CN101955945A - Clone, transformation and application of TeWKS1 genes of thinopyrum elongatum - Google Patents
Clone, transformation and application of TeWKS1 genes of thinopyrum elongatum Download PDFInfo
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Abstract
The invention relates to clone, transformation and application of TeWKS1 genes of a thinopyrum elongatum and belongs to the technical field of molecular biology and biotechnology. In the invention, wheat stripe rust disease candidate resistance genes TeWKS1 which are TeWKS1-1 and TeWKS1-2W are obtained from decaploid thinopyrum elongatum by utilizing a homology cloning method, the NO.1294 thymine (T) of the TeWKS1-2W gene is replaced into a guanine (G), the NO.1294-NO.1296 nucleotide sequences of the TeWKS1-2W gene are changed from 'TAG' (Terminator Codon) to 'GAG' (Glutamic Acid Codon), and the gene is named TeWKS1-2M after being transformed. The opening reading frame parts of TeWKS1-1 and TeWKS1-2M gene transcription products mRNA have 1935 bps and can code albumen with 644 amino acids, and contain two conserved sequences of Kinase and START. By applying the two genes in the wheat, the broad-spectrum resistance of the wheat to stripe rust germs can be improved.
Description
(1) invention field
The present invention relates to clone, transformation and the application of long fringe couchgrass TeWKS1 gene, belong to molecular biology and biological technical field.
(2) technical background
Wheat family (Triticeae) is important population in the grass, comprises wheat crops and many important forage grass such as wheat, barley and rye.Wheat accounts for 16% of global cultivated area, and about 23% (FAOSTATdata 2005) of global edible dry-matter are provided.In China, wheat is the second largest food crop that are only second to paddy rice, and sown area surpasses 3.5 hundred million mu, and the stable and high yields that ensures wheat is to ensureing staple food supply, guaranteeing that grain-production safety is significant.
Stripe rust of wheat is commonly called as " jaundice ", is to be caused by stripe rust bacterium (Puccinia striiformis Westend.f.sp.tritici).The stripe rust bacterium has characteristics such as propagation is far away, popular soon, harm Da Heda district is popular by airborne transmission.In the general popular time, stripe rust causes 30% left and right sides underproduction, and the underproduction is up to 50-75% (Conner and Kuzyk 1988 during serious time; Roelfset al.1992), serious harm world wheat grain-production.In China, stripe rust of wheat generally takes place, and throughout the year hazard area 6000-8000 is ten thousand mu, and is and increases the weight of trend year by year.
In recent years, stripe rust bacterium dominant races toxicity broadens, and causes stripe rust control difficulty to strengthen.At present, most Cultivars are to dominant races, Su-14 and CYR32, and apparent altitude infects (Wan et al.2007).On producing, though use sterilant control strip rust that certain effect is arranged, economic benefit is low, causes drug residue, environmental pollution, harm humans health.Excavate or utilize potential Stripe Rust Resistance Gene, combine with biotechnology by the conventional breeding means, cultivation has the new variety of wheat of high anti-bar rust property, becomes the means of the most economic actual effect of control strip rust evil, and becomes an important goal of modern wheat breeding.
At present, 41 of the wheat stripe rust resistance genes of having named (McIntosh et al.2008), wherein genes such as Yr5, Yr9, Yr15 and Yr26 on producing, be applied (Li Fengqi etc. 2008).Recently, two high Stripe Rust Resistance Gene of wide spectrum of Yr18 and Yr36 are cloned (Fu et al.2009; Krattinger et al.2009).The Yr36 gene finds in wild emmer (Triticum turgidum ssp.dicoccoides) that at first stripe rust of wheat is had resistance of wide spectrum, can effectively reduce stripe rust harm, reduces production loss (Fu et al.2009; Uauy et al.2005).The novel disease-resistant albumen of this genes encoding one class carries 2 with defensive raction relevant Kinase and START conserved regions, therefore is named as WHEATKINASE-START 1 (WKS1) gene (Fu et al.2009).Studies have shown that the WKS1 gene only is present in the wild wheat of part family germplasm, and loses in the cultivated wheat kind.For this reason, excavating the excellent WKS1 gene of other wheat family germplasm, in order to improve the stripe rust resistance of wheat crops, is the free-revving engine that this invention is created.
Long fringe couchgrass (Thinopyrum elongatum, Syn:Agropyron elongatum) has another name called Peng's ladder card couchgrass, Peng's ladder card thin ice grass etc., is the important perennial grass plant of wheat family.The disease resistance assay certificate, long fringe couchgrass is high anti-to immunity to stripe rust of wheat, to sick immunity fully such as wheat leaf rust, black rust, Powdery Mildew, is the important germ plasm resource of wheat crops genetic improvement.China has obtained the achievement of attracting attention in the world aspect the couchgrass resource utilizing.From the fifties in last century, Li Zhenshengs etc. have been carried out the distant hybirdization work of common wheat and couchgrass, excellent gene in the long fringe couchgrass of decaploid is changed in the wheat, and having bred to lay down for a short time No. 6 is high-quality, high yield, the disease-resistant serial new variety of wheat of laying down for a short time (Li et al.2008) of representative.For a short time lay down No. 6 in Shaanxi, province accumulative total popularizing areas such as Shanxi, Henan reach 1.5 hundred million mu, increase production 4,000,000,000 kilograms.Recent two decades comes, derive more than 70 of breedings of the system of laying down for a short time, and the accumulative total popularizing area is more than 300,000,000 mu, and the volume increase wheat has surpassed 7,500,000,000 kilograms.Therefore, long fringe couchgrass is the potential clone high Stripe Rust Resistance Gene of wide spectrum (WKS1 gene), in order to improve the desirable genetic resources of wheat crops stripe rust resistance, also is the purpose of this research.
