc cagaagttga 1020

tatttggagt gctggagtaa ttctatacat cttactttgt ggtgtcccac cattttgggc 1080

agaaactgag caaggagttg cacaagcaat tatacgatct gttgttgatt tcaaaaggga 1140

tccatggcca aaagtttccg ataatgctaa agaccttgtg aagaagatgc tagatcctga 1200

cccaaggcga cgacttactg cccaggaagt gttagatcat ccgtggttac aaaatgcaaa 1260

gaaagctccc aatgtttcgt taggagaaac agttagagca aggctcaagc aattttccgt 1320

aatgaacaag cttaagaaga gagctttgag ggtgattgca gagcatttgt cggttgaaga 1380

agctgctgga ctaaaagagg gattcaaggt tatggataca agcaacaaag gcaagattaa 1440

cattgatgaa ctacgagtag ggttgcttaa actaggccat caaattcctg acgcagatgt 1500

ccaaattctt atggaagctg gtgatgtaga ccgggatggg tacctagatt atggggagta 1560

tgtagccatt tctgttcatc tgagaaagat gggaaatgat gagcaccttc acaaagcctt 1620

tgaatttttt gatgagaatc aaagtgggta tattgagatt gacgagctgc gcaatgccat 1680

atctgatgaa gttgaaacaa acagtgaaga agccattaat gcaattatgc atgatgtgga 1740

cacagacaag gatggaagga taagttatga ggaatttgct acaatgatga aggctggcac 1800

agattggaga aggcatcaag gcagtattcc cgagagaggt ttaacaatct aagcctgaaa 1860

ttgatgaagg atgggtcatt ccaagtaaac aatgaaaaac aatgacatta gatgacttta 1920

gaaatgccaa attgtacttt tggaaaaaag aaaacatgat aaagaagtct attatttttt 1980

atttttaacc ttctgatctt cattgtaatt cttttctcca ataccccaat ttttgggtga 2040

aaaaaaaata ggatattggc cccaaaaaaa aaaa 2074

<210> 3

<211> 1566

<212> DNA

<213> Caragana inermedia

<400> 3

atgggtaact gttgcgcaac ccctccttct gttgcgggag aagaaacgaa aaagaagaaa 60

aacaaaaagg ggaaaaagga aaaccctttc gcaatcgact acgccttcaa caacaacaac 120

aacataaaca ccgggtcaaa actcaccgtt ttgaaaaacc caacagggaa agagatcgag 180

gttcggtacg agctaggtcg ggaactaggt cggggtgagt tcgggataac gtacctgtgt 240

acggataagg agacagggga agagctagcg tgcaagtcga tatcgaagaa gaagcttaga 300

actgcgatag atattgaaga tgtgaggaga gaagttgaga tcatgagaca catgcctaaa 360

caccctaata ttgtgacttt gaaggatacc tatgaagatg acaatgccgt tcaccttgtt 420

atggagcttt gtgagggtgg tgagcttttt gatcgaatcg tggcgcgtgg acactacacc 480

gaacgcgccg ctgctgctgt caccaaaacc atagttgaag ttgttcagat gtgccacaaa 540

catggtgtga tgcataggga tcttaagcct gagaactttt tgtttgcgaa taagaaggaa 600

acagcacctc tgaaagctat agattttgga ttgtcagtgt tctttaaacc aggggaaaga 660

tttaatgaga tagttggaag tccatattac atggctcctg aggtattgaa gcgaaattat 720

ggcccagaag ttgatatttg gagtgctgga gtaattctat acatcttact ttgtggtgtc 780

ccaccatttt gggcagaaac tgagcaagga gttgcacaag caattatacg atctgttgtt 840

gatttcaaaa gggatccatg gccaaaagtt tccgataatg ctaaagacct tgtgaagaag 900

atgctagatc ctgacccaag gcgacgactt actgcccagg aagtgttaga tcatccgtgg 960

ttacaaaatg caaagaaagc tcccaatgtt tcgttaggag aaacagttag agcaaggctc 1020

aagcaatttt ccgtaatgaa caagcttaag aagagagctt tgagggtgat tgcagagcat 1080

ttgtcggttg aagaagctgc tggactaaaa gagggattca aggttatgga tacaagcaac 1140

aaaggcaaga ttaacattga tgaactacga gtagggttgc ttaaactagg ccatcaaatt 1200

cctgacgcag atgtccaaat tcttatggaa gctggtgatg tagaccggga tgggtaccta 1260

gattatgggg agtatgtagc catttctgtt catctgagaa agatgggaaa tgatgagcac 1320

cttcacaaag cctttgaatt ttttgatgag aatcaaagtg ggtatattga gattgacgag 1380

ctgcgcaatg ccatatctga tgaagttgaa acaaacagtg aagaagccat taatgcaatt 1440

