CN102643856B - Applications of At-ACA8 gene of arabidopsis thaliana for enhancing plant stress resistance and regulating plant growing development - Google Patents

Abstract

The invention provides an application of an arabidopsis thaliana calcium ion transported-ATP (Adenosine Tri-Phosphate) enzyme gene At-ACA8. The application comprises an application of the gene on aspects of regulating plant growing development and resisting adverse circumstance stress, and an application of the gene in an aspect of an adverse circumstance stress resisting transgenic plant variety. A T-DNA (Transforming-Deoxyribonucleic Acid) inserting mutant aca8 of the At-ACA8 gene obtained from an arabidopsis thaliana biological resource center is utilized as a target and an experiment result confirms that the mutant has obvious resistances on adverse circumstances of low temperature, high temperature, penetration compelling and the like; a seed germination phase is sensitive to whether sucrose exists or not; the germination of seeds is seriously inhibited under the condition of not adding the sucrose and a growing phase is more sensitive to the raising of the concentration of the sucrose; the seed germination phase is sensitive to a hormone ABA (Abscisic Acid); and less ABA is applied to obviously inhibiting the germination of the seeds. The invention discloses responding functions of regulating the plant growing development and resisting stress of the calcium ion conduction system and provides a good-quality gene for the variety improvement of the plants; the invention represents a new method and a mechanism which are used for improving the resisting adverse circumstance resisting capability and have good resisting variety and important economic meanings and application values.

Description

The application of Arabidopis thaliana At-ACA8 gene in strengthening stress resistance of plant and regulating growth of plants

Technical field:

The invention belongs to plant genetic engineering field, particularly, relate to the utilization of a kind of Arabidopis thaliana calcium ion transport-ATP zymoprotein Gene A t-ACA8, the mutant aca8 that relates to Gene A t-ACA8 is in the application improving aspect Genes For Plant Tolerance low temperature, high temperature, osmotic stress and hormone response and on regulating growth of plants, meanwhile, also relate to this Gene A t-ACA8 cultivation have various abiotic stress resistance, to without sucrose and high concentration sucrose sensitivity and seed germination stage to the application aspect the transgenic plant kind of hormone ABA sensitivity.

Background technology:

Growing of plant is closely related with their existing environment.Occurring in nature, due to many-sided reasons such as different geographical position, weather condition and mankind's activities, has caused various adverse circumstances, and plant is everlasting under hostile environment condition and grows, as arid, waterlogging, salt damage, low temperature, high temperature, disease and pest etc.Concerning agriculture production, various adverse circumstances are to affect crop yield and the most direct, the most important factor of quality.Therefore, strengthen the research of physiological responses of plants to anti-environment, verify the vital movement rule of plant under adverse circumstance artificial adjustment in addition, cultivate the improved seeds with opposing poor environment proterties, to improve the yield and quality of crop, for obtaining, agricultural year stable yields is significant.

Plant has the ability to environmental change quick sensing and active adaptation.Plant experiences adverse circumstance signal, conduction adverse circumstance by cell to be stimulated, activate a series of molecular pathways and regulates and controls the expression of genes involved and 3 stages such as physiological response adapt to adverse circumstances, obtains resistance in adverse circumstance.Resistant gene is the identification of stress factors to external world, needs signalling system accurate and responsive in cell for basis.Some researchs show, have the signal thing of systemic transmission of information under adverse circumstance in plant materials, and the main signal molecule of finding at present has Ca ²⁺, protein kinase, H ⁺(pH), ABA, ROS and NO etc., they are as signal transmitter, the degeneration-resistant reaction of involved in plant.

Ca ²⁺as the second messenger in plant cell signaling transduction process, extensively approved.In vegetable cell, outside cytoplasmic matrix and born of the same parents or between calcium stores (some organoid), there is a very large Ca ²⁺concentration extreme difference.Studies confirm that, various born of the same parents' external stimulus signal, as Drought and salt is coerced, heat shock, low temperature, oxidative stress and anoxic etc. all may cause Free Ca in vegetable cell ²⁺the variation of concentration, causes Ca ²⁺in intracellular Gradient distribution or distributed areas.Free Ca in born of the same parents ²⁺the variation of concentration is mainly to pass through Ca ²⁺cross-film running or the adjusting of calcium chelate realize, on plasma membrane, vacuole skin, endoplasmic reticulum, found calcium ion pump, calcium channel and Ca ²⁺/ H ⁺the existence of exchange system.Direct or indirect these calcium haulage system of adjusting of various born of the same parents' external stimulus signals possibility, thus Free Ca in born of the same parents caused ²⁺the variation of concentration, and cause different cell responses.

Calcium ion pump (calcium pump) is also called Ca ²⁺-ATP enzyme (Ca ²⁺-ATPase), for Ca in cell ²⁺the conduction of signal is extremely important, belong to P type ATP enzyme supergene family, according to protein sequence homology principle, be divided into again two unique IIA of calcium pump family and IIB gene family, the member in Zhe Liangge family comprises respectively endoplasmic reticulum membranous type calcium ion pump (ECA type) and the calcium ion of inhibition type certainly pump (ACA type).In Arabidopis thaliana, 4 ECA type calcium ion pump encoding genes and 10 ACA type calcium ion pump encoding genes have altogether been found at present.