(3) summary of the invention
The present invention utilizes the method for homologous clone, has been cloned into two the similar genes of TeWKS1, i.e. TeWKS1-1 and TeWKS1-2W in the long fringe couchgrass of widely used decaploid (2n=10X=70, genome StStStStEeEeEbEbExEx) from China's breeding.Sequential analysis shows that TeWKS1-1 normally reads over; And initial clone's TeWKS1-2W gene carries precocious terminator, its translation product is compared with TeWKS1-1,213 amino acid that lack TeWKS1-1 albumen carbon teminal, cause START conserved regions disappearance, sequential analysis shows, precocious terminator mooring points sudden change is (due to the G → T), TeWKS1-2W 1294-1296 position nucleotide sequence is the TAG terminator, for guaranteeing the gene activity of TeWKS1-2W, the present invention carries out the point mutation reparation to precocious terminator, the 1294th thymus pyrimidine (T) is replaced into guanine (G), the 1294-1296 position sports L-glutamic acid codon GAG by terminator signal TAG, repair improved TeWKS1-2W gene through sudden change, called after TeWKS1-2M, this reparation makes 644 amino acid of TeWKS1-2M genes encoding.
Wherein the TeWKS1-1 gene nucleotide series is shown in SEQ ID NO 1, and aminoacid sequence is shown in SEQ ID NO 2; The TeWKS1-2M gene nucleotide series is shown in SEQ ID NO 3, and aminoacid sequence is shown in SEQ ID NO 4.
TeWKS1-1 and each 1935bp of TeWKS1-2M two gene transcript mRNA open reading frame part push away to such an extent that codified has 644 amino acid whose albumen thus, carry two conserved sequences separately, Kinase and START.TeWKS1-1 and TeWKS1-2M gene all have 99% homology on nucleic acid and coded product Argine Monohydrochloride level.Bioinformatic analysis shows that it is 97% that long fringe couchgrass TeWKS1-1 becomes the nucleic acid sequence homology of strain resistant gene Yr36 with TeWKS1-2M two genes with the stripe rust of wheat high temperature of having delivered; Amino acid identity 95% has the high stripe rust resisting function of potential wide spectrum.
Another object of the present invention is the application of above-mentioned two genes in the sick breeding of wheat stripe rust resisting, as mentioned above, TeWKS1-1 and TeWKS1-2M two genes have the high stripe rust resisting function of potential wide spectrum, be transformed in the cultivated wheat by technique means such as gene transformation or distant hybirdization, can be as Yr36 gene (Fu et al.2009; Uauy et al.2005) the same, improve the resistance of wide spectrum of wheat to stripe rust, effectively reduce stripe rust harm, reduce production loss.And the gene that derives from long fringe couchgrass is described as technical background, and there is not the first-class problem of technology in widespread use in the wheat breed improvement.
Gene order of the present invention is as follows:
(1) information of SEQ ID NO 1
(a) sequence signature
* length: 1935 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: long fringe couchgrass
(f) sequence description: SEQ IN NO.1
1 ATGGAGCTCC?CACGAAACAA?ACTTGCAGAT?TTGAACCAAG?GAAATGAAAA?TTTAAAGGCA 60