atgcatgatg tggacacaga caaggatgga aggataagtt atgaggaatt tgctacaatg 1500

atgaaggctg gcacagattg gagaaggcat caaggcagta ttcccgagag aggtttaaca 1560

atctaa 1566

Claims

1. The CiCPK32 gene of the caragana intermedia is characterized in that the nucleotide sequence of the CiCPK32 gene is shown as SEQ ID NO.2, and the coding sequence thereof is shown as SEQ ID NO. 1.

2. The recombinant expression vector is inserted with a CiCPK32 gene, wherein the CiCPK32 gene is a nucleotide sequence shown as SEQ ID NO.2, or a nucleotide sequence which is complementary and matched with the SEQ ID NO.2, or a nucleotide sequence which codes the SEQ ID NO. 1.

3. The transient and stable expression vector is inserted with CiCPK32 gene, the CiCPK32 gene is the nucleotide sequence shown in SEQ ID NO.2, or the nucleotide sequence complementary and matched with SEQ ID NO.2, or the nucleotide sequence coding SEQ ID NO. 1.

4. The overexpression vector is inserted with a CiCPK32 gene, and the CiCPK32 gene is a nucleotide sequence shown as SEQ ID NO.2, or a nucleotide sequence which is complementary and matched with the SEQ ID NO.2, or a nucleotide sequence which codes the SEQ ID NO. 1.

5. Use of the CiCPK32 gene of claim 1, the expression vector of any one of claims 2-3 for modulating stress tolerance in a plant; the plant is arabidopsis thaliana or caragana intermedia; the stress resistance refers to resistance to osmotic stress, drought stress and salt stress.

6. A biological agent for improving the stress resistance of plants is characterized in that the active component is a CiCPK32 gene, and the CiCPK32 gene is a nucleotide sequence shown as SEQ ID NO.2, or a nucleotide sequence which is complementary and matched with the SEQ ID NO.2, or a nucleotide sequence which codes the SEQ ID NO. 1; the plant is arabidopsis thaliana or caragana intermedia; the stress resistance refers to resistance to osmotic stress, drought stress and salt stress.

7. A method for improving stress resistance of plants is characterized in that the method comprises the overexpression of a CiCPK32 gene, wherein the CiCPK32 gene is a nucleotide sequence shown as SEQ ID NO.2, or a nucleotide sequence which is complementary and matched with the SEQ ID NO.2, or a nucleotide sequence which codes SEQ ID NO. 1; the plant is arabidopsis thaliana or caragana intermedia; the stress resistance refers to resistance to osmotic stress, drought stress and salt stress.

8. Use of the CiCPK32 gene of claim 1, or the recombinant expression vector of claim 2, or the transient and stable expression vector of claim 3, or the overexpression vector of claim 4, for genetic breeding for improved plant stress resistance, wherein the CiCPK32 gene is the nucleotide sequence set forth in SEQ ID No.2, or a nucleotide sequence complementary paired with SEQ ID No.2, or a nucleotide sequence encoding SEQ ID No. 1; the plant is arabidopsis thaliana or caragana intermedia; the stress resistance refers to osmotic stress resistance, drought stress resistance and salt stress resistance.