At-ACA8 gene is an important member in ACA type calcium ion pump gene family, is positioned on plasma membrane, exercises by utilizing the energy of ATP hydrolysis release by intracytoplasmic Ca ²⁺the function of pump to born of the same parents, and have higher sequence homology with two other member At-ACA9 and At-ACA10 gene in family.Research before finds, At-ACA8 and At-ACA10 gene have expression in all organs of Arabidopis thaliana, and At-ACA9 gene is only being spent middle expression, apply hormone ABA can quick active seedling in the expression of At-ACA8 and At-ACA10 gene; The disappearance of Arabidopis thaliana At-ACA9 gene function, can uphold and generating portion male sterile by remarkably influenced pollen tube; At-ACA10 gene has has regulated and controled the elongation growth in vegetation growth of plant stage, and the normal development of inflorescence structure is played a decisive role.About mainly concentrating on N end, the research of At-ACA8 gene forms and from inhibit feature, the cytobiology effect in its regulating growth of plants stage did not also have report from the amino acid of arrestin structural domain.

Summary of the invention:

The object of the invention is to provide the application of a kind of Arabidopis thaliana calcium ion transport-ATP enzyme Gene A t-ACA8 aspect regulating growth of plants and anti-coercing.

The present invention also aims to provide the application of a kind of arabidopsis gene At-ACA8 aspect cultivation resistant transgenic plant.

Simultaneously, the present invention also aims to provide a kind of arabidopsis gene At-ACA8 and Arabidopis thaliana At-ACA8 gene T-DNA insertion mutation body aca8 to have responsive without sucrose and high concentration sucrose in cultivation, the seed germination stage is responsive to hormone ABA, applies the application that a small amount of ABA significantly suppresses the transgenic plant aspect of seed germination.

Technical scheme of the present invention is achieved like this:

The application of calcium ion transport-ATP enzyme Gene A t-ACA8 of Arabidopis thaliana in improving Genes For Plant Tolerance adverse circumstance ability.

The application of calcium ion transport-ATP enzyme Gene A t-ACA8 of Arabidopis thaliana in regulating growth of plants.

The application of arabidopsis gene At-ACA8 in cultivating cryophylactic transgenic plant.

The application of arabidopsis gene At-ACA8 in the transgenic plant of cultivating high temperature resistance.

The application of arabidopsis gene At-ACA8 in cultivating drought-resistant transgenic plant of coercing.

Arabidopsis gene At-ACA8 is cultivating the application in the transgenic plant without sucrose and high concentration sucrose sensitivity.

Arabidopsis gene At-ACA8 is to cultivate the seed germination stage responsive to hormone ABA, and lower concentration ABA significantly suppresses the application in the transgenic plant of seed germination.

The application of Arabidopis thaliana At-ACA8 gene T-DNA insertion mutation body aca8 in cultivating cryophylactic transgenic plant.

The application of Arabidopis thaliana At-ACA8 gene T-DNA insertion mutation body aca8 in the transgenic plant of cultivating high temperature resistance.

The application of Arabidopis thaliana At-ACA8 gene T-DNA insertion mutation body aca8 in cultivating drought-resistant transgenic plant of coercing.

Arabidopis thaliana At-ACA8 gene T-DNA insertion mutation body aca8 is cultivating the application in the transgenic plant without sucrose and high concentration sucrose sensitivity.

Arabidopis thaliana At-ACA8 gene T-DNA insertion mutation body aca8 is to cultivate the seed germination stage responsive to hormone ABA, and lower concentration ABA significantly suppresses the application in the transgenic plant of seed germination.

In above-mentioned application, described transgenic plant are by the At-ACA8 gene in Arabidopis thaliana and the homologous gene that derives from other plant, complete clone or wherein part fragmentation, and by being connected on different sorts carrier after replacement, increase and decrease base, and transform by different expression vectors the transgenic plant that obtain different resistances.

Technical scheme of the present invention is that the principle based on following draws: a kind of T-DNA mutant aca8 of Arabidopis thaliana At-ACA8 gene has remarkable tolerance effect to multiple environment-stress (low temperature, high temperature and osmotic stress); To having or not the sucrose sensitive of sucrose and high density; The seed germination stage is responsive to hormone ABA, applies a small amount of ABA and can significantly suppress seed germination.Find out thus, At-ACA8 gene all plays extremely important regulating and controlling effect to the vital movement of plant under multiple environment-stress, study it as the effect of the degeneration-resistant border of calcium ion pump regulating plant ability, further from molecular level, illustrate the mechanism of calcium signal transduction system involved in plant resistance reaction, the mechanism of experiencing various environment stresses to disclosing plant provides has substantive value, and agriculture production and crop improvement are had to important realistic meaning.

The variation of genetic expression on three typical phases that the present invention's application Affymetrix ATH1 chip detection Arabidopis thaliana response low temperature freezing-disaster is coerced (cold tame and docile, freeze, melt) transcriptional level.Screening is found at this three phases Arabidopis thaliana Ca ²⁺all there is remarkable change in the expression amount of-ATP enzyme Gene A t-ACA8, especially the expression amount at freeze thawing stage gene obviously rises, and according to before to Ca ²⁺the elaboration of signaling molecule function, illustrates that this gene may be the key gene of plant responding low temperature stress, at Ca ²⁺in the low-temperature signal conduction path of mediation, play a significant role.

The numbering of At-ACA8 gene in GenBank is AT5G57110.1 and AT5G57110.2, and CDS sequence is long is 3225bp, 1074 amino acid of encoding.With SMART programanalysis prediction At-ACA8 albumen, this albumen contains following important structural domain, as shown in Figure 1: Ca ²⁺-ATP enzyme N end from inhibitory character structural domain (Ca ²⁺-ATPase N terminal autoinhibitory domain, 27-72 position), be present in eukaryote, by approximately 50 amino acid, formed, contain conservative RRFR sequential element, Phe and Trp are two conservative amino acid that enforcement plays a decisive role to function; Cation transfer body/ATP enzyme N end structure territory (Cation transporter/ATPase N terminus, 135-209 position); E1-E2ATP enzyme (223-472 position), also claims P type ATP enzyme, and major function is the various ions of energy transmembrane transport and the phosphatide that utilizes ATP hydrolysis to produce; Lytic enzyme (Hydrolase, 476-797 position) structural domain; Cation transfer body/ATP enzyme C end structure territory (Cation transporter/ATPase C terminus, 867-1045 position), the spirane structure that 5 cross-films are contained in this family.