61 AAGGCAAAGT?GGACACCCAA?TGCCAGGAGG?TTCACAGAAC?ATCAGATTAA?AAGAATTACT 120
121 AAGAACTATA?GAACTCATGT?TGGCAAAGGT?GCCTTTGGTG?AGGTTTTCCG?AGGTTTTCTT 180
181 GACGATGGCA?GTCCAGTTGC?AGTGAAGAAA?TACATCAACC?AGAATATGAA?AGAAGGGTTT 240
241 GACAAAGAGA?TAACTATCCA?TTGCCAAGTC?AACCACAAGA?ACATAGTCAA?GCTTTTGGGT 300
301 TATTGTTCAG?AGGGAAATGC?CTTGACGATG?GTCACTGAGT?ACATTCCTAG?AGGAAACCTC 360
361 AAAGACCTCC?TTCATGGAAG?TGATGATCCC?ATTTCTTTTG?AGGCAAGATT?GTGTATTGCT 420
421 ATAGATTGTG?CAGAGGCATT?AGCTTTCATG?CATTCAAAGG?ATCCGCCAAT?CATTCACGGT 480
481 GACATCAAAC?CTGACAATAT?ACTCTTGGAT?GATAACTTGG?GTGCAAAATT?ATCTGACTTT 540
541 GGAATATCAA?GGTTGCTTTC?TATGGAGAAT?AGTTATTTTA?CTAATAATGT?AATAGGAAGC 600
601 AGAGGTTACA?TGGATCCAGA?ACACATTCAG?ACTGGCCGCG?TTGATCCTAA?GAATGATGTT 660
661 TACAGTTTTG?GGGTTGTTTT?GGTAGAACTA?GTTACCAGAG?CTATGGCAGC?TGAGAATGGG 720
721 ACATGTAGCG?ACCTTGCAAA?AAAATTCATT?GAAGCTTTCC?TCCAAAAAAA?TATTTTTTTG 780
781 AAAGTTTTTG?GAAAGCAAAA?AAAGGCAAGA?GAGATGTTTG?ATACCCAGAT?AGCAAATGCG 840
841 AGCAACATGG?AGGTTCTAGA?AAAAATTGGA?GAGCTGGCAA?TTGAGTGTCT?TAGAAGGGAT 900
901 ATCAAGAAAC?GTCCTGAAAT?GAATCATGTT?GTAGAACGTC?TTCGAATGCT?TGGTAAAGAT 960
961 CATGAAAAAA?GACAAAATAG?AAAAGCAGAA?AAGCAACATG?GAGTGGTGCC?TCCTGATGGT 1020
1021 AATACAACCT?TTAGCTCTTC?GTGGCCAAAG?GGTAGATTAG?AAAATGGAAG?GAAATCTTCT 1080
1081 TCCTCTGATG?CCGAGAGTTT?GTTCAGTCAT?AGGGAGGTGG?AAGCAGTAGA?CGAAGAAAAT 1140
1141 CAACATCCAT?TATTGCGAAG?AAAATCAATT?GGAAATGGTC?CTCCGGAATC?ATTTCATGAT 1200
1201 TGGACCTATG?GGAACGACAT?AGGAGGCCCT?GACGAAGTAT?TCTCTAGAGG?ATACTGGCAT 1260
1261 CTTCTCGGAT?GCCAGAATGG?CCTCCACATT?TTTGAGCCCC?TCGAGTATGG?TGATTACCTT 1320
1321 GTAAGAGCCG?TTGGCAAAGT?GATGAAGGCC?GTTGGTGTGA?TTGAGGCACC?TTGTGAGGCT 1380
1381 ATATTTCAGC?TTCTCATGAG?CATTGACGCT?AGCCGTTATG?AGTGGGACTG?CAGCTTCATG 1440
1441 TATGGTAGTC?TAGTGGAGGA?GCTAGATGGC?CACACTGCAA?TACTATACCA?TAGGCTACAC 1500
1501 CTAGATTGGT?TCTTGACGTT?TGTTTGGCCT?CGTGATCTTT?GTTATGTACG?ATATTGGCAG 1560
1561 CGTAATGACG?ACGGAGGTTA?CGTGGTGTTG?TTCCAATCCA?GAGAGCACCC?AAACTGTGGT 1620
1621 CCACAGCCAG?GATTTGTGAG?GGCACACATT?GAGATTGGCG?GGTTCAAAAT?TTCTCCACTG 1680
1681 AAAACCCGTA?ATGGAAGAAC?TCGAACACAA?GTACAATACC?TTATGAAGAT?GGATTTGAAG 1740
1741 GGTTGGGGCG?TTGGTTACTT?ATCCTCGTTT?CAGCAGCATT?GCGTCCTCCG?CATGCTGAAC 1800
1801 ACTATTGCTG?GGCTCAGGGA?ATGGTTTTCA?CGAAGTGATG?AAATTCCAAC?TTCGATGGAT 1860
1861 CAAAGTAGAT?ATTCTACAAT?GCTTGAAGAG?GAGTCAGATG?AAGACGAGTT?GTCTTCAGGA 1920
1921 AGTGATGAAA?GTTGA 1935
(2) information of SEQ IN NO.2
(a) sequence signature
* length: 644 amino acid
* type: amino acid
* chain: strand
* topological framework: linearity
(b) molecule type: protein
(c) sequence description: SEQ IN NO.2
MET?Glu?Leu?Pro?Arg?Asn?Lys?Leu?Ala?Asp?Leu?Asn?Gln?Gly?Asn
1 5 10 15
Glu?Asn?Leu?Lys?Ala?Lys?Ala?Lys?Trp?Thr?Pro?Asn?Ala?Arg?Arg
16 20 25 30
Phe?Thr?Glu?His?Gln?Ile?Lys?Arg?Ile?Thr?Lys?Asn?Tyr?Arg?Thr
31 35 40 45
His?Val?Gly?Lys?Gly?Ala?Phe?Gly?Glu?Val?Phe?Arg?Gly?Phe?Leu
46 50 55 60
Asp?Asp?Gly?Ser?Pro?Val?Ala?Val?Lys?Lys?Tyr?Ile?Asn?Gln?Asn
61 65 70 75
MET?Lys?Glu?Gly?Phe?Asp?Lys?Glu?Ile?Thr?Ile?His?Cys?Gln?Val
76 80 85 90
Asn?His?Lys?Asn?Ile?Val?Lys?Leu?Leu?Gly?Tyr?Cys?Ser?Glu?Gly
91 95 100 105
Asn?Ala?Leu?Thr?MET?Val?Thr?Glu?Tyr?Ile?Pro?Arg?Gly?Asn?Leu
106 110 115 120
Lys?Asp?Leu?Leu?His?Gly?Ser?Asp?Asp?Pro?Ile?Ser?Phe?Glu?Ala
121 125 130 135
Arg?Leu?Cys?Ile?Ala?Ile?Asp?Cys?Ala?Glu?Ala?Leu?Ala?Phe?MET
136 140 145 150
His?Ser?Lys?Asp?Pro?Pro?Ile?Ile?His?Gly?Asp?Ile?Lys?Pro?Asp
151 155 160 165
Asn?Ile?Leu?Leu?Asp?Asp?Asn?Leu?Gly?Ala?Lys?Leu?Ser?Asp?Phe
166 170 175 180
Gly?Ile?Ser?Arg?Leu?Leu?Ser?MET?Glu?Asn?Ser?Tyr?Phe?Thr?Asn
181 185 190 195
Asn?Val?Ile?Gly?Ser?Arg?Gly?Tyr?MET?Asp?Pro?Glu?His?Ile?Gln
196 200 205 210
Thr?Gly?Arg?Val?Asp?Pro?Lys?Asn?Asp?Val?Tyr?Ser?Phe?Gly?Val
211 215 220 225
Val?Leu?Val?Glu?Leu?Val?Thr?Arg?Ala?MET?Ala?Ala?Glu?Asn?Gly
226 230 235 240
Thr?Cys?Ser?Asp?Leu?Ala?Lys?Lys?Phe?Ile?Glu?Ala?Phe?Leu?Gln
241 245 250 255
Lys?Asn?Ile?Phe?Leu?Lys?Val?Phe?Gly?Lys?Gln?Lys?Lys?Ala?Arg
256 260 265 270
Glu?MET?Phe?Asp?Thr?Gln?Ile?Ala?Asn?Ala?Ser?Asn?MET?Glu?Val
271 275 280 285
Leu?Glu?Lys?Ile?Gly?Glu?Leu?Ala?Ile?Glu?Cys?Leu?Arg?Arg?Asp
286 290 295 300
Ile?Lys?Lys?Arg?Pro?Glu?MET?Asn?His?Val?Val?Glu?Arg?Leu?Arg
301 305 310 315
MET?Leu?Gly?Lys?Asp?His?Glu?Lys?Arg?Gln?Asn?Arg?Lys?Ala?Glu
316 320 325 330
Lys?Gln?His?Gly?Val?Val?Pro?Pro?Asp?Gly?Asn?Thr?Thr?Phe?Ser
331 335 340 345
Ser?Ser?Trp?Pro?Lys?Gly?Arg?Leu?Glu?Asn?Gly?Arg?Lys?Ser?Ser
346 350 355 360
Ser?Ser?Asp?Ala?Glu?Ser?Leu?Phe?Ser?His?Arg?Glu?Val?Glu?Ala
361 365 370 375
Val?Asp?Glu?Glu?Asn?Gln?His?Pro?Leu?Leu?Arg?Arg?Lys?Ser?Ile
376 380 385 390
Gly?Asn?Gly?Pro?Pro?Glu?Ser?Phe?His?Asp?Trp?Thr?Tyr?Gly?Asn
391 395 400 405
Asp?Ile?Gly?Gly?Pro?Asp?Glu?Val?Phe?Ser?Arg?Gly?Tyr?Trp?His
406 410 415 420
Leu?Leu?Gly?Cys?Gln?Asn?Gly?Leu?His?Ile?Phe?Glu?Pro?Leu?Glu
421 425 430 435
Tyr?Gly?Asp?Tyr?Leu?Val?Arg?Ala?Val?Gly?Lys?Val?MET?Lys?Ala
436 440 445 450
Val?Gly?Val?Ile?Glu?Ala?Pro?Cys?Glu?Ala?Ile?Phe?Gln?Leu?Leu
451 455 460 465
MET?Ser?Ile?Asp?Ala?Ser?Arg?Tyr?Glu?Trp?Asp?Cys?Ser?Phe?MET
466 470 475 480
Tyr?Gly?Ser?Leu?Val?Glu?Glu?Leu?Asp?Gly?His?Thr?Ala?Ile?Leu
481 485 490 495
Tyr?His?Arg?Leu?His?Leu?Asp?Trp?Phe?Leu?Thr?Phe?Val?Trp?Pro
496 500 505 510
Arg?Asp?Leu?Cys?Tyr?Val?Arg?Tyr?Trp?Gln?Arg?Asn?Asp?Asp?Gly
511 515 520 525
Gly?Tyr?Val?Val?Leu?Phe?Gln?Ser?Arg?Glu?His?Pro?Asn?Cys?Gly
526 530 535 540
Pro?Gln?Pro?Gly?Phe?Val?Arg?Ala?His?Ile?Glu?Ile?Gly?Gly?Phe
541 545 550 555
Lys?Ile?Ser?Pro?Leu?Lys?Thr?Arg?Asn?Gly?Arg?Thr?Arg?Thr?Gln
556 560 565 570
Val?Gln?Tyr?Leu?MET?Lys?MET?Asp?Leu?Lys?Gly?Trp?Gly?Val?Gly
571 575 580 585
Tyr?Leu?Ser?Ser?Phe?Gln?Gln?His?Cys?Val?Leu?Arg?MET?Leu?Asn
586 590 595 600
Thr?Ile?Ala?Gly?Leu?Arg?Glu?Trp?Phe?Ser?Arg?Ser?Asp?Glu?Ile
601 605 610 615
Pro?Thr?Ser?MET?Asp?Gln?Ser?Arg?Tyr?Ser?Thr?MET?Leu?Glu?Glu
616 620 625 630
Glu?Ser?Asp?Glu?Asp?Glu?Leu?Ser?Ser?Gly?Ser?Asp?Glu?Ser
631 635 640 644
(3) information of SEQ ID NO 3
(a) sequence signature
* length: 1935 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: long fringe couchgrass
(f) sequence description: SEQ IN NO.3
1 ATGGAGCTCC?CACGAAACAA?ACTTGCAGAT?TTGAACCAAG?GAAATGAAAA?TTTAAAGGCA 60
61 AAGGCAAAGT?GGACACCCAA?TGCCAGGAGG?TTCACAGAAC?ATCAGATTAA?AAGAATTACT 120
121 AAGAACTATA?GAACTCATGT?TGGCAAAGGT?GCCTTTGGTG?AGGTTTTCCG?AGGTTTTCTT 180
181 GACGATGGCA?GTCCAGTTGC?AGTGAAGAAA?TACATCAACC?AGAATATGAA?AGAAGGGTTT 240
