CN101509006B - Uses of arabidopsis thaliana gene AIK1 in regulating and controlling vegetation growth, and in stress resistance aspect - Google Patents
Uses of arabidopsis thaliana gene AIK1 in regulating and controlling vegetation growth, and in stress resistance aspect Download PDFInfo
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Abstract
The invention relates to function identification and application of an Arabidopsis thaliana gene AIKI, in particular to application of the gene in regulation of plant growth, application of the gene in regulation of plant adverse stress tolerance and application of the gene in cultivating transgenic plant variety with adverse stress tolerance. The identification and application establish the action of AIKI in ABA regulating growth of root and seedling, action of AIKI in plant stress tolerance and regulation mechanism thereof through separation of the AIKI gene and function analysis identification, and provide important evidence for studying ABA regulated plant seedling, in particular to root growth mechanism, so that early growth of seedling can be controlled by the gene in agriculture production to make the growth of the seedling tolerate adverse stress better, and theory and production practical foundation for cultivating new variety of transgenic crops are laid. Compared with wild Arabidopsisthaliana, growth of seedling and root of the AIKI gene function deletion mutant seedling presents reduced single sensibility on ABA.
Description
Technical field
The present invention relates to Function Identification and the application of a kind of arabidopsis gene AIK1, relate in particular to the application of this gene in regulating growth of plants, also relate to simultaneously the application of this gene aspect coercing in the degeneration-resistant border of regulating plant, and the application of this gene aspect the stress transgenic plant variety of the degeneration-resistant border of cultivation, belong to the genetically engineered field.
Background technology
Root system of plant is as important vegetative organ, not only bearing the vital role that absorbs moisture and mineral nutrition, and be that plant experiences that soil moisture stress, saline and alkaline, nutrition are deficient to be lacked and the important receptor the during environment-stress such as soil disease and pest, be the important factor of restriction agriculture production.Studies show that, Abscisic Acid ABA (abscisic acid) is a kind of general, the important root adverse circumstance signal of plant when experiencing environment-stress, when experiencing water stress such as plant, the tip of a root (root-hair zone is to root cap) is by synthetic a large amount of ABA, growing of regulation and control root, promote well developed root system (comprising the growth that suppresses main root, the growth that promotes the root hair etc.), thereby promote suction and the exudation speed of root system, guarantee that root is to the service discharge of over-ground part.
Abscisic Acid ABA is considered to the most important hormone of coercing in the plant materials, and existing ABA causes that in several minutes pore closes fast in the Under Drought Stress in Plant.Simultaneously, ABA can suppress the biosynthesizing of nucleic acid and protein as growth inhibitor, suppresses the germination of seed and the growth of plant.Although the finite concentration Exogenous ABA as plant important coerce hormone and growth inhibitor, the physiological phenomenon that can suppress growth (comprising the growth of seed germination and root) has been found that more than 30 year, has comprised the mechanism that suppresses root growth and solves seldom but suppress growth for ABA.Roots of plants is to the impression of ABA signal, the inter-level of signal transduction and to suppress in the growth and development process cell mechanism etc. be the important topic that the scientists expectation solves always.Key factor in many signal transduction processes is such as Ca
2+, pH, cADP ribose, H
2O
2Suppress seed germination and regulate in the signal transduction of stomatal movement to play an important role at ABA with MAPK etc.Whether these middle elements play regulating and controlling effect too in root and seedling development, how to act on; Which gene has been brought into play effect in the perception of ABA and the signal transduction, all becomes interesting problem.Understand fully that root and different tissues in the growth and development of plants experience the signal transduction mechanism of ABA, the mechanism of not only experiencing various environment stresses to disclosing plant provides has substantial theory value, control the root system development of plant, and agriculture production and crop improvement are had important practical significance.
Summary of the invention
The object of the present invention is to provide the application of a kind of arabidopsis gene AIK1 aspect the ABA regulating growth of plants, to expand the range of application of Gene A IK1.
Simultaneously, the object of the invention also is to provide a kind of arabidopsis gene AIK1 regulating plant anti-application of coercing the aspect.
Further, the present invention also aims to provide the application of a kind of arabidopsis gene AIK1 aspect cultivation resistant transgenic plant.
To achieve these goals, technical scheme of the present invention has adopted the application of a kind of arabidopsis gene AIK1 in regulating growth of plants.
Described Gene A IK1 is numbered At2G18470 in Genebank, the CDS length of this gene is 1902bp, 633 the amino acid whose albumen of encoding.