Arabidopis thaliana Ca of the present invention ²⁺the albumen of-ATP enzyme Gene A t-ACA8 coding is mainly positioned on cytoplasmic membrane, and this gene is mainly exercised Ca on cytoplasmic membrane ²⁺function by intracellular transport to born of the same parents.

The present invention utilizes the At-ACA8 gene T-DNA insertion mutation body aca8 (seed is numbered CS859889) obtaining from Arabidopis thaliana Biological resources center ABRC (Arabidopsis Biological Resource Center) as research object, find that this mutant has the resistibility of the wild-type of being significantly higher than to adverse environmental factors such as low temperature, high temperature, osmotic stresses, to responsive without sucrose and high concentration sucrose, the seed germination stage is responsive to hormone ABA, applies a small amount of ABA and significantly suppresses seed germination.

Positively effect of the present invention is:

Arabidopis thaliana Ca of the present invention ²⁺the relevant physiological of-ATP enzyme Gene A t-ACA8 experimental results show that, this gene involved in plant grows and regulates and the process such as stress response, and the present invention understands calcium singal transduction system and aspect regulating growth of plants and stress response, having substantial value deep; Because aca8 mutant has significant resistibility to low temperature, high temperature, osmotic stress, and studies have shown that in a large number in the past be take most of functional genes that Arabidopis thaliana identified as material and can be applied in the research of other plant, therefore the present invention is also for the improvement of plant variety provides a Fineness gene, set forth a kind of new raising plant opposing adverse circumstance ability and cultivated method and the mechanism with good resistant variety, having there is important economic implications and utilization prospect.

Accompanying drawing explanation:

The structural domain of SMART programanalysis prediction At-ACA8 albumen for Fig. 1;

The evaluation of Fig. 2 At-ACA8 gene T-DNA insertion mutation body; The insertion point of 2A, T-DNA insertion mutation body aca8 (CS889) and PCR primers designed design diagram; The homozygote PCR of 2B, T-DNA insertion mutation body aca8 (CS889) identifies electrophorogram; In the wild-type of 2C, 22 ℃ of normal growths and mutant aca8 blade, At-ACA8 gene transcription level sxemiquantitative RT-PCR identifies;

Fig. 3 low temperature stress is processed the Analysis of Resistance of mutant aca8; 3A low temperature stress is processed aseptic seedling Col-0 and aca8 phenotype and the maximum Photochemical quantum yield of Photosystem I I (Fv/Fm) and is changed; 3B, low temperature stress are processed earth culture seedling Col-0 and aca8 phenotype; 3C, low temperature stress process earth culture seedling Col-0 and the maximum Photochemical quantum yield of aca8 blade Photosystem I I (Fv/Fm) changes; 3D, low temperature stress are processed low temperature responsive genes transcriptional level semi-quantitative RT-PCR analysis in Col-0 and aca8 earth culture seedling leaf;

Fig. 4 high temperature stress is processed the Analysis of Resistance of mutant aca8; 4A, high temperature stress are processed Col-0 and aca8 aseptic seedling phenotype and the maximum Photochemical quantum yield of Photosystem I I (Fv/Fm) and are changed; 4B, high temperature stress are processed Col-0 and aca8 earth culture seedling phenotype; 4C, high temperature stress process Col-0 and the maximum Photochemical quantum yield of aca8 earth culture seedling leaf Photosystem I I (Fv/Fm) changes;

Fig. 5 drought stress is processed the Analysis of Resistance of mutant aca8; 5A, 22 ℃ long-term dewater Col-0 and aca8 earth culture seedling phenotypes; The PEG simulating drought of 5B. different concns is processed Col-0 and aca8 phenotype;

The ABA of Fig. 6 different concns processes seed germination situation and the green bud rate of Col-0 and aca8 and analyzes;

The seed germination situation of the saccharose treatment Col-0 of Fig. 7 different concns and aca8 and green bud rate are analyzed.

Embodiment:

Below in conjunction with accompanying drawing, with embodiments of the invention, further illustrate essentiality content of the present invention, but with this, do not limit the present invention.

Embodiment 1:

The screening of At-ACA8 homozygous mutation body aca8 and gene expression amount detect:

(1) screening of At-ACA8 homozygous mutation body aca8:

The Arabidopis thaliana T-DNA insertion mutation body (seed is numbered CS859889) of At-ACA8 gene (sequence table is as shown in table 1, table 2, table 3), by PCR, identify homozygous mutation strain, as shown in Figure 2 A, the primer is as follows: 5 ' end primer: 5 '-CTTCAACGCCTTTGTTCTCTG-3 '; 3 ' end primer: 5 '-CCAGTAATCCAAAGTTGCTCG-3 '; LBpROK2:5 '-ATTTTGCCGATTTCGGAA C-3 ', further sequence verification finds that this mutant comes from T-DNA and inserts in the 34th exon, and causes transgenation.Result as shown in Figure 2 B, identifies that CS889 is homozygote.

(2) in Col-0 and mutant aca8 blade, the sxemiquantitative RT-PCR of At-ACA8 gene transcription level identifies:

The total RNA that extracts the approximately 3 weeks big or small Arabidopis thaliana earth culture seedling leafs of growing at normal 22 ℃, carries out semi-quantitative RT-PCR analysis.At-ACA8 gene primer used is: F:5 '-GATGCTCCTGCATTGCACGA-3 ', R:5 '-CGAGAGCGGCGACATTGACT-3 '; Actin gene primer is: F:5 '-TGTGCCAATCTACGAGGGTTT-3 ', R:5 '-TTTCCCGCTCTGCTGTTGT-3 '.Result as shown in Figure 2 C, under different high and low temperature treatment condition, in aca8 and Col-0 there is significant difference in the expression amount of At-ACA8 gene, show the insertion of T-DNA, cause the transcriptional level expression of At-ACA8 gene in mutant that the change of wild-type has occurred to be different from, thereby determined the response process that At-ACA8 gene involved in plant is coerced high and low temperature.

In this test, there are two transcripts in Gene A t-ACA8, the numbering in GenBank is AT5G57110.1 and AT5G57110.2, and CDS sequence is long is 3225bp, 1074 amino acid of encoding, its nucleotide sequence and aminoacid sequence are as shown in SEQ ID No 1 and SEQ ID No 2.The Arabidopsis Mutants aca8 that the At-ACA8 gene T-DNA that utilization obtains from Arabidopis thaliana Biological resources center ABRC (Arabidopsis Biological Resource Center) inserts is as research object, the seed of mutant strain is numbered CS859889, one section of T-DNA that mutant aca8 comes from carrier pROK2 inserts in the 34th exon of Gene A t-AC48, cause transgenation, the flanking sequence at insertion point place is as shown in SEQ ID No 3.

Table 1At-ACA8 gene nucleotide series table

1 ATGACGAGTC TCTTGAAGTC ATCGCCTGGA CGACGCCGTG GAGGCGATGT

51 GGAGTCTGGG AAGAGTGAAC ACGCAGACTC TGATAGCGAC ACTTTCTACA

101 TCCCATCGAA GAATGCTTCT ATTGAGCGGC TTCAACAGTG GAGAAAAGCT

151 GCACTGGTTC TCAATGCGTC CCGGAGATTC CGGTACACCT TGGACTTGAA

201 GAAGGAGCAA GAAACGAGGG AAATGAGACA GAAGATCAGA AGTCATGCTC

251 ATGCTCTTTT GGCGGCGAAT CGTTTTATGG ATATGGGACG TGAGTCAGGA

301 GTTGAAAAAA CAACTGGGCC TGCAACTCCG GCTGGTGATT TTGGAATTAC

351 TCCTGAACAG CTTGTGATAA TGTCAAAGGA TCACAACAGT GGTGCCCTGG

401 AGCAATATGG AGGGACTCAA GGATTGGCCA ATTTGCTCAA GACAAATCCA

451 GAGAAAGGTA TCAGTGGCGA TGATGATGAC TTGCTAAAGC GCAAGACCAT

501 TTATGGATCA AACACATATC CTCGCAAGAA AGGGAAAGGG TTTCTGAGGT

551 TTCTTTGGGA TGCTTGCCAT GATCTCACCT TGATCATTTT AATGGTTGCT

601 GCTGTAGCGT CTTTAGCACT TGGGATAAAA ACAGAGGGTA TCAAAGAAGG

651 ATGGTATGAT GGAGGAAGCA TTGCATTTGC TGTGATTCTT GTGATTGTTG

701 TGACGGCTGT CAGTGACTAC AAACAGTCGC TCCAGTTTCA AAACTTGAAT

751 GATGAAAAGA GAAACATACA TCTGGAGGTG TTAAGAGGTG GAAGACGAGT

801 GGAGATTTCG ATCTATGACA TAGTGGTGGG TGATGTCATT CCTCTCAACA

851 TTGGCAATCA GGTTCCTGCT GATGGAGTAC TGATATCTGG CCACTCTCTC

901 GCCCTTGATG AATCTAGCAT GACTGGAGAG AGCAAAATTG TTAACAAAGA

951 CGCTAACAAG GACCCATTCT TAATGTCTGG CTGTAAAGTG GCAGATGGAA

1001 ATGGTTCTAT GCTGGTTACT GGTGTTGGAG TCAACACTGA ATGGGGATTG

1051 CTGATGGCCA GTATTTCTGA AGACAATGGT GAAGAAACTC CCTTGCAGGT

1101 GCGCTTAAAT GGGGTAGCTA CTTTTATTGG TTCAATTGGC TTAGCGGTGG

1151 CTGCTGCTGT GCTAGTGATT CTTCTGACTC GATATTTCAC TGGTCACACT

1201 AAAGACAATA ATGGAGGTCC TCAATTCGTT AAAGGCAAGA CAAAAGTCGG

1251 TCATGTTATT GATGATGTGG TCAAAGTCCT TACCGTAGCG GTTACAATTG

1301 TCGTAGTGGC AGTGCCTGAG GGTCTTCCTT TGGCTGTTAC TTTAACCCTT

1351 GCCTATTCAA TGAGGAAAAT GATGGCAGAT AAGGCTTTGG TGCGGAGGCT

1401 ATCTGCTTGC GAGACAATGG GCTCTGCCAC CACTATTTGC AGCGATAAAA

1451 CTGGAACACT AACTCTGAAT CAGATGACTG TGGTTGAGTC TTATGCTGGG

1501 GGCAAGAAAA CAGATACTGA ACAATTGCCA GCAACTATCA CTTCGCTAGT

1551 AGTTGAAGGA ATATCTCAAA ACACAACTGG TAGTATTTTT GTCCCAGAGG

1601 GTGGAGGTGA TTTAGAGTAC TCTGGTTCAC CAACAGAAAA GGCCATTCTT

1651 GGCTGGGGAG TTAAGCTGGG AATGAATTTC GAGACAGCCC GATCGCAGTC

1701 TTCTATTCTT CATGCTTTTC CATTTAACTC AGAGAAGAAA CGTGGTGGTG

1751 TTGCTGTAAA AACGGCTGAT GGTGAAGTTC ATGTTCACTG GAAAGGAGCT

1801 TCTGAAATTG TCCTGGCATC ATGCCGAAGC TACATCGATG AGGATGGTAA

1851 TGTGGCACCA ATGACTGACG ACAAGGCATC GTTTTTCAAG AATGGCATTA

1901 ATGATATGGC TGGAAGAACC TTACGATGTG TTGCACTCGC CTTTAGAACC

1951 TATGAGGCTG AAAAGGTCCC AACAGGCGAA GAGCTCTCAA AGTGGGTACT

2001 CCCAGAGGAT GATCTTATAT TATTAGCTAT AGTGGGCATA AAGGATCCAT

2051 GTAGACCAGG AGTCAAAGAT TCAGTTGTGT TGTGTCAAAA TGCTGGTGTC

2101 AAGGTCCGTA TGGTTACTGG TGACAACGTC CAAACTGCCA GGGCTATTGC

2151 CTTGGAATGT GGAATACTAA GTTCAGATGC AGATCTCTCC GAGCCTACTC

2201 TCATTGAAGG AAAATCATTC CGTGAGATGA CTGATGCAGA AAGGGACAAG

2251 ATTAGTGACA AAATATCGGT TATGGGCCGA TCATCTCCCA ATGACAAACT

2301 TCTGCTGGTA CAATCTCTGA GACGACAAGG GCATGTTGTT GCTGTCACCG

2351 GGGATGGGAC AAATGATGCT CCTGCATTGC ACGAGGCAGA TATTGGTCTT

2401 GCTATGGGAA TAGCAGGAAC AGAAGTGGCT AAGGAGAGTT CGGACATCAT

2451 CATCTTGGAT GACAACTTTG CTTCAGTTGT CAAGGTTGTT CGATGGGGGC

2501 GATCAGTGTA TGCCAACATT CAGAAGTTCA TCCAATTTCA GCTCACAGTC

2551 AATGTCGCCG CTCTCGTAAT TAATGTTGTA GCTGCTATTT CGAGTGGTGA

2601 CGTTCCACTT ACCGCTGTGC AGCTTCTCTG GGTTAATCTG ATAATGGACA

2651 CTCTTGGAGC ATTGGCTTTG GCTACTGAAC CACCCACTGA TCACCTCATG

2701 GGGAGGCCTC CAGTTGGCAG AAAAGAACCT CTTATTACCA ACATCATGTG

2751 GAGAAACTTG CTGATCCAGG CTATTTATCA AGTGAGTGTA TTGTTAACAC

2801 TCAATTTCCG AGGCATAAGC ATACTTGGCC TGGAGCATGA AGTGCATGAA

2851 CACGCCACTA GAGTGAAGAA CACAATAATC TTCAACGCCT TTGTTCTCTG

2901 CCAAGCTTTC AATGAGTTCA ATGCTCGTAA ACCAGATGAG AAGAACATCT

2951 TCAAAGGTGT GATCAAGAAT CGTCTCTTTA TGGGAATAAT AGTCATAACT

3001 CTTGTGCTCC AGGTTATCAT TGTTGAGTTC CTTGGTAAAT TCGCTTCCAC

3051 GACGAAACTA AACTGGAAGC AATGGCTTAT CTGTGTTGGC ATCGGTGTTA

3101 TCAGTTGGCC TCTTGCTTTG GTCGGGAAAT TCATTCCGGT GCCTGCAGCT

3151 CCTATAAGCA ACAAATTGAA AGTACTAAAA TTCTGGGGCA AGAAGAAAAA

3201 TTCTTCTGGA GAAGGTTCAC TCTGA(Length：3225bp)

Table 2At-ACA8 aminopeptidase gene acid sequence table

1 Met Thr Ser Leu Leu Lys Ser Ser Pro Gly Arg Arg Arg Gly Gly Asp Val Glu Ser Gly

21 Lys Ser Glu His Ala Asp Ser Asp Ser Asp Thr Phe Tyr Ile Pro Ser Lys Asn Ala Ser

41 Ile Glu Arg Leu Gln Gln Trp Arg Lys Ala Ala Leu Val Leu Asn Ala Ser Arg Arg Phe

61 Arg Tyr Thr Leu Asp Leu Lys Lys Glu Gln Glu Thr Arg Glu Met Arg Gln Lys Ile Arg

81 Ser His Ala His Ala Leu Leu Ala Ala Asn Arg Phe Met Asp Met Gly Arg Glu Ser Gly

101 Val Glu Lys Thr Thr Gly Pro Ala Thr Pro Ala Gly Asp Phe Gly Ile Thr Pro Glu Gln

121 Leu Val Ile Met Ser Lys Asp His Asn Ser Gly Ala Leu Glu Gln Tyr Gly Gly Thr Gln

141 Gly Leu Ala Asn Leu Leu Lys Thr Asn Pro Glu Lys Gly Ile Ser Gly Asp Asp Asp Asp

161 Leu Leu Lys Arg Lys Thr Ile Tyr Gly Ser Asn Thr Tyr Pro Arg Lys Lys Gly Lys Gly

181 Phe Leu Arg Phe Leu Trp Asp Ala Cys His Asp Leu Thr Leu Ile Ile Leu Met Val Ala

201 Ala Val Ala Ser Leu Ala Leu Gly Ile Lys Thr Glu Gly Ile Lys Glu Gly Trp Tyr Asp

221 Gly Gly Ser Ile Ala Phe Ala Val Ile Leu Val Ile Val Val Thr Ala Val Ser Asp Tyr

241 Lys Gln Ser Leu Gln Phe Gln Asn Leu Asn Asp Glu Lys Arg Asn Ile His Leu Glu Val

261 Leu Arg Gly Gly Arg Arg Val Glu Ile Ser Ile Tyr Asp Ile Val Val Gly Asp Val Ile

281 Pro Leu Asn Ile Gly Asn Gln Val Pro Ala Asp Gly Val Leu Ile Ser Gly His Ser Leu

301 Ala Leu Asp Glu Ser Ser Met Thr Gly Glu Ser Lys Ile Val Asn Lys Asp Ala Asn Lys

321 Asp Pro Phe Leu Met Ser Gly Cys Lys Val Ala Asp Gly Asn Gly Ser Met Leu Val Thr

341 Gly Val Gly Val Asn Thr Glu Trp Gly Leu Leu Met Ala Ser Ile Ser Glu Asp Asn Gly

361 Glu Glu Thr Pro Leu Gln Val Arg Leu Asn Gly Val Ala Thr Phe Ile Gly Ser Ile Gly

381 Leu Ala Val Ala Ala Ala Val Leu Val Ile Leu Leu Thr Arg Tyr Phe Thr Gly His Thr

401 Lys Asp Asn Asn Gly Gly Pro Gln Phe Val Lys Gly Lys Thr Lys Val Gly His Val Ile

421 Asp Asp Val Val Lys Val Leu Thr Val Ala Val Thr Ile Val Val Val Ala Val Pro Glu

441 Gly Leu Pro Leu Ala Val Thr Leu Thr Leu Ala Tyr Ser Met Arg Lys Met Met Ala Asp

461 Lys Ala Leu Val Arg Arg Leu Ser Ala Cys Glu Thr Met Gly Ser Ala Thr Thr Ile Cys

481 Ser Asp Lys Thr Gly Thr Leu Thr Leu Asn Gln Met Thr Val Val Glu Ser Tyr Ala Gly

501 Gly Lys Lys Thr Asp Thr Glu Gln Leu Pro Ala Thr Ile Thr Ser Leu Val Val Glu Gly

521 Ile Ser Gln Asn Thr Thr Gly Ser Ile Phe Val Pro Glu Gly Gly Gly Asp Leu Glu Tyr

541 Ser Gly Ser Pro Thr Glu Lys Ala Ile Leu Gly Trp Gly Val Lys Leu Gly Met Asn Phe

561 Glu Thr Ala Arg Ser Gln Ser Ser Ile Leu His Ala Phe Pro Phe Asn Ser Glu Lys Lys

581 Arg Gly Gly Val Ala Val Lys Thr Ala Asp Gly Glu Val His Val His Trp Lys Gly Ala

601 Ser Glu Ile Val Leu Ala Ser Cys Arg Ser Tyr Ile Asp Glu Asp Gly Asn Val Ala Pro

621 Met Thr Asp Asp Lys Ala Ser Phe Phe Lys Asn Gly Ile Asn Asp Met Ala Gly Arg Thr

641 Leu Arg Cys Val Ala Leu Ala Phe Arg Thr Tyr Glu Ala Glu Lys Val Pro Thr Gly Glu

661 Glu Leu Ser Lys Trp Val Leu Pro Glu Asp Asp Leu Ile Leu Leu Ala Ile Val Gly Ile

681 Lys Asp Pro Cys Arg Pro Gly Val Lys Asp Ser Val Val Leu Cys Gln Asn Ala Gly Val

701 Lys Val Arg Met Val Thr Gly Asp Asn Val Gln Thr Ala Arg Ala Ile Ala Leu Glu Cys

721 Gly Ile Leu Ser Ser Asp Ala Asp Leu Ser Glu Pro Thr Leu Ile Glu Gly Lys Ser Phe

741 Arg Glu Met Thr Asp Ala Glu Arg Asp Lys Ile Ser Asp Lys Ile Ser Val Met Gly Arg

761 Ser Ser Pro Asn Asp Lys Leu Leu Leu Val Gln Ser Leu Arg Arg Gln Gly His Val Val

781 Ala Val Thr Gly Asp Gly Thr Asn Asp Ala Pro Ala Leu His Glu Ala Asp Ile Gly Leu

801 Ala Met Gly Ile Ala Gly Thr Glu Val Ala Lys Glu Ser Ser Asp Ile Ile Ile Leu Asp

821 Asp Asn Phe Ala Ser Val Val Lys Val Val Arg Trp Gly Arg Ser Val Tyr Ala Asn Ile

841 Gln Lys Phe Ile Gln Phe Gln Leu Thr Val Asn Val Ala Ala Leu Val Ile Asn Val Val

861 Ala Ala Ile Ser Ser Gly Asp Val Pro Leu Thr Ala Val Gln Leu Leu Trp Val Asn Leu

881 Ile Met Asp Thr Leu Gly Ala Leu Ala Leu Ala Thr Glu Pro Pro Thr Asp His Leu Met

901 Gly Arg Pro Pro Val Gly Arg Lys Glu Pro Leu Ile Thr Asn Ile Met Trp Arg Asn Leu

921 Leu Ile Gln Ala Ile Tyr Gln Val Ser Val Leu Leu Thr Leu Asn Phe Arg Gly Ile Ser

941 Ile Leu Gly Leu Glu His Glu Val His Glu His Ala Thr Arg Val Lys Asn Thr Ile Ile

961 Phe Asn Ala Phe Val Leu Cys Gln Ala Phe Asn Glu Phe Asn Ala Arg Lys Pro Asp Glu

981 Lys Asn Ile Phe Lys Gly Val Ile Lys Asn Arg Leu Phe Met Gly Ile Ile Val Ile Thr

1001 Leu Val Leu Gln Val Ile Ile Val Glu Phe Leu Gly Lys Phe Ala Ser Thr Thr Lys Leu

1021 Asn Trp Lys Gln Trp Leu Ile Cys Val Gly Ile Gly Val Ile Ser Trp Pro Leu Ala Leu

1041 Val Gly Lys Phe Ile Pro Val Pro Ala Ala Pro Ile Ser Asn Lys Leu Lys Val Leu Lys

1061 Phe Trp Gly Lys Lys Lys Asn Ser Ser Gly Glu Gly Ser Leu(Length：1074aa)

Table 3At-ACA8 gene mutation body flanking sequence table

SEQ ID No：3

1 ATTGAAAGTA CTAAAATTCT GGGGCAAGAA GAAAAATTCT TCTGGAGAAG

51 GTAATAGTAA CTTACAGCTT GTGATTATCN ATGAACTAAA TTCTTAAGAA

101 AAGTTTGGGG TGGGCAACTT GTGTAATAAA ACTTATCCTG AATTTAAACT

151 GTAATAAGGA TTTGGTTTAA GCT(Length：173bp)

Embodiment 2:

The resistance of mutant aca8 to low temperature stress:

(1) the low temperature stress phenotypic assay of wild-type plant and mutant aca8:

By Col-0 and aca8 seed, subregion after sterilization is sowed in containing the MS substratum of 1.5% sucrose concentration, in 4 ℃ of vernalization 3 days, carry out afterwards normal germination and growth (22 ℃ of temperature, humidity 50%-60%, photoperiod 10h illumination/14h is dark), the aseptic seedling of cultivating after 14 days is carried out the tame and docile frozen process experiment of low temperature cold.Treatment condition are: 4 ℃ of cold taming and dociling 3 days, fast cooling freezes 3.5h-4h to-6 ℃, is warmed up to 4 ℃ of renewal cultivations one day, is placed in afterwards 22 ℃ of normal cultivations, can be observed the phenotypic difference of Col-0 and aca8 after about 3-5 days.As shown in Figure 3A, after subzero treatment, the aseptic seedling survival rate of aca8 is higher, and result shows that the aseptic seedling of mutant aca8 is more low temperature resistant and coerces.

Also use the earth culture seedling of approximately 3 weeks sizes to carry out low temperature stress experiment, treatment condition are simultaneously: 4 ℃ of cold taming and dociling 7 days, fast cooling freezes 1h to-6 ℃, is warmed up to 4 ℃ of renewal cultivations one day, is placed in afterwards 22 ℃ of normal cultivations.The variation of the maximum Photochemical quantum yield of Photosystem I I (PSII) (Fv/Fm) by Real-Time Monitoring blade, checks injured degree and the recovery situation of plant leaf.As shown in Fig. 3 B and 3C, after processing, the earth culture seedling leaf of aca8 has stronger fluorescent value, and fluorescence restorability is better than Col-0, has higher survival rate, and result shows more low temperature resistant the coercing of earth culture seedling of mutant aca8.

(2) transcriptional expression of low temperature response genes involved in wild-type and aca8 mutant under subzero treatment:

The Arabidopis thaliana earth culture seedling of approximately 3 weeks sizes is carried out to low temperature stress processing, respectively normal 22 ℃ of cultivations, 4 ℃ cold tame and docile 7 days (4 ℃ of 7d), 4 ℃ cold tame and docile 7 days after fast cooling to-6 ℃, freeze 1h (6 ℃ of 1h CA), not coldly tame and docile direct fast cooling and to-6 ℃, freeze four points of 1h (6 ℃ of 1h D) and sample, the total RNA that extracts blade carries out sxemiquantitative RT-PCR, analyzes the transcriptional expression level of low temperature responsive genes LTI78.LTI78 gene primer is: F:5 '-GTTGTCAGTTTCTCCGCC-3 ', R:5 '-CTTTGACTCTGTTCTCGGT-3 '; ACTIN gene primer is: F:5 '-TGTGCCAATCTACGAGGGTTT-3 ', R:5 '-TTTCCCGCTCTGCTGTTGT-3 '.As shown in Figure 3 D, the expression level of low temperature stress responsive genes LTI78, in Col-0 treatment stage of Different hypothermia and aca8, there is significant difference, the sudden change of At-AC48 gene, affected the expression pattern of genes involved in plant low temperature signal transduction path, again directly proved that At-AC48 gene is relevant to the anti-low temperature stress ability of plant.

Embodiment 3:

The resistance of mutant aca8 to high temperature stress:

(1) the high temperature stress phenotypic assay of wild-type plant and mutant aca8:

By Col-0 and aca8 seed, subregion after sterilization is sowed in containing the MS substratum of 1.5% sucrose concentration, in 4 ℃ of vernalization 3 days, then normal germination and growth (22 ℃ of temperature, humidity 50%-60%, photoperiod 10h illumination/14h is dark), the aseptic seedling of cultivating after 14 days is carried out the warm tame and docile heat shock processing of height, condition is: 38 ℃ of heat are tamed and dociled 2h, be placed in 22 ℃ of room temperatures and recover 1h, be warmed up to 45 ℃ of heat shock 3-4h, be placed in afterwards 22 ℃ of renewal cultivations.After about 3-5 days, can be observed the phenotypic difference of wild-type Col-0 and mutant aca8, as shown in Figure 4 A, after pyroprocessing, aca8 aseptic seedling survival rate is higher, shows that the aseptic seedling of mutant aca8 is more high temperature resistant, and the restorability after heat shock is better than wild-type.

Also use the earth culture seedling of approximately 3 weeks sizes also to carry out high temperature stress experiment simultaneously, condition is: 38 ℃ of heat are tamed and dociled 2h, be placed in 22 ℃ of room temperatures and recover 1h, be warmed up to 45 ℃ of gradient heat shock 2.5-4h, be placed in afterwards 22 ℃ of renewal cultivations, the variation of the maximum Photochemical quantum yield of Photosystem I I (Fv/Fm) by Real-Time Monitoring blade, checks injured degree and the recovery situation of plant.As shown in Figure 4 B and 4C, under same treatment condition, the survival rate of aca8 earth culture seedling is higher, and different high-temperature gradients is processed aca8 and all than Col-0, shown stronger fluorescent value, weak and the good restorability of the degree that is hurt, result shows more high temperature resistant the coercing of earth culture seedling of mutant aca8.

Embodiment 4:

The resistance of mutant aca8 to drought stress:

(1) the dehydration rehydration phenotypic assay of wild-type plant and mutant aca8 earth culture seedling:

By earth culture seedling Col-0 and the aca8 of 3 weeks sizes of about normal growth, continuous drought was processed after 14 days, find Col-0 and the aca8 plant symptom that here all occurs withering, Col-0 withers here degree is more serious, water and recover to find afterwards for 4 days, aca8 plant 100% can restore normal growth, and Col-0 plant only has 75% can restore normal growth, as shown in Figure 5A, this shows the more resistance to water deficit of aca8 plant.

(2) PEG simulating drought is processed the phenotypic assay of wild-type and mutant aca8 Rooted Cuttings:

In order to compare Col-0 and aca8 to arid susceptibility, also use PEG treatment of simulated drought condition, observe the drought-resistant ability of Col-0 and aca8 Rooted Cuttings, (concentration is respectively 0% respectively the Rooted Cuttings of Col-0 and aca8 to be placed in to the PEG water planting liquid of different concns, 2%, 5% and 8%), simulation arid situation in various degree.As shown in Figure 5 B, after 4 days, observe better that aca8 plant grows than Col-0, more drought-resistant coercing.

Embodiment 5:

The susceptibility of mutant aca8 to ABA:

Dormin ABA is considered to a kind of growth-inhibiting type plant hormone conventionally, such as suppressing seed germination, suppressing growth promotion dormancy etc.Wherein inhibition of seed germination experiment is a kind of very useful method, can show intuitively the difference of range gene mutant to ABA susceptibility, thereby whether proof gene function is relevant with the effect of hormone ABA.In order to compare Col-0 and the sensitivity differences of aca8 to ABA, the seed of Col-0 and aca8 is broadcast on the MS substratum that comprises different ABA concentration (being respectively 0 μ M, 0.25 μ M, 0.5 μ M, 1 μ M, 3 μ M and 5 μ M) and sprouted.Sprout and can find out obvious difference after 7 days, as shown in Figure 6, when ABA concentration is equal to or higher than 0.25 μ M, Col-0 compares with aca8, aca8 plant is more responsive to ABA, the elongation of its seed germination and root is all subject to severe inhibition, finds out thus, in expression and the ABA signal correction of seed germination stage A t-ACA8 gene.

Embodiment 6:

The susceptibility that mutant aca8 changes sucrose concentration:

Aseptic seedling culture is common a kind of mode that vegetable material is provided in plant physiology experiment, the main carbon source that relies on sucrose to provide plant seed germination and early growth period to need in the MS substratum using, and more general sucrose working concentration is 1%-1.5%.But the present invention's discovery, the seed germination of mutant aca8 and primary growth are more responsive to the variation of sucrose concentration.

The seed of Col-0 and aca8 is broadcast and (is respectively 0% comprising different sucrose, 0.5%, 1%, 5% and 7%) on MS substratum, sprout, when sucrose concentration is 0%, the Seed Germination and growth of aca8 is subject to severe inhibition, after adding sucrose, just can recover and sprouting and growth pattern that Col-0 is same, still, when sucrose concentration is higher than 5% time, the growth of aca8 plant will be subject to certain inhibition, when sucrose concentration is 7%, inhibition is more remarkable, as shown in Figure 7.Therefore, compare with wild-type, sucrose is to guarantee the normal key component of sprouting of aca8, and aca8 is more responsive to the variation of sucrose concentration, especially under high concentration sucrose condition, also can affect growing of plant.

At-ACA8 participates in the plant stress-resistance reaction process such as low temperature, high temperature, osmotic stress, and mutant strain aca8 is better than wild-type plant to above several resistivities of coercing.

Mutant strain aca8 is to having or not applying of sucrose in substratum, and the rising of sucrose concentration is more responsive, and not applying in the situation of sucrose can severe inhibition aca8 seed germination, and growth phase causes growing suppressed compared with the rising of easy perception sucrose concentration; Mutant strain aca8 also has response, external source to apply significantly inhibition seed germination of micro-ABA to hormone dormin (ABA) in the seed germination stage, belongs to ABA responsive type mutant strain.

Arabidopis thaliana Ca ²⁺the relevant physiological of-ATP enzyme Gene A t-ACA8 experimental results show that, this gene involved in plant grows and regulates and the process such as stress response, and the present invention understands calcium singal transduction system and aspect regulating growth of plants and stress response, having substantial value deep; Because aca8 mutant has significant resistibility to low temperature, high temperature, osmotic stress, and studies have shown that in a large number in the past be take most of functional genes that Arabidopis thaliana identified as material and can be applied in the research of other plant, therefore the present invention is also for the improvement of plant variety provides a Fineness gene, set forth a kind of new raising plant opposing adverse circumstance ability and cultivated method and the mechanism with good resistant variety, having there is important economic implications and utilization prospect.

Finally, it is also to be noted that, what more than enumerate is only wherein several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many improvement.The improvement that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.

Claims (1)

1. the arabidopsis mutant strain aca8 application in the Arabidopis thaliana of cultivating anti-low temperature or high temperature resistance, described mutant strain aca8 is the mutant strain that the seed of Arabidopis thaliana Biological resources center ABRC is numbered CS859889, and its At-ACA8 gene is suddenlyd change because T-DNA inserts.

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