CN102154236B - Wheat prematurity associated protein TaMAPK1 and coding gene and use thereof - Google Patents
Wheat prematurity associated protein TaMAPK1 and coding gene and use thereof Download PDFInfo
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- CN102154236B CN102154236B CN 201110071338 CN201110071338A CN102154236B CN 102154236 B CN102154236 B CN 102154236B CN 201110071338 CN201110071338 CN 201110071338 CN 201110071338 A CN201110071338 A CN 201110071338A CN 102154236 B CN102154236 B CN 102154236B
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Abstract
The invention relates to the field of genetic engineering, in particular to a wheat prematurity associated protein TaMAPK1 and a coding gene and use thereof. The amino sequence of the protein is represented by SEQ ID No.1, and the sequence of the gene is represented by SEQ ID No.2. The protein and gene, which are provided by the invention, have obvious significance for breeding premature crops.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, the present invention relates to the precocious GAP-associated protein GAP TaMAPK1 of a grow wheat and encoding sox and application.
Background technology
The former activated protein kinase of mitogen family plays an important role in all eukaryotic signal conducting paths.When receiving from extracellular environmental stimulus; MAPKKK activates MAPKK through the phosphorylation in serine-threonine site; And then MAPKK makes the Serine and the tyrosine site phosphorylation of T-x-Y motif among the MAPK protein-active loop, activated MAPK and then make other protein kinases of downstream, metabolic enzymes, transcription factor and cytoskeleton composition exercise the different cells function.
There are at least 60 MAPKKK in the arabidopsis gene group, 10 MAPKK and 20 MAPK, but the signal pathway major part of present these kinase whose functions and participation thereof is not clear.Ichimura etc. are divided into four types of A～D with plant MAPK protein family; A, B, the proteic loop ring of three types of MAPK of C have the TEY motif; And the kinase whose loop ring of D proteinoid has the TDY motif; Simultaneously, the C of D class MAPK end lacks the specific anchoring structure of the decision interact protein territory (CD domain) that is made up of the electronegative amino-acid residue of cluster.The MAPK cascade of plant is being coerced environment and non-ambient, and plant self grows, play an important role in the necrocytosis.Existing that the proteic functional description of all kinds of MAPK of Arabidopis thaliana is following.Arabidopis thaliana category-A MAPK comprises AtMAPK3, AtMAPK6, AtMAPK10, and on function, there are functional redundancy in AtMAPK3, AtMAPK6.For example, AtMAPK3/6 participates in ethene, phytoalexin camalexin is synthetic; AtMAPK3/6 promotes cell fission in the anther development process; The AtMAPK3/6 involved in plant is to the allergy of ozone.The expression amount of AtMAPK10 seldom infers that it possibly be non-functional.Category-B MAPKs comprises AtMAPK4～5, AtMAPK11～13.Studying maximum is AtMAPK4, it can with AtMAPEKK1, AtMAPKK1/AtMAPKK2 constitutes a complete MAPKKK-MAPKK-MAPK signal path, and innate immune defense and the necrocytosis of plant played the negative regulation effect; Also involved in plant response that low temperature, dehydration, touch, wound, height are oozed.The kinase whose research of C class is very limited, but the expression of microarray analysis discovery AtMAPK7 receives circadian regulation and control; AtMAPK7 possibly participate in the cause of disease signal transduction path of PR1 genetic expression; AtMAPK1/2 come to harm, active rising when the fragrant acid of Whitfield's ointment, jasmine, hydrogen peroxide.The genome of Arabidopis thaliana has 6 D class MAPK, and the research of such AtMAPK does not also appear in the newspapers, can be by fungi and wounding signal abduction delivering but distinguish with Arabidopis thaliana D class homologous paddy rice (Oryza sativa) BWMK1 and alfalfa TDY1 gene.
At present, have been found that the existence of a large amount of MAPK in some plants, but the present MAPK evaluation comparatively small amt that comes to light in the wheat, the research of the precocious relevant gene M APK of wheat does not appear in the newspapers as yet.Kind length breeding time always is an important goal proterties in the wheat breeding, changes breeding time of all kinds of wheats through breeding method, particularly shortens the breeding time of kind, no matter be in the past and in the future, development all has crucial meaning to Wheat Production.Play crucial effects in the prematureness of MAPK raising plant.This produces huge pushing effect and economic benefit to the agriculture prodn meeting.Therefore, utilize precocious relevant MAPK gene genetically modified crops to have extremely important theory and practice meaning.
Summary of the invention
The purpose of this invention is to provide the precocious GAP-associated protein GAP TaMAPK1 of a grow wheat.
A purpose more of the present invention provides the encoding sox of the precocious GAP-associated protein GAP TaMAPK1 of the above-mentioned wheat of coding.
Another object of the present invention provides the recombinant vectors that comprises said gene.
Another object of the present invention provides precocious GAP-associated protein GAP TaMAPK1 of above-mentioned wheat and application thereof.
The precocious GAP-associated protein GAP TaMAPK1 of wheat provided by the present invention derives from wheat BS20, and its aminoacid sequence is shown in SEQ ID NO.1.
SEQ?ID?NO.1：
MTHGGRFLLYNIFGNQFEITAKYQPPIMPIGRGAYGIVCSVMNFETREMVAIKKIANAFDNNMDAKRTLREIKLLRHLDHENIVGLRDVIPPATPQSFNDVYIATELMDTDLHHIIRSNQELSEEHCQYFLYQLLRGLKYIHSANVIHRDLKPGNLLLNANCDLKICDFGLARPSSESDMMTEYVVTRWYRAPELLLNSTDYSAAIDVWSVGCIFMELINRAPLFPGRDHMHQMRLITEVIGTPTDDDLGFIRNEDARRYMRHLPQFPRRSFPGQFPKVQPAALDLIERMLTFNPLQRITVEEALEHPYLERLRDVADEPICTDPFSFDFEQHPLTEDQMKQLIFNEALELNPNFRY.
According to the above-mentioned albumen of TaMAPK1 genes encoding of the present invention, can have nucleotide sequence shown in SEQ ID NO.2, TaMAPK1 ORFs (ORF) length is 1074bp.
SEQ?ID?NO.2：
atgacgcacg?gcggccgctt?cctcctctac?aacatattcg?gcaaccagtt?cgagatcacg 60
gccaagtacc?agccgccgat?catgcccatc?ggccgcggcg?cctacgggat?cgtctgctcg 120
gtgatgaact?tcgagacgag?ggagatggtg?gcaatcaaga?agatcgcaaa?cgctttcgac 180
aacaacatgg?acgccaagcg?cacgctccgg?gagatcaagc?tcctgaggca?cctcgaccac 240
gagaacatag?taggcctccg?agatgtgatc?ccgccggcga?ccccgcagtc?cttcaacgac 300
gtctacatcg?ccaccgagct?catggacacg?gacctccacc?acatcatccg?ctccaaccaa 360
gaactctcgg?aagaacactg?ccagtacttc?ctgtaccagc?tgctgcgcgg?cctcaagtac 420
atccactcgg?cgaacgtgat?ccaccgcgac?ctcaagccgg?gcaacctgct?gctgaacgcc 480
aactgcgacc?tcaagatctg?cgacttcggc?ctggcgcggc?cgtcgtccga?gagcgacatg 540
atgacggagt?acgtggtcac?gcggtggtac?cgggccccgg?agctgctgct?caactccacc 600
gactactccg?cggccatcga?cgtctggtcc?gtcggctgca?tcttcatgga?gctcatcaac 660
cgcgcgccgc?tcttcccggg?gagggaccac?atgcaccaga?tgcggctcat?cacggaggtg 720
atcggcaccc?ccaccgacga?cgacctgggc?ttcatccgga?acgaggacgc?caggaggtac 780
atgaggcacc?tgccgcagtt?ccctcgccgg?tccttcccgg?gacagttccc?caaggtgcag 840
cccgccgcgc?tggacctcat?cgagaggatg?ctcaccttca?acccgctgca?gaggatcaca 900
gttgaagagg?cgctggagca?cccataccta?gagcggcttc?gcgacgtcgc?cgacgagccc 960
atctgcacgg?accccttctc?cttcgacttc?gaacagcacc?cactgacgga?agaccagatg 1020
aagcagctca?tattcaacga?agccctggag?ttgaacccca?acttccgata?ctag 1074
Another object of the present invention provides a kind of method of cultivating quick grower.
The method of cultivation quick grower provided by the present invention is that the above-mentioned recombinant expression vector that any contains the TaMAPK1 gene is imported in the vegetable cell, obtains quick grower.
The present invention is an experiment material with wheat BS20 (Triticum aestivum L.), has obtained the precocious genes involved TaMAPK1 of wheat, and it is imported tobacco, and the discovery transgene tobacco is bloomed than wild-type and shifted to an earlier date 20 days.
Description of drawings
Fig. 1 shows the growing state of transgene tobacco and Nicotiana gossei, and A is bimestrial growing state; B, C are 4 four months growing state.
Embodiment
Make the experimental methods of molecular biology specify in following examples, all carry out, perhaps carry out according to test kit and product description with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
The clone of embodiment 1, TaMAPK1 gene and sequence motifs analysis
To the growth wheat leaf blade in flowering period, extract the total RNA of Xinjiang couchgrass with Trizol.Use 5 ' RACE test kit (5 ' RACE System for Rapid Amplification of cDNA Ends Kit) (GIBCOBRL; CAT.NO.18374-058) and 3 ' RACE test kit (3 ' RACE System for Rapid Amplification ofcDNA Ends Kit) (GIBCOBRL CAT.NO.18373-019) obtains the full length sequence 1074bp of TaMAPK1 gene.
Extract total RNA of Xinjiang couchgrass seedling with Trizol, acquire cDNA with the reverse transcription of superscript II (invitrogen) ThermoScript II.According to TaMAPK1 gene coding region sequences Design primer P1 and P2.The cDNA that obtains with reverse transcription is a template, carries out pcr amplification with primer P1 and P2.The sequence of primer P1 and P2 is following:
P1：5’-atgacgcacggcggccgctt-3’，
P2：5’-aaccccaacttccgatactag-3’。
The PCR product is carried out 0.8% agarose gel electrophoresis detect, obtain molecular weight and be about the band about 1kb, conform to expected results.Reclaim test kit (TIANGEN) with sepharose and reclaim this fragment.Should reclaim fragment is connected with pGEM-T Easy (Promega); Method (Proc Natl Acad Sci with reference to Cohen etc.; 69:2110); To connect product transformed into escherichia coli DH5 α competent cell,, obtain containing the segmental recombinant plasmid of recovery according to the acillin resistance marker screening positive clone on the pGEM-T Easy carrier.With T7 on this recombinant plasmid vector and SP6 promoter sequence is that primer carries out nucleotide sequencing to it; The ORFs (ORF) that sequencing result shows the TaMAPK1 gene that increases for SEQ ID No.2 from 5 ' terminal the 1st to 894 deoxyribonucleotide, encoding amino acid sequence is the protein of SEQ ID No.1.The recombinant vectors called after pTE-ErNAC7 that will contain TaMAPK1 gene shown in the sequence SEQ ID No.3, its cDNA clone result is as shown in Figure 1.
The sequence of TaMAPK1 gene is compared on Genabnk, and MAPK has higher homology in this gene and the wheat, but gene function is unknown.
The expression characteristic of the tobacco of embodiment 2, commentaries on classics TaMAPK1 gene
The TaMAPK1 gene is connected to the 35S promoter downstream of PBI121 double base plant expression vector, makes up the overexpression carrier, and be transformed in the tobacco W38 strain system through the tobacco leaf disc conversion method of agrobacterium tumefaciens C58C1 mediation.Transplant to flowerpot after the transgenic tobacco plant of treating the PCR test positive grows into certain altitude, observe and find that transgene tobacco is bloomed than wild-type and shifted to an earlier date 20 days.
Claims (7)
1. the precocious GAP-associated protein GAP TaMAPK1 of a grow wheat is characterized in that its aminoacid sequence is shown in SEQ ID NO.1.
2. the precocious genes involved TaMAPK1 of a grow wheat is characterized in that, the described albumen of coding claim 1.
3. the precocious genes involved TaMAPK1 of wheat as claimed in claim 2 is characterized in that its base sequence is shown in SEQ ID NO.2.
4. the recombinant vectors that comprises claim 2 or the precocious genes involved TaMAPK1 of 3 said wheats.
5. the application of the precocious GAP-associated protein GAP TaMAPK1 of the said wheat of claim 1.
6. the application of the precocious genes involved TaMAPK1 of the said wheat of claim 2.
7. method of cultivating quick grower, said method comprise the recombinant expression vector that contains the precocious genes involved TaMAPK1 of the said wheat of claim 2 are imported in the vegetable cell, obtain the step of quick grower.
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| CN1285872A (en) * | 1998-01-16 | 2001-02-28 | 分子农业生物学院 | Gene associated with disease resistance in plants |
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| CN1285872A (en) * | 1998-01-16 | 2001-02-28 | 分子农业生物学院 | Gene associated with disease resistance in plants |
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