Described Gene A IK1 participates in plant seedlings and the root growth growth course of environment stress hormone ABA regulation and control; Gene A IK1 a kind of receptor albuminoid kinase that is positioned cytolemma of encoding, the expression of this gene is subjected to inducing of ABA.
Simultaneously, technical scheme of the present invention has also adopted a kind of arabidopsis gene AIK1 in regulating plant is anti-aspect coercing application.
Described Gene A IK1 is numbered At2G18470 in Genebank, the CDS length of this gene is 1902bp, 633 the amino acid whose albumen of encoding.Described Gene A IK1 participates in plant seedlings and the root growth growth course of environment stress hormone ABA regulation and control; The seedling of the AIK1 gene T-DNA insertion mutation body (SALK_026953, SALK_034666) that obtains from Arabidopis thaliana resource center (ABRC, Ohio State University) and the growth fraction wild-type of root are to the Reduced susceptibility of adverse circumstance hormone ABA.
A kind of receptor albuminoid kinase that is positioned cytolemma of described Gene A IK1 coding, the expression of this gene is subjected to inducing of ABA.
Say that further technical scheme of the present invention has adopted the application of a kind of arabidopsis gene AIK1 aspect the anti-stress transgenic plant of cultivation; With the AIK1 gene or wherein the part segment be connected on the different sorts carrier, transform by different expression vector and obtain degeneration-resistant relevant transgenic plant.
The present invention utilizes the AIK1 gene T-DNA insertion mutation body that obtains from Arabidopis thaliana resource center, and research finds that the growth of mutant (aik1-1, aik1-2) seedling and root shows obvious Reduced susceptibility to ABA.The clone has obtained the AIK1 gene, and this gene of overexpression can recover to lack plant to the susceptibility of ABA.Establish AIK1 by molecular biological analysis and only be subjected to inducing of hormone ABA.
The present invention utilizes cytobiology technology to disclose AIK1 and mainly works by the elongation growth of regulating cell in the growing of ABA regulating plant root.The clone has obtained this Gene A IK1, by adopting the cellular localization method of GUS tissue positioned and GFP fusion rotein, analyzes cell and the tissue expression of AIK1 gene.
The present invention utilizes patch clamp technique, and it is by Ca in the regulation and control downstream signaling molecule born of the same parents that calcium imaging technique and transgenic technology have been established AIK1
2+Concentration and participated in the root development of the seedling of ABA regulation and control.Utilize simultaneously hybridization technique, laser co-focusing, the technology such as quantitative PCR have also been established H
2O
2Also tool has certain effect in the seedling root development that AIK1 participates in regulating and control.
The present invention utilize yeast vivoexpression carrier with the AIK1 gene protein at vivoexpression and purifying, analysis of Phosphorylation has disclosed the effect of substrate phosphorylation and the autophosphorylation of albumen, and Ca
2+With the remarkable kinase activity of Enhancin of ABA.
The present invention is numbered receptoroid protein kinase of AIK1 (ABA Insensitive Kinase1) genes encoding of At2G18470, be positioned on the cytolemma, have important effect in the signal transduction of plant to adverse circumstance signal ABA, the relative growth that has participated in the plant stress response is grown.By be associated with the cell walls perception or transmit extraneous ABA signal of its extracellular region, regulate courier Ca in the born of the same parents by the kinases district in the born of the same parents
2+Content so that plant is made corresponding reaction to the adverse circumstance hormone.Lack the adverse-resistant characteristic that this gene can improve plant, the adverse circumstance hormone is shown significant Reduced susceptibility.The present invention identifies by separation and functional analysis to the AIK1 gene, established effect and effect in plant stress-resistance and the regulation mechanism thereof of AIK1 in the growing of ABA regulation and control root and seedling, for understand ABA regulating plant seedling especially root growth mechanism important evidence is provided, utilize this gene can control the early growth of seedling in agriculture production, make the growth of seedling more tolerate environment stress, establish theoretical and the production practice basis for cultivating the genetically modified crops new variety.Compare with the wild-type Arabidopis thaliana, the growth of AIK1 gene function deletion mutant seedling and root growth present single-minded susceptibility to ABA and descend.
Description of drawings
Fig. 1 is deletion mutant aik1 and the phenotype analytical of overexpression AIK1 gene Arabidopis thaliana root growth on the ABA substratum;
Fig. 2 deletion mutant aik1-1 seedling is containing 17 days the seedling of substratum growth of ABA;
Fig. 3 AIK1 is by Ca in the regulation and control born of the same parents
2+And participated in ABA growing to roots of plants.
Wherein, among Fig. 1: (A) expression of sxemiquantitative pcr analysis AIK1 gene in wild-type, aik1-1 and aik1-2 mutant and overexpression (aik1/AIK1) plant; (B) ABA processes the growth of lower root.
Among Fig. 3, (A) calcium ion carrier A 23187, CaCl
2With the impact of ockers nifedipine on Arabidopis thaliana aik1-1 and aik1-2 seedling root growth; (B) the extended length statistics of processing root after 3 days.
Embodiment
The screening of mutant, phenotype and genetic analysis
Ask for T-DNA insertion mutation body SALK_026953 and SALK_034666 from Arabidopis thaliana resource center (Arabidopsis Biological Resource Center), it is carried out homozygote identify; Utilize the T-DNA screened to insert the homozygous mutation body, analyze and obtain as shown in Figure 1 the transcriptional level gene and do not express in deletion mutant, the growth phase of root obviously shows as the Reduced susceptibility of ABA to wild-type in the ABA processing; And the growth of seedling also shows as the Reduced susceptibility to ABA as shown in Figure 2.Show that the AIK1 gene regulated the growth of seedling in environment stress.
The AIK1 gene cloning, vector construction, the selection of transfer-gen plant
Promoter region sequence and the CDS sequence of from the arabidopsis gene group, utilizing pcr amplification to separate the AIK1 that obtains, the clone obtains the GUS report carrier of pCAMBIA1381 and the GFP cellular localization carrier of pEGAD, and overexpression vector; Agrobacterium mediation converted obtains the transgenic positive plant.
Search the whole genome sequence of AIK1 from Genbank, then swim over to seek the terminator codon of a upper gene from initiator codon ATG and contain the sequences such as TATA box, CAAT box, then nearly 1381bp designs primer.According to the multiple clone site of pCMBIA1381 Vector, select EcoRI and PstI restriction enzyme site design primer.With the wild-type arabidopsis thaliana genomic dna as template, the promotor of pcr amplification AIK1.And simultaneously according to Arabidopis thaliana AIK1 CDS sequences Design specific amplification primer, and add restriction enzyme site to introduce overexpression vector pBIB at the primer two ends.The full length sequence of amplification CDS is connected into the pBIB carrier after enzyme is cut.Utilize simultaneously the CDS of the pEGAD-GFP-AIK1 primer PCR amplification AIK1 of design, enzyme is cut and is connected into the pEGAD carrier, construction recombination plasmid, and cut evaluation by PCR and enzyme.
Change the carrier that builds over to Agrobacterium GV3101, screening positive clone transforms to be used.Inoculate positive Agrobacterium GV3101 and (contain 50 μ g/mL Rif in the 10mLLB substratum, 50 μ g/mL Kan) in, 28 ℃ of 200rpm shaking culture are spent the night, in 1: 50 ratio be forwarded to 200mL contain in the identical antibiotic LB substratum enlarged culturing to OD600 be 1.2-1.6, the centrifugal 15min of 5000rpm collects thalline.Be resuspended in and infiltrate damping fluid (0.25 * MS, 5% (W/V) sucrose, 0.03%silwet L-77), regulate OD600 to 0.8.Treat that Arabidopis thaliana grows to bud stage, ready Agrobacterium be positioned in the cuvette with the flowerpot caliber size, contaminate 3s after, with preservative film whole plant cover is got up, lie in (attention moisturizing) in the vinyl disc.After 10h is placed in the room temperature dark place, open again film and be positioned over the cultivation of illumination cultivation chamber.After one week, treated that new bud grows, can repeat to transform.The seed that obtains is identified.
The pCMBIA1381 that builds and the Agrobacterium-mediated Transformation wild-type Arabidopis thaliana of pEGAD recombinant vectors carrier will be carried; The T-DNA that carries the Agrobacterium-mediated Transformation evaluation of the pBIB carrier that builds inserts the homozygous mutation body.
Carrier pBIB and pCMBIA1381 are with Hygromycin B resistant gene, with the transgenic plant of the MS screening culture medium acquisition that contains Hygromycin B.F1 is sowed on the MS screening culture medium that contains 25mg/L Totomycin (hygromycin B) for transgenic plant seed, until behind the 15d complete normotrophic plantlet of transplant in Nutrition Soil, Deng restoreing normal growth, extract again genomic dna, carry out PCR and detect.
Carrier pEGAD has the resistance to weedicide Bastar with Bastar resistance protein gene.When obtaining F1 and growing to two true leaves after for planting seed, spray the Bastar screening resistant plant of 35mg/L, spray once every three days, spray altogether 3 times.Well-grown resistance seedling is shifted out.Screen to such an extent that F2 is used for the analyzing proteins location for seed.The seedling of size takes out with 2 week of growth on the substratum, the flush away substratum, get root and carry out the Fluirescence observation observation, use laser confocal microscope (Laser Scanningconfocal microscope, LSCM), observe GFP fluorescence at Green channel (488nm excitation, 510-550nm emission).
Embodiment 3
The expression analysis of AIK1 gene
Utilize sxemiquantitative pcr analysis AIK1 gene pairs hormon reaction, disclose and only to be subjected to inducing of ABA, and be not subjected to other hormone-treated impact.The transgenic plant that utilization obtains show that by the GUS histochemical stain AIK1 mainly is distributed in the root of plant, floral organ and spire, this with this gene under ABA processes the control root grow consistent.The protein localization that GFP fusion rotein positioning analysis represents this genes encoding is on cytolemma, and the Degrading experiment by cell walls discloses with cell walls and has certain cognation.This shows that also AIK1 plays a role in the early stage transduction of adverse circumstance signal.
Embodiment 4
The analysis of AIK1 protein kinase function
Utilize the existing yeast expression system in laboratory to make up the vivoexpression carrier, the external source inducible protein is expressed, purifying protein.Detect protein kinase activity, detected result shows that AIK1 albumen has the function that son is given birth to phosphorylation and substrate phosphorylation, and at external use ABA and Ca
2+Kinase activity can obviously strengthen after processing.Further specifying AIK1 should play a role in the signal transduction of adverse circumstance hormone ABA, and has close relationship with second messenger's calcium ion.
Embodiment 5
The Analysis on Mechanism of AIK1 gene regulating root growth
Utilizing the cytology methods analyst to disclose AIK1 mainly is by having regulated the elongation growth of root cells to the regulation and control that the relative ABA of seedling root grows.Utilize the Laser scanning confocal microscopy technology, the calcium imaging technique is analyzed ABA and is processed H in the lower root cells
2O
2And Ca
2+Dynamic change; Ca on the Root cell membrane after utilizing patch clamp technique monitoring ABA to process
2+The activity change of ionic channel, the present invention utilizes again Calcium ionophore and ockers that root growth is made phenotype analytical (such as Fig. 3), is to have regulated the response of root to adverse circumstance by the length that the change of regulating cellular calcium concentration has affected cell thereby net result discloses AIK1 on the reaction of hormone ABA.
Claims (4)
1. arabidopsis gene
AIK1Application in regulating growth of plants is characterized in that: described gene
AIK1Be numbered At2G18470 in Genebank, the CDS length of this gene is 1902bp, 633 the amino acid whose albumen of encoding.
2. application according to claim 1 is characterized in that: described gene
AIK1Participate in plant seedlings and the root growth growth course of environment stress hormone ABA regulation and control.
3. arabidopsis gene
AIK1Application aspect the anti-stress transgenic plant of cultivation, described gene
AIK1Be numbered At2G18470 in Genebank, the CDS length of this gene is 1902bp, 633 the amino acid whose albumen of encoding.
4. arabidopsis gene according to claim 3
AIK1Application aspect the stress transgenic plant of the degeneration-resistant border of cultivation is characterized in that: will
AIK1Gene is connected on the different sorts carrier, transforms by different expression vectors and obtains degeneration-resistant relevant transgenic plant, described gene
AIK1Be numbered At2G18470 in Genebank, the CDS length of this gene is 1902bp, 633 the amino acid whose albumen of encoding.
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| CN 200910064420 CN101509006B (en) | 2009-03-19 | 2009-03-19 | Uses of arabidopsis thaliana gene AIK1 in regulating and controlling vegetation growth, and in stress resistance aspect |
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| CN 200910064420 CN101509006B (en) | 2009-03-19 | 2009-03-19 | Uses of arabidopsis thaliana gene AIK1 in regulating and controlling vegetation growth, and in stress resistance aspect |
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| CN116590314B (en) * | 2023-07-11 | 2023-09-29 | 烟台吉恩生物科技发展有限公司 | Gene for improving salt and alkali tolerance of wheat and application thereof |
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