241 GACAAAGAGA?TAACTATCCA?TTGCCAAGTC?AACCACAAGA?ACATAGTCAA?GCTTTTGGGT 300
301 TATTGTTCAG?AGGAAAATGC?CTTGACGATG?GTCACTGAGT?ACATTCCTAG?AGGAAACCTC 360
361 AAAGACCTCC?TTCATGGAAG?TGATGATCCC?ATTTCTTTTG?AGGCAAGATT?GTGTATTGCT 420
421 ATAGATTGTG?CAGAGGCATT?AGCTTTCATG?CATTCAAAGG?ATCCGCCAAT?CATTCACGGT 480
481 GACATCAAAC?CTGACAATAT?ACTCTTGGAT?GATAACTTGG?GTGCAAAATT?ATCTGACTTT 540
541 GGAATATCAA?GGTTGCTTTC?TATGGAGAAT?AGTTATTTTA?CTAATAATGT?AATAGGAAGC 600
601 AGAGGTTACA?TGGATCCAGA?ACACATTCAG?ACTGGCCGCG?TTGATCCTAA?GAATGATGTT 660
661 TACAGTTTTG?GGGTTGTTTT?GGTAGAACTA?GTTACCAGAG?CTATGGCAGC?TCAGAATGGG 720
721 ACATGTAGCG?ACCTTGCAAA?AAAATTCATT?GAAGCTTTCC?TCCAAAAGAA?TATTTTTTTG 780
781 AAAGTTTTTG?GAAAGCAAAA?AAAGGCAAGA?GAGATGTTTG?ATACCCAGAT?AGCAAATGCG 840
841 AGCAACATGG?AGGTTCTAGA?AAAAATTGGA?GAGCTGGCAA?TTGAGTGTCT?TAGAAGGGAT 900
901 ATCAAGAAAC?GTCCTGAAAT?GAATCATGTT?GTAGAACGTC?TTCGAATGCT?TGGTAAAGAT 960
961 CATGAAAAAA?GACAAAATAG?AAAAGCAGAA?AAGCAACATG?GAGTGGTGCC?TCCTGATGGT 1020
1021 AATACAACCT?TTAGCTCTTC?GTGGCCAAAG?GGTAGATTAG?AAAATGGAAG?GAAATCTTCT 1080
1081 TCCTCTGATG?CCGAGAGTCT?GTTCAGTCAT?AGGGAGGTGG?AAGCAGTAGA?CGAAGAAAAT 1140
1141 CAACATCCAT?TATTGCGAAG?AAAATCAATT?GGAAATGGTC?CTCCGGAATC?ATTTCATGAT 1200
1201 TGGACCTATG?GGAACGACAT?AGGAGGCCCT?GACGAAGTAT?TCTCTAGAGG?ATACTGGCAT 1260
1261 CTTCTCGGAT?GCCAGAATGG?CCTCCACATT?TTTGAGCCCC?TCGAGTATGG?TGATTACCTT 1320
1321 GTAAGAGCCG?TTGGCAAAGT?GATGAAGGCC?GTTGGTGTGA?TTGAGGCACC?TTGTGAGGCT 1380
1381 ATATTTCAGC?TTCTCATGAG?CATGGACGCT?AGCCGTTATG?AGTGGGACTG?CAGCTTCATG 1440
1441 TATGGTAGTC?TAGTGGAGGA?GCTAGATGGC?CACACTGCAA?TACTATACCA?TAGGCTACAC 1500
1501 CTAGATTGGT?TCTTGACGTT?TGTTTGGCCT?CGTGATCTTT?GTTATGTACG?ATATTGGCAG 1560
1561 CGTAATGACG?ACGGAGGTTA?CGTGGCGTTG?TTCCAATCCA?GAGAGCACCC?AAACTGTGGT 1620
1621 CCACAGCCAG?GATTTGTGAG?GGCACACATT?GAGATTGGCG?GGTTCAAAAT?TTCTCCACTG 1680
1681 AAAATCCGTA?ATGGAAGAAC?TCGAACACAA?GTACAATACC?TTATGAAGAT?GGATTTGAAG 1740
1741 GGTTGGGGCG?TTGGTTACTT?ATCCTCGTTT?CAGCAGCATT?GCGTCCTCCG?CATGCTGAAC 1800
1801 ACTATTGCTG?GGCTCAGGGA?ATGGTTTTCA?CGAAGTGATG?AAATTCCAAC?TTCGATGGAT 1860
1861 CAAAGTAGAT?ATTCTACAAT?GCTTGAAGAG?GAGTCAGATG?AAGACGAGTT?GTCTTCAGGA 1920
1921 AGTGATGAAA?GTTGA 1935
(4) information of SEQ IN NO.4
(a) sequence signature
* length: 644 amino acid
* type: amino acid
* chain: strand
* topological framework: linearity
(b) molecule type: protein
(c) sequence description: SEQ IN NO.4
MET?Glu?Leu?Pro?Arg?Asn?Lys?Leu?Ala?Asp?Leu?Asn?Gln?Gly?Asn
1 5 10 15
Glu?Asn?Leu?Lys?Ala?Lys?Ala?Lys?Trp?Thr?Pro?Asn?Ala?Arg?Arg
16 20 25 30
Phe?Thr?Glu?His?Gln?Ile?Lys?Arg?Ile?Thr?Lys?Asn?Tyr?Arg?Thr
31 35 40 45
His?Val?Gly?Lys?Gly?Ala?Phe?Gly?Glu?Val?Phe?Arg?Gly?Phe?Leu
46 50 55 60
Asp?Asp?Gly?Ser?Pro?Val?Ala?Val?Lys?Lys?Tyr?Ile?Asn?Gln?Asn
61 65 70 75
MET?Lys?Glu?Gly?Phe?Asp?Lys?Glu?Ile?Thr?Ile?His?Cys?Gln?Val
76 80 85 90
Asn?His?Lys?Asn?Ile?Val?Lys?Leu?Leu?Gly?Tyr?Cys?Ser?Glu?Glu
91 95 100 105
Asn?Ala?Leu?Thr?MET?Val?Thr?Glu?Tyr?Ile?Pro?Arg?Gly?Asn?Leu
106 110 115 120
Lys?Asp?Leu?Leu?His?Gly?Ser?Asp?Asp?Pro?Ile?Ser?Phe?Glu?Ala
121 125 130 135
Arg?Leu?Cys?Ile?Ala?Ile?Asp?Cys?Ala?Glu?Ala?Leu?Ala?Phe?MET
136 140 145 150
His?Ser?Lys?Asp?Pro?Pro?Ile?Ile?His?Gly?Asp?Ile?Lys?Pro?Asp
151 155 160 165
Asn?Ile?Leu?Leu?Asp?Asp?Asn?Leu?Gly?Ala?Lys?Leu?Ser?Asp?Phe
166 170 175 180
Gly?Ile?Ser?Arg?Leu?Leu?Ser?MET?Glu?Asn?Ser?Tyr?Phe?Thr?Asn
181 185 190 195
Asn?Val?Ile?Gly?Ser?Arg?Gly?Tyr?MET?Asp?Pro?Glu?His?Ile?Gln
196 200 205 210
Thr?Gly?Arg?Val?Asp?Pro?Lys?Asn?Asp?Val?Tyr?Ser?Phe?Gly?Val
211 215 220 225
Val?Leu?Val?Glu?Leu?Val?Thr?Arg?Ala?MET?Ala?Ala?Gln?Asn?Gly
226 230 235 240
Thr?Cys?Ser?Asp?Leu?Ala?Lys?Lys?Phe?Ile?Glu?Ala?Phe?Leu?Gln
241 245 250 255
Lys?Asn?Ile?Phe?Leu?Lys?Val?Phe?Gly?Lys?Gln?Lys?Lys?Ala?Arg
256 260 265 270
Glu?MET?Phe?Asp?Thr?Gln?Ile?Ala?Asn?Ala?Ser?Asn?MET?Glu?Val
271 275 280 285
Leu?Glu?Lys?Ile?Gly?Glu?Leu?Ala?Ile?Glu?Cys?Leu?Arg?Arg?Asp
286 290 295 300
Ile?Lys?Lys?Arg?Pro?Glu?MET?Asn?His?Val?Val?Glu?Arg?Leu?Arg
301 305 310 315
MET?Leu?Gly?Lys?Asp?His?Glu?Lys?Arg?Gln?Asn?Arg?Lys?Ala?Glu
316 320 325 330
Lys?Gln?His?Gly?Val?Val?Pro?Pro?Asp?Gly?Asn?Thr?Thr?Phe?Ser
331 335 340 345
Ser?Ser?Trp?Pro?Lys?Gly?Arg?Leu?Glu?Asn?Gly?Arg?Lys?Ser?Ser
346 350 355 360
Ser?Ser?Asp?Ala?Glu?Ser?Leu?Phe?Ser?His?Arg?Glu?Val?Glu?Ala
361 365 370 375
Val?Asp?Glu?Glu?Asn?Gln?His?Pro?Leu?Leu?Arg?Arg?Lys?Ser?Ile
376 380 385 390
Gly?Asn?Gly?Pro?Pro?Glu?Ser?Phe?His?Asp?Trp?Thr?Tyr?Gly?Asn
391 395 400 405
Asp?Ile?Gly?Gly?Pro?Asp?Glu?Val?Phe?Ser?Arg?Gly?Tyr?Trp?His
406 410 415 420
Leu?Leu?Gly?Cys?Gln?Asn?Gly?Leu?His?Ile?Phe?Glu?Pro?Leu?Glu
421 425 430 435
Tyr?Gly?Asp?Tyr?Leu?Val?Arg?Ala?Val?Gly?Lys?Val?MET?Lys?Ala
436 440 445 450
Val?Gly?Val?Ile?Glu?Ala?Pro?Cys?Glu?Ala?Ile?Phe?Gln?Leu?Leu
451 455 460 465
MET?Ser?MET?Asp?Ala?Ser?Arg?Tyr?Glu?Trp?Asp?Cys?Ser?Phe?MET
466 470 475 480
Tyr?Gly?Ser?Leu?Val?Glu?Glu?Leu?Asp?Gly?His?Thr?Ala?Ile?Leu
481 485 490 495
Tyr?His?Arg?Leu?His?Leu?Asp?Trp?Phe?Leu?Thr?Phe?Val?Trp?Pro
496 500 505 510
Arg?Asp?Leu?Cys?Tyr?Val?Arg?Tyr?Trp?Gln?Arg?Asn?Asp?Asp?Gly
511 515 520 525
Gly?Tyr?Val?Ala?Leu?Phe?Gln?Ser?Arg?Glu?His?Pro?Asn?Cys?Gly
526 530 535 540
Pro?Gln?Pro?Gly?Phe?Val?Arg?Ala?His?Ile?Glu?Ile?Gly?Gly?Phe
541 545 550 555
Lys?Ile?Ser?Pro?Leu?Lys?Ile?Arg?Asn?Gly?Arg?Thr?Arg?Thr?Gln
556 560 565 570
Val?Gln?Tyr?Leu?MET?Lys?MET?Asp?Leu?Lys?Gly?Trp?Gly?Val?Gly
571 575 580 585
Tyr?Leu?Ser?Ser?Phe?Gln?Gln?His?Cys?Val?Leu?Arg?MET?Leu?Asn
586 590 595 600
Thr?Ile?Ala?Gly?Leu?Arg?Glu?Trp?Phe?Ser?Arg?Ser?Asp?Glu?Ile
601 605 610 615
Pro?Thr?Ser?MET?Asp?Gln?Ser?Arg?Tyr?Ser?Thr?MET?Leu?Glu?Glu
616 620 625 630
Glu?Ser?Asp?Glu?Asp?Glu?Leu?Ser?Ser?Gly?Ser?Asp?Glu?Ser
631 635 640 644
(4) description of drawings
Fig. 1: the conserved regions synoptic diagram of TeWKS1-1 proteins encoded
Fig. 2: TeWKS1-2 genetic modification synoptic diagram
Among Fig. 1: the albumen of TeWKS1-1 and TeWKS1-2M genes encoding carries Kinase and START conserved regions, and this is the characteristic feature (Fu et al.2009) of nearest clone's wheat stripe rust resistance genes Yr36.Preliminary supposition, the Kinase zone relates to signal transduction, and the START zone is relevant with lipid molecule combination and proteic conformational change.Because the albumen and the wheat stripe rust resistance genes YR36 coded product of TeWKS1 genes encoding have 95% homology, the TeWKS1 gene can provide excellent stripe rust resistance.
Among Fig. 2: TeWKS1-1 and TeWKS1-2 gene are represented by the different background pattern respectively.Two genes have 99% homology, and in the 124bp scope between BspEI and XhoI two restriction enzyme sites, both unique differences are to cause the point mutation (shown in the red line) of TeWKS1-2W premature termination.Substitute the respective segments of TeWKS1-1W with the respective segments of BspEI and XhoI double digestion TeWKS1-1, and then obtain the TeWKS1-2M gene.
(5) concrete invention embodiment
Embodiment 1: the acquisition of long fringe couchgrass TeWKS1 gene cDNA
The present invention uses Trizol reagent, and (Invitrogen, CA USA) extract the total RNA of long fringe couchgrass, use SuperScript
II reverse transcription test kit (Invitrogen) synthesizes cDNA.RNA extraction and cDNA synthetic method are according to the said firm's test kit supporting technology.The present invention has designed forward special primer: 5 '-CACCATGGAGCTCCCACGAAACAAAC-3 ' and the long fringe couchgrass TeWKS1 gene of reverse special primer: 5 '-AGTACTTCGATGAAACAGATGGAA-3 ' amplification.Amplified reaction uses high-fidelity DNA polymerase Phusion
TM(USA), reaction system is with reference to product description for New EnglandBiolabs, MA; Reaction conditions is: the pre-sex change of 98 degree 30 seconds, and 98 degree sex change 10 seconds, 58 degree renaturation 30 seconds, 72 degree extend 60 seconds (30 circulations), and 72 degree extended 10 minutes eventually, 4 degree standbies.Amplified production separates through 0.8% agarose gel electrophoresis, and (Qiagen, CA USA) reclaim to use DNA glue to reclaim test kit.Reclaim product cloning to carrier pENTR/D-TOPO
(Invitrogen) on, carry out sequencing by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
By 22 cDNA clones are checked order, find that wherein 19 belong to the TeWKS1-1 type, 3 belong to the TeWKS1-2 type.Utilize universal sequencing primer thing (the forward and reverse primer of M13) and TeWKS1 gene primer on the cloning vector, the representativeness clone is carried out total order measure, obtain the cDNA segment of 1985bp, wherein 1935bp is an encoding sequence.Utilize BLAST software that the corresponding sequence in isolated cDNA sequence and translation product aminoacid sequence and the gene pool is compared, determine that they are the genes that become strain resistant gene Yr36 to have high homology with stripe rust of wheat high temperature.
Embodiment 2: the transformation of long fringe couchgrass TeWKS1-2M gene
TeWKS1-1 and TeWKS1-2 gene have 99% homology, and in the 124bp scope between BspEI and XhoI two restriction enzyme sites, both unique differences be to cause the TeWKS1-2W premature termination point mutation (Fig. 2. shown in the red line).Because pENTR/D-TOPO
(Invitrogen) BspEI and XhoI are single restriction enzyme site on the cloned plasmids, therefore substitute the respective segments of TeWKS1-1W with the respective segments of BspEI and XhoI double digestion TeWKS1-1, the 1294th Nucleotide of TeWKS1-2W gene is thymus pyrimidine (T) before transforming, and is terminator with its three initial company's codons (TAG); The 1294th Nucleotide of improved long fringe couchgrass TeWKS1-2M gene is guanine (G), initial three connects codons (GAG) coding L-glutamic acid (Glu) with it, and this reparation makes 644 amino acid of TeWKS1-2M genes encoding.And then acquisition TeWKS1-2M gene.Improved TeWKS1-2M gene is delivered Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and is checked order, and sequencing result shows that sequence is errorless, transforms successfully.
Two genes of embodiment 3:TeWKS1-1 and TeWKS1-2M are in Application in Wheat
By gene transformation technology, above-mentioned two genes are changed in the cultivated wheat kind, can improve the resistance of wide spectrum of wheat to stripe rust, effectively reduce stripe rust harm.
＜110〉Shandong Agricultural University
＜120〉clone, transformation and the application of long fringe couchgrass TeWKS1 gene
<160>4
<210>1
<211>1935
<212>DNA
＜213〉long fringe couchgrass (Thinopyrum elongatum, Syn:Agropyron elongatum)
<400>1
1 atggagctcc?cacgaaacaa?acttgcagat?ttgaaccaag?gaaatgaaaa?tttaaaggca 60
61 aaggcaaagt?ggacacccaa?tgccaggagg?ttcacagaac?atcagattaa?aagaattact 120
121 aagaactata?gaactcatgt?tggcaaaggt?gcctttggtg?aggttttccg?aggttttctt 180
181 gacgatggca?gtccagttgc?agtgaagaaa?tacatcaacc?agaatatgaa?agaagggttt 240
241 gacaaagaga?taactatcca?ttgccaagtc?aaccacaaga?acatagtcaa?gcttttgggt 300
301 tattgttcag?agggaaatgc?cttgacgatg?gtcactgagt?acattcctag?aggaaacctc 360
361 aaagacctcc?ttcatggaag?tgatgatccc?atttcttttg?aggcaagatt?gtgtattgct 420
421 atagattgtg?cagaggcatt?agctttcatg?cattcaaagg?atccgccaat?cattcacggt 480
481 gacatcaaac?ctgacaatat?actcttggat?gataacttgg?gtgcaaaatt?atctgacttt 540
541 ggaatatcaa?ggttgctttc?tatggagaat?agttatttta?ctaataatgt?aataggaagc 600
601 agaggttaca?tggatccaga?acacattcag?actggccgcg?ttgatcctaa?gaatgatgtt 660
661 tacagttttg?gggttgtttt?ggtagaacta?gttaccagag?ctatggcagc?tgagaatggg 720
721 acatgtagcg?accttgcaaa?aaaattcatt?gaagctttcc?tccaaaaaaa?tatttttttg 780
781 aaagtttttg?gaaagcaaaa?aaaggcaaga?gagatgtttg?atacccagat?agcaaatgcg 840
841 agcaacatgg?aggttctaga?aaaaattgga?gagctggcaa?ttgagtgtct?tagaagggat 900
901 atcaagaaac?gtcctgaaat?gaatcatgtt?gtagaacgtc?ttcgaatgct?tggtaaagat 960
961 catgaaaaaa?gacaaaatag?aaaagcagaa?aagcaacatg?gagtggtgcc?tcctgatggt 1020
1021 aatacaacct?ttagctcttc?gtggccaaag?ggtagattag?aaaatggaag?gaaatcttct 1080
1081 tcctctgatg?ccgagagttt?gttcagtcat?agggaggtgg?aagcagtaga?cgaagaaaat 1140
1141 caacatccat?tattgcgaag?aaaatcaatt?ggaaatggtc?ctccggaatc?atttcatgat 1200
1201 tggacctatg?ggaacgacat?aggaggccct?gacgaagtat?tctcta9agg?atactggcat 1260
1261 cttctcggat?gccagaatgg?cctccacatt?tttgagcccc?tcgagtatgg?tgattacctt 1320
1321 gtaagagccg?ttggcaaagt?gatgaaggcc?gttggtgtga?ttgaggcacc?ttgtgaggct 1380
1381 atatttcagc?ttctcatgag?cattgacgct?agccgttatg?agtgggactg?cagcttcatg 1440
1441 tatggtagtc?tagtggagga?gctagatggc?cacactgcaa?tactatacca?taggctacac 1500
1501 ctagattggt?tcttgacgtt?tgtttggcct?cgtgatcttt?gttatgtacg?atattggcag 1560
1561 cgtaatgacg?acggaggtta?cgtggtgttg?ttccaatcca?gagagcaccc?aaactgtggt 1620
1621 ccacagccag?gatttgtgag?ggcacacatt?gagattggcg?ggttcaaaat?ttctccactg 1680
1681 aaaacccgta?atggaagaac?tcgaacacaa?gtacaatacc?ttatgaagat?ggatttgaag 1740
1741 ggttggggcg?ttggttactt?atcctcgttt?cagcagcatt?gcgtcctccg?catgctgaac 1800
1801 actattgctg?ggctcaggga?atggttttca?cgaagtgatg?aaattccaac?ttcgatggat 1860
1861 caaagtagat?attctacaat?gcttgaagag?gagtcagatg?aagacgagtt?gtcttcagga 1920
1921 agtgatgaaa?gttga 1935
＜110〉Shandong Agricultural University
＜120〉clone, transformation and the application of long fringe couchgrass TeWKS1 gene
<210>2
<211>644
<212>PRT
＜213〉long fringe couchgrass (Thinopyrum elongatum, Syn:Agropyron elongatum)
<400>2
MET?Glu?Leu?Pro?Arg?Asn?Lys?Leu?Ala?Asp?Leu?Asn?Gln?Gly?Asn
1 5 10 15
Glu?Asn?Leu?Lys?Ala?Lys?Ala?Lys?Trp?Thr?Pro?Asn?Ala?Arg?Arg
16 20 25 30
Phe?Thr?Glu?His?Gln?Ile?Lys?Arg?Ile?Thr?Lys?Asn?Tyr?Arg?Thr
31 35 40 45
His?Val?Gly?Lys?Gly?Ala?Phe?Gly?Glu?Val?Phe?Arg?Gly?Phe?Leu
46 50 55 60
Asp?Asp?Gly?Ser?Pro?Val?Ala?Val?Lys?Lys?Tyr?Ile?Asn?Gln?Asn
61 65 70 75
MET?Lys?Glu?Gly?Phe?Asp?Lys?Glu?Ile?Thr?Ile?His?Cys?Gln?Val
76 80 85 90
Asn?His?Lys?Asn?Ile?Val?Lys?Leu?Leu?Gly?Tyr?Cys?Ser?Glu?Gly
91 95 100 105
Asn?Ala?Leu?Thr?MET?Val?Thr?Glu?Tyr?Ile?Pro?Arg?Gly?Asn?Leu
106 110 115 120
Lys?Asp?Leu?Leu?His?Gly?Ser?Asp?Asp?Pro?Ile?Ser?Phe?Glu?Ala
121 125 130 135
Arg?Leu?Cys?Ile?Ala?Ile?Asp?Cys?Ala?Glu?Ala?Leu?Ala?Phe?MET
136 140 145 150
His?Ser?Lys?Asp?Pro?Pro?Ile?Ile?His?Gly?Asp?Ile?Lys?Pro?Asp
151 155 160 165
Asn?Ile?Leu?Leu?Asp?Asp?Asn?Leu?Gly?Ala?Lys?Leu?Ser?Asp?Phe
166 170 175 180
Gly?Ile?Ser?Arg?Leu?Leu?Ser?MET?Glu?Asn?Ser?Tyr?Phe?Thr?Asn
181 185 190 195
Asn?Val?Ile?Gly?Ser?Arg?Gly?Tyr?MET?Asp?Pro?Glu?His?Ile?Gln
196 200 205 210
Thr?Gly?Arg?Val?Asp?Pro?Lys?Asn?Asp?Val?Tyr?Ser?Phe?Gly?Val
211 215 220 225
Val?Leu?Val?Glu?Leu?Val?Thr?Arg?Ala?MET?Ala?Ala?Glu?Asn?Gly
226 230 235 240
Thr?Cys?Ser?Asp?Leu?Ala?Lys?Lