CN110184253A

CN110184253A - Application of the Caragana intermedia CiCPK32 gene in regulation stress resistance of plant

Info

Publication number: CN110184253A
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: 2019-08-30
Anticipated expiration: 2039-06-06
Also published as: CN110184253B

Abstract

The present invention relates to biological gene engineering fields, in particular to Caragana intermedia CiCPK32 gene, while being related to the functional study of the gene and the application in regulation stress resistance of plant.The present invention utilizes the transcript profile database of Caragana intermedia, clone obtains CiCPK32 gene relevant to abiotic stress from Caragana intermedia, after being overexpressed in arabidopsis, carry out the Stress treatments such as arid, with high salt and ABA, and its function is disclosed, being applied to from now on for it, which improves agricultural grass Plant Tolerance adverse circumstance ability, provides theoretical foundation and support.

Description

Application of the Caragana intermedia CiCPK32 gene in regulation stress resistance of plant

Technical field

The present invention relates to biological gene engineering fields to relate to simultaneously in particular to Caragana intermedia CiCPK32 gene And the gene functional study and regulation stress resistance of plant application.

Background technique

The environmental conditions such as low temperature, arid, saline Land are to influence the main environment stress factor of plant growth and development, are planted Physiological acoustic signals of the object under environment stress and be research hotspot in recent years to the research of adverse circumstance adaptability.It explores such as The resistance what improves plant has become critical issue urgently to be solved.Plant is understood in molecule, carefully to adapt to adverse circumstance environment Timely adjusting is made in the level such as born of the same parents, organ, Physiology and biochemistry.With the evolution of biology, complicated signal is produced in plant Transmission system, to improve the anti-adversity ability of plant.Calcium ion is second messenger intracellular, is a kind of important in plant Signal transduction factor participates in plant growth and development, immune defense and hormone control, and has sound to light and various stress signals It answers.Currently, having calmodulin, Calcium-dependent protein kinase (CDPK) and calcineurin B-like proteins these three calcium receptors in plant Albumen, wherein Calcium-dependent protein kinase is the hot spot studied at present.Calcium-dependent protein kinase is main primary calcium ion impression One of device, while being also Ca²⁺Response protein, it is widely distributed in plant, play a significant role during Plant calcium signal transduction, It plays an important role in plant response adverse circumstance signal transduction process simultaneously.

CDPK is that plant and protist be specific, still not found a kind of serine/threonine type in animal body Protein kinase, its full name are as follows: protein kinase (the Calcium-dependent and that calcium relies on and calmodulin does not depend on Calmodulin-independent protein kinase) or class calmodulin structural domain protein kinase (Calmodulin- Like protein kinase), it is a kind of special calcium ion-binding protein.The albumen is for the first time in pea (Pisum Sativum report in), and purified and identified for the first time in soybean (Glycine max).Not with the research to CDPK It is disconnected to go deep into, it has been found that CDPK is a big gene family, currently, in rice (Oryza sativa), wheat (Triticum Aestivum), arabidopsis (Arabidopsis thaliana), poplar (populus trichocarpa) and grape (Vitis ) etc. vinifera CDPK albumen is identified in various plants, for example, having identified in model plant arabidopsis gene group 34 CDPK family members are located on five chromosomes, and wherein CPK2/17/20/34 is proved have with pollen tube growth It closes；And show that two the genes PiCPK1 and PiCPK2 of petunia may also be participated in pollen tube growth in early stage research Adjusting, arabidopsis CPK28 gene is considered as gibberellin (Gibberellic acid, GA) and jasmonic (Jasmonic Acid, JA) adjusting gene, CPK4 and CPK11 play an important role in the biosynthesis of ethylene, and CPK21 and CPK23 exist The salt tolerant of plant and drought-enduring approach play negative regulation, which enhances the tolerance to corresponding abiotic stress, and The strain of overexpression then shows opposite phenotype.In rice, 31 CDPK family members, soybean have been identified so far 50 CDPK gene members of middle discovery, middle clone of corn (Zea mays) obtains 40 CDPK genes, in common wheat and poplar 20 CDPK genes are found respectively, identify 25 CDPK genes in M. truncatula (Medicago truncatula) genome, 29 CDPK genes are found in millet (Setaria italica), identify 21 in potato (Solanum tuberosum) CDPK gene, in addition, in muskmelon (cucumis melo), apple (Malus pumila Millapple), grape and peanut CDPK gene is identified in (Arachis hypogaea).With the continuous deepening of research, discovery CDPK participate in flowering regulation, The synthesis of plant root system development and hormone and the adjusting of signal path, while also being sent out in regulating cell differentiation, programmed death Critical function is waved, as StCDPK5 transgenic potato enhances the resistance to late blight encephalapthy agent.

Caragana intermedia (Caragana inermedia) belongs to the perennial shrub of pulse family Caragana.Root system is more developed, Nearly 5 meters of subterraneous root, stem uprightly or obliquely, 1-2 meters of plant height；Pinnate compound leaf, leaflet 12-16 piece, obovate or ellipse are more There is coat；Caragana intermedia starts to grow mid-April, and the florescence is mid-May, and corolla butterfly, yellow, fruiting period is June, seed For kidney shape, there are two kinds of colors of pale green brown and yellowish-brown.It is extensive in Area distributions such as China Inner Mongol, Shanxi, Ningxia and Shaanxi, It is the excellent plant that arid, Desert Area check winds and fix drifting sand and conserve water and soil with drought-enduring, high temperature resistant, saline-alkali tolerant and the characteristics such as cold-resistant Object；In addition, Caragana intermedia is also used as feed, fuel, papermaking, fertilizer and plate raw material, while also there is medical value. Other extreme environments such as arid, with high salt and freezing drastically influence the yield of the growth and development and crops of plant.Plant is not As animal flexibly can initiatively go to hide various environment stresses, therefore, in order to adapt to various bad growing environments, plant also by Progressiveization forms the degeneration-resistant mechanism of a series of complex, to enhance the resistance of plant itself.CDPKs is considered as cytoplasm The important regulating and controlling factor of calcium ion downstream various plants signal transduction pathway may play the role of enhancing stress resistance of plant.This Invention is cloned from Caragana intermedia obtains one and degeneration-resistant relevant CiCPK32 gene, and tentatively grind to its function Study carefully, probes into its degeneration-resistant mechanism from each level such as molecule and physiology.

Summary of the invention

The present invention utilizes the transcript profile database of Caragana intermedia, and clone obtains and abiotic stress from Caragana intermedia Relevant CiCPK32 gene after being overexpressed in arabidopsis, carries out the Stress treatments such as arid, with high salt and abscisic acid (ABA), and Its function is disclosed, being applied to from now on for it, which improves agricultural grass Plant Tolerance adverse circumstance ability, provides theoretical foundation and support.

The Caragana intermedia CiCPK32 gene that can increase stress resistance of plant the first purpose of the invention is to provide one and It encodes albumen.

Caragana intermedia CiCPK32 gene set forth in the present invention, coding amino acid sequence such as sequence table in SEQ Shown in ID NO.1, gene nucleotide series are as shown in SEQ ID NO.2 in sequence table.

It is a further object of the present invention to provide Caragana intermedia CiCPK32 to improve plant to the application in resistance.It is special Not, the resistance refers to Osmotic Stress Tolerance, drought resisting stress, salt stress-resistant and anti-ABA stress.

Recombinant expression carrier inserted with CiCPK32 gene, the CiCPK32 gene are as shown in SEQ ID NO.2 Nucleotide sequence, or be the nucleotide sequence with SEQ ID NO.2 complementary pairing, or be coding SEQ ID NO.1 protein Nucleotide sequence.

Instantaneous and stable expressed vector inserted with CiCPK32 gene, the CiCPK32 gene are such as SEQ ID NO.2 Shown in nucleotide sequence, or be the nucleotide sequence with SEQ ID NO.2 complementary pairing, or be coding SEQ ID NO.1 Nucleotide sequence.

Overexpression and interference expression vector inserted with CiCPK32 gene, the CiCPK32 gene are such as SEQ ID Nucleotide sequence shown in NO.2, or be the nucleotide sequence with SEQ ID NO.2 complementary pairing, or be coding SEQ ID The nucleotide sequence of NO.1.

It is a further object of the present invention to provide application of the CiCPK32 gene in plant breeding.

CiCPK32 gene or its recombinant expression carrier or its transient expression and stable expressed vector or its overexpression and Interference expression vector is such as SEQ ID improveing the application in genetic breeding of the plant to resistance, the CiCPK32 gene Nucleotide sequence shown in NO.2, or be the nucleotide sequence with SEQ ID NO.2 complementary pairing, or be coding SEQ ID The nucleotide sequence of NO.1.

Above-mentioned Caragana intermedia CiCPK32 gene is applied to plant genetic engineering it is another object of the present invention to a kind of In, the overexpression of CiCPK32 and the transgenic plant of interference are obtained, it is particularly, described degeneration-resistant for changing the resistance of plant Property refer to Osmotic Stress Tolerance, drought resisting stress, salt stress-resistant and anti-ABA stress.

It is particularly, described degeneration-resistant it is a further object of the present invention to provide a kind of biological agent of resistance for improving plant Property refer to Osmotic Stress Tolerance, drought resisting stress, salt stress-resistant and anti-ABA stress, preferably refer to drought resisting stress, salt stress-resistant and Anti- ABA stress.

A kind of biological agent for the resistance improving plant, active constituent derive from the recombinant expression carrier of CiCPK32 Or overexpression and interference expression vector or its active constituent contain the biological products of regulation CiCPK32 gene expression, it is described CiCPK32 gene is the nucleotide sequence as shown in SEQ ID NO.2, or is the nucleotide with SEQ ID NO.2 complementary pairing Sequence, or to encode the nucleotide sequence of SEQ ID NO.1.

It is a further object of the present invention to provide a kind of methods of the resistance of regulation plant, and particularly, the resistance is Refer to Osmotic Stress Tolerance, drought resisting stress, salt stress-resistant and anti-ABA stress.

A method of the resistance of regulation plant, this method include regulation CiCPK32 gene expression, the CiCPK32 Gene is the nucleotide sequence as shown in SEQ ID NO.2, or is the nucleotide sequence with SEQ ID NO.2 complementary pairing, or For the nucleotide sequence for encoding SEQ ID NO.1.

The present invention constructs the overexpression and interference expression vector of CiCPK32, and the genetic transformation for passing through agrobacterium-mediated transformation Overexpression and interference expression vector are converted to normal plants kind, finally obtain the overexpression and interference table of CiCPK32 by method The genetically modified plants reached.

Particularly, the plant is preferably terrestrial plant.The terrestrial plant can be planted for dicotyledon and/or unifacial leaf Object.The dicotyledon concretely crucifer；The crucifer can be arabidopsis.It is specific real at one It applies in scheme, plant is preferably pulse family caragana plant, and the pulse family caragana plant can be Caragana intermedia.

In the present invention, we obtain a Caragana intermedia CiCPK32 and carry out application exploration, elaborate the base Because of following application mode:

(1) it clones to obtain Caragana intermedia CiCPK32 gene using RACE technology.

(2) CiCPK32 gene is by arid, the stress-inducing of NaCl, ABA.

(3) under 100mM NaCl, 400mM mannitol or 0.4 μ Μ ABA processing, CiCPK32 is overexpressed strain seed and sprouts Hair rate is higher than control.

(4) under dehydration, the percentage of water loss of each strain of CiCPK32 gene is overexpressed significantly lower than control；After Osmotic treatment, It is overexpressed strain survival rate and is apparently higher than wild type.

(5) under ABA processing, the expression quantity of stress-related genes in CiCPK32 gene strain is overexpressed obviously higher than open country Raw type.

Beneficial effects of the present invention are as follows: clone obtains CiCPK32 relevant to abiotic stress from Caragana intermedia Gene, and its degeneration-resistant sexual function is disclosed, being applied to from now on for it, which improves agricultural grass Plant Tolerance adverse circumstance ability, provides direction.

Detailed description of the invention

Fig. 1: Technology Roadmap of the invention.

Fig. 2: CiCPK32 full length gene cDNA clone electrophoretogram (3 ' RACE B:CiCPK32 of A:CiCPK32,5 ' RACE C:CiCPK32ORF)。

Fig. 3: CiCPK32 and the evolutionary relationship of the CDPK of other species (are amino acid accession number in bracket；Number in branch Word indicates the percentage of the confidence level based on 1000 repetition nodes in Bootstrap verifying；Scale represents evolution distance).

Fig. 4: CiCPK32 (branch of different colours represents different subfamilies from the phylogenetic analysis of arabidopsis CDPKs Member, blue: I subfamily；Purple: II subfamily；Green: III subfamily；It is red: IV subfamily).

Fig. 5: the CiCPK32 expression pattern analysis under different disposal.

The screening of Fig. 6: CiCPK32 transgenic homozygous body seedling.

The identification of Fig. 7: CiCPK32 transgenic line expression.

Fig. 8: control and CiCPK32 are overexpressed the detection of strain germination rate under treatment with mannitol；A: on normal 1/2MS culture medium The statistics of the germination rate of each strain；B: the statistics of the germination rate of each strain on the 1/2MS culture medium containing 400mM Mannitol.

The lower control of Fig. 9: NaCl processing and CiCPK32 are overexpressed the detection of strain germination rate；A and C: normal 1/2MS culture medium The sprouting situation of upper each strain and the statistics (photo of growth 2d) of germination rate；B and D: the 1/2MS culture containing 100mM NaCl The statistics (photo of growth 2d) of the sprouting situation of each strain and germination rate on base.

Figure 10: control and CiCPK32 are overexpressed strain drought resistance and percentage of water loss analysis；A: drought stress handles first 2 weeks big Seedling (above), Osmotic treatment 10 days seedling (middle) and the seedling (following figure) after rehydration 3 days；B: the survival rate of each strain Statistics；C: each strain aerial part percentage of water loss statistics；* indicates P < 0.01.

Figure 11: ABA handles lower wild type and CiCPK32 overexpression strain germination rate detection；A and C: normal 1/2MS culture The statistics (photo of growth 7d) of the sprouting situation of each strain and germination rate on base；B and D: the 1/2MS training containing 0.4 μM of ABA Support the statistics (photo of growth 7d) of the sprouting situation of each strain and germination rate on base.

Lower CiCPK32 Arabidopsis thaliana Seedlings and the control seedling main root length of being overexpressed of Figure 12: ABA processing compares；A and C: normal The root long situation of each strain and statistics (photo of growth 10d) on 1/2MS culture medium；B and D: the 1/2MS training containing 12 μM of ABA Support the root long situation of each strain and statistics (photo of growth 10d) on base；* indicate that P < 0.05, * * indicate that P < 0.01, * * * indicate P <0.001。

Figure 13: arabidopsis difference strain ABA signal pathway gene and the detection of stress response gene expression amount.

Specific embodiment

The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified Conventional method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.Specifically Technology Roadmap is as shown in Figure 1.

The clone of 1. Caragana intermedia CiCPK32 encoding gene of embodiment

1.1 RACE methods expand 3 ' terminal sequence of CiCPK32 gene

Screening obtains a CiCPK32 homologous with arabidopsis CPK32 from Caragana intermedia dehydration transcription group database The est sequence of gene, fragment length 566bp have found the gene order 3 ' end with 5 ' ends not in NCBI by sequence alignment Completely.Design Outer and Inner primer: CiCDPK32-3 '-out and CiCDPK32-3 '-is required according to 3 ' RACE kits In, specific primer sequence are as follows:

1.2 RACE methods expand CiCPK32 gene 5 ' terminal sequence

According to the requirement of Smarter RACE cDNA Amplicaion Kit kit, 5 ' RACE primers, primer are designed Sequence is as follows:

In kit have 5 ' UPM primers, 5' end sequence expand the step of be carried out according to kit specification.

The clone of 1.3 gene coding regions CiCPK32

By 1.1 and 1.2 RACE amplification through Shanghai Sheng Gong biotech firm sequencing after with Vector NTI10 software into Row splicing, obtains CiCPK32 gene cDNA full length sequence, and analysis obtains complete open reading encoder block (ORF) sequence.According to ORF sequence, design primer: HA-CiCPK32-sense and HA-CiCPK32-anti:

Using the cDNA of Caragana intermedia as template, high fidelity enzyme PrimeSTAR is utilized^HSDNA Polymerase is to purpose base The ORF of cause is expanded.Wherein annealing temperature is set as 63 DEG C, and extension of time is set as 1.5min, reaction cycle number 30.

As it can be seen that expanding to obtain the 5 ' terminal sequences of 1196bp by 5 ' RACE, 3 ' RACE expand to obtain the 3 ' terminal sequences of 854bp (Fig. 2).After sequencing, the sequence that RACE technology expands is spliced by Vector NTI10 with the est sequence screened, is obtained Obtain cDNA full length sequence.Using Caragana intermedia cDNA as template, specific primer is designed, the ORF of the gene, sequence verification are cloned Show that splicing is correct.

The sequence of 1.4 CiCPK32 genes is analyzed

Application software obtains cDNA to amplification in 1.3 and carries out analysis finding, the cDNA of the CiCPK32 gene expanded is complete A length of 2074bp (SEQ ID NO.2), by open reading frame (ORF) 1566bp (SEQ ID NO.3), the end 3' noncoding region and 5' Hold noncoding region composition.The gene encodes 522 amino acid (SEQ ID NO.1), initiation codon ATG, termination codon altogether Son is TAA.Making discovery from observation has Gly (G) residue in CiCPK32 second, it may be possible to the site of myristoylation, and the Four and the 5th position is occupied by Cys (C) residue, it may be possible to the site of palmitoylation, it is known that protein myristoylation promotes albumen Matter and film or protein-protein interact, this structure plays an important role in CiCPK32 film position fixing process.

1.5 gene biological bioinformatics analysis

In order to further appreciate that the evolutionary relationship of CiCPK32 with the CDPK of other species, CiCDPK32 gene is encoded Amino acid sequence carries out BLAST sequence alignment in NCBI, chooses soybean, chick-pea, M. truncatula, plum blossom (Prunus through comparing Mume), apple, grape, cotton, cocoa chocolate tree (Theobroma cacao), arabidopsis, sweet orange (Citrus SinensisL.Osb.), tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum), in potato CDPK albumen, with CiCPK32 protein sequence by constructing chadogram (Fig. 3) together with MAGE7.0, it has been found that itself and arabidopsis AtCPK32 (AT3G57530) consistency reaches 77%.It is analyzed by sequence, Caragana intermedia CiCPK32 and chick-pea, puncture vine Clover, the affiliation of soybean are closer, gather in a big branch, confidence level reaches 99%.

In order to further appreciate that the function of CiCPK32 gene, will in the gene and arabidopsis have been found that 34 CPK bases It because the protein sequence of coding has carried out phylogenetic analysis, and constructs phylogenetic tree (Fig. 4), finds using MAGE 7.0 CiCPK32 is closer with the gene affiliation of the IIIth subtribe of arabidopsis, and cluster together, illustrates that the gene belongs to the of arabidopsis III subfamily.And according to research reports, plant can be enhanced to drought stress in the AtCDPK8 and AtCDPK10 of the IIIth subtribe of arabidopsis Resistance, and AtCPK32 control pollen tube polar growth, provide direction for the follow-up study to target gene.

The expression characterization analysis of embodiment 2, Caragana intermedia CiCPK32 gene

In order to further determine CiCDPK32 gene whether by abiotic stress induction, by Caragana intermedia respectively de- After being handled under the conditions of water, salt, ABA, using the variation of qRT-PCR technology detection destination gene expression amount under Different stress processing Situation.

It selects that seed is full, seed of free from insect pests in Caragana intermedia seed, is seeded in and is placed with composite soil (nutrition Soil: vermiculite=1:3) plant culturing pot, condition of culture are as follows: 22 DEG C, 16h illumination/8h is dark, intensity of illumination 7000- 8000lux.Choose that growth conditions are consistent and well-developed Caragana intermedia seedlings carry out abscisic acid (ABA), salt and the dehydration side of body Compel processing, as the raw material of research purpose gene pairs Different stress response condition.

Testing result shows that the expression of CiCPK32 is induced rapidly after dehydration, and the expression quantity of target gene reaches when 3h To highest, 2.2 times when being untreated, expression quantity is held essentially constant when 6h, is declined slightly trend later, but be still higher than not Expression quantity when processing；After salt treatment, the expression quantity of target gene is substantially reduced, and processing 3h expression quantity is rapidly decreased to untreated When half, reach minimum when handling 6h, expression quantity when 12h is still significantly lower than untreated；After ABA processing, mesh when 6h 4 times when being increased to untreated of the expression quantity of gene.The above testing result illustrates that CiCPK32 gene may participate in dehydration, salt With the response (Fig. 5) of ABA stress.

Embodiment 3, the acquisition for turning CiCPK32 gene Arabidopsis plant and resistance identification

The buildings of 3.1 Caragana intermedia CiCPK32 expression vectors and arabidopsis thaliana transformation

(1) restriction enzyme site for including in primer when cloning the ORF of CiCPK32 gene is Sal I and Sac I, more than Two kinds of restriction enzymes cut target gene fragment from pEASY-Blunt Simple recombinant vector, verified correct, It is connect with the linear expression vector pCanG-HA after double digestion, for being overexpressed the building of strain.

(2) connection product in (1) is transformed into bacillus coli DH 5 alpha competent cell, is containing 20mgmL^-1Card Screening positive clone bacterium on the LB culture medium of that mycin carries out plasmid extraction, carries out double digestion verifying using Sal I and Sac I.

(3) Agrobacterium GV3101 competent cell is prepared, and the correct plasmid of verifying in (2) is converted by electrotransformation Agrobacterium GV3101.The over-express vector pCanG-CiCPK32 built is converted into wildtype Arabidopsis thaliana (Col- with flower-dipping method 0) mature T, is collected₁The seed in generation, after seed dries, by T₁It is sowed for seed and (contains 25mgL on 1/2MS culture medium^- ¹Kan the screening of transgenic seedling) is carried out.Green seedlings (Fig. 6 A) in culture medium are moved on to equipped with composite soil (vermiculite: Nutrition Soil =3:1) culturing pot in cultivated, single plant is collected after seed is mature, the transgenosis T of acquisition₂For seed.Continue T₂Generation kind Son sowing (contains 25mgL on 1/2MS culture medium^-1Kan it) is screened, Mendel's law of segregation will be met on culture medium Green seedlings transplant seedlings to equipped with composite soil culturing pot in cultivated (Fig. 6 B).After plant maturation, single plant collects T₃Generation kind Son, the T that will be collected₃(contain 25mgL on 1/2MS culture medium for seed sowing^-1Kan), seedling on 7 days or so observation plates Upgrowth situation, Quan Lvmiao be homozygote strain (Fig. 6 C).

By screening and identification, 12 CiCPK32 transgenic arabidopsis homozygote strains are obtained altogether.The T that screening is obtained₃Generation Homozygote strain detects the expression (Fig. 7) of CiCPK32 by qRT-PCR.In subsequent experimental, OE-2, OE-6 and OE- are chosen 12 homozygote strains do Phenotypic examination.

The detection of seed germination rate under 3.2 different disposals

The stage is sprouted to the tolerance of osmotic stress in order to detect CiCPK32 in seed, will convert pCanG-HA empty carrier Homozygous lines (as control) and 3 overexpression homozygote strain OE-2, OE-6, OE-12 seed program requests contain 0mM and On the 1/2MS culture medium of 400mM mannitol, 55 seeds of each strain.In the culture medium containing 0mM mannitol, control and The germination rate that CiCPK32 is overexpressed arabidopsis seed does not have significant difference (Fig. 8 A).And in the culture medium containing 400mM mannitol In, CiCPK32 is overexpressed the germination rate of strain obviously higher than wildtype Arabidopsis thaliana, for example, being overexpressed when sprouting the 5th day The germination rate of strain is above 92.1%, and wildtype Arabidopsis thaliana germination rate is only 74.5% (Fig. 8 B), shows to be overexpressed CiCPK32 enhances arabidopsis to the tolerance of osmotic stress.

3.3, which are overexpressed CiCPK32 gene Seed Germination of Arabidopsis Pumila, detects Salt Stress Tolerance

It is overexpressed arabidopsis in order to detect control and CiCPK32 and sprouts the stage to the tolerance of salt stress, by 3 in seed Strain and control each 55 program requests of arabidopsis seed are overexpressed respectively on the 1/2MS culture medium containing 0mM and 100mM NaCl. In the culture medium without NaCl, the germination rate of control and CiCPK32 overexpression strain is not different (Fig. 9 A and C)；And containing In the culture medium for having 100mM NaCl, the germination rate that 3 CiCPK32 are overexpressed strain is apparently higher than wild type (Fig. 9 B and D).Example Such as, when seed sprouts the 2nd day, the germination rate of wild type is 60.0%, and the germination rate of 3 overexpression strains is above 87.3%, illustrate that its ability for being resistant to salt stress can be improved in Seed Germination of Arabidopsis Pumila stage CiCPK32 overexpression.

3.4 are overexpressed CiCPK32 gene arabidopsis drought stress phenotype and percentage of water loss detection

Control and CiCPK32 in order to further probe into function of the CiCPK32 gene in drought stress, to growth 3 weeks The aerial part for being overexpressed arabidopsis has carried out the detection of percentage of water loss, and the percentage of water loss that discovery is overexpressed strain is below control (figure 10C).It is found to normal growth control in 2 weeks and after being overexpressed strain progress Osmotic treatment, after stopping watering 10d, wild type leaf Piece dehydration is serious, and blade is in curling and wilted condition, and the wilting phenomenon of transgenic line blade obviously relatively compares light (figure 10A).To the plant rehydration of Osmotic treatment, survival rate is counted after 3d, the survival rate of control is 56.3%, and CiCPK32 is overexpressed Strain OE-2, OE-6 and OE-12 survival rate is respectively 99.3%, 100% and 99.6%, obviously higher than wild type (Figure 10 B). In conclusion CiCPK32 improves the drought resistance of arabidopsis after being overexpressed.

3.5, which are overexpressed CiCPK32 gene Seed Germination of Arabidopsis Pumila, detects the tolerance of ABA

It is whether related with ABA in order to detect the regulating and controlling effect that CiCPK32 sprouts the stage in seed, by control and CiCPK32 Strain difference program request is overexpressed on the 1/2MS culture medium containing 0 and 0.4 μM of ABA, counts the germination rate of seed.Do not adding On the culture medium of ABA, control and the germination rate being overexpressed are without significant difference (Figure 11 A and C)；And in the training containing 0.4 μM of ABA It supporting on base, CiCPK32 is overexpressed the germination rate of strain obviously higher than control (Figure 11 B and D), for example, when sprouting the 7th day, The germination rate of wild type is 83.0%, and the average value for being overexpressed strain germination rate is 95.8%, illustrates that being overexpressed CiCPK32 improves Arabidopsis seed is in the stage of sprouting to ABA tolerance.

Influence of 3.6 ABA to CiCPK32 gene Arabidopsis thaliana Seedlings root long is overexpressed

In order to further appreciate that whether CiCPK32 gene has an impact the growth and development of Arabidopsis thaliana Seedlings root system, use 12 μM of ABA handle the Arabidopsis thaliana Seedlings for sprouting 5d, observe and record seedling root growth situation.It will be cultivated in 1/2MS The Arabidopsis thaliana Seedlings that 5d is grown in base move on on the 1/2MS culture medium containing 0 and 12 μM of ABA, and the length for measuring and recording root is L₁, it is L that root long is measured and recorded after vertical culture 10₂, then the extended length of root is denoted as L (L=L₁-L₂), as a result such as Figure 12 institute Show, be overexpressed the root long elongation slightly longer than control (Figure 12 A and C) of strain after 10d is grown on 1/2MS culture medium, and is containing 12 μ The root long elongation for being overexpressed strain on the culture medium of M ABA after growth 10d is apparently higher than control (Figure 12 B and D), and is overexpressed The growth conditions of the blade of strain obviously than be overexpressed it is good, illustrate the gene seedling period equally can be improved root system of plant and Tolerance of the aerial part to ABA.

3.7 ABA signal path genes and the detection of stress response gene expression amount

By real-time fluorescence quantitative PCR, have detected under normal growth state and the processed control of 100 μM of ABA and The expression quantity of following 10 genes in CiCPK32 transgenosis Rooted Cuttings.(Figure 13) as the result is shown, 4 selected ABA signal paths ABF1, ABF2, ABI1, ABI2 and 5 stress response gene Ms YC2, MYB2, KIN1, COR15A, RD29A etc. are normal in gene Growth conditions does not have significant difference in each strain, and expresses all lower；After ABA processing, the expression quantity for being detected cls gene is all aobvious It writes and improves, illustrate that the processing of ABA is effective；And each tested cls gene is overexpressed the expression quantity in strain in CiCPK32 It is above wild type.After illustrating that CiCPK32 is overexpressed in arabidopsis, not to the influence of these genes in normal growth Greatly；When plant, which encounters arid, salt and ABA, coerces, the expression of these genes can be induced, to resist the shadow of extraneous poor environment It rings.

SEQUENCE LISTING

<110>applicant

<120>application of the Caragana intermedia CiCPK32 gene in regulation stress resistance of plant

<130>application of the Caragana intermedia CiCPK32 gene in regulation stress resistance of plant

<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. a kind of Caragana intermedia CiCPK32 gene, which is characterized in that the nucleotide sequence such as SEQ of the CiCPK32 gene Shown in ID NO.2, the amino acid sequence as shown in SEQ ID NO.1 is encoded.

2. the recombinant expression carrier inserted with CiCPK32 gene, the CiCPK32 gene is the core as shown in SEQ ID NO.2 Nucleotide sequence, or be the nucleotide sequence with SEQ ID NO.2 complementary pairing, or to encode the nucleotides sequence of SEQ ID NO.1 Column.

3. the instantaneous and stable expressed vector inserted with CiCPK32 gene, the CiCPK32 gene is such as SEQ ID NO.2 institute The nucleotide sequence shown, or be the nucleotide sequence with SEQ ID NO.2 complementary pairing, or to encode the core of SEQ ID NO.1 Nucleotide sequence.

4. overexpression and interference expression vector inserted with CiCPK32 gene, the CiCPK32 gene is such as SEQ ID NO.2 Shown in nucleotide sequence, or be the nucleotide sequence with SEQ ID NO.2 complementary pairing, or be coding SEQ ID NO.1 Nucleotide sequence.

5. the expression vector of any CiCPK32 gene of CiCPK32 gene described in claim 1, claim 2-4 is being adjusted It controls in plant to the application in resistance.

6. a kind of biological agent for the resistance for improving plant, characterized in that its active constituent derives from the recombination table of CiCPK32 Contain the biological products of regulation CiCPK32 gene expression, institute up to carrier or overexpression and interference expression vector or its active constituent Stating CiCPK32 gene is the nucleotide sequence as shown in SEQ ID NO.2, or is the nucleosides with SEQ ID NO.2 complementary pairing Acid sequence, or to encode the nucleotide sequence of SEQ ID NO.1.

7. a kind of method of regulation stress resistance of plant, characterized in that this method includes regulation CiCPK32 gene expression, described CiCPK32 gene is the nucleotide sequence as shown in SEQ ID NO.2, or is the nucleotide with SEQ ID NO.2 complementary pairing Sequence, or to encode the nucleotide sequence of SEQ ID NO.1.

8. described in recombinant expression carrier described in CiCPK32 gene described in claim 1 or claim 2 or claim 3 Instantaneous and stable expressed vector or overexpression as claimed in claim 4 and interference expression vector in improvement stress resistance of plant Application in genetic breeding, the CiCPK32 gene is the nucleotide sequence as shown in SEQ ID NO.2, or is and SEQ ID The nucleotide sequence of NO.2 complementary pairing, or to encode the nucleotide sequence of SEQ ID NO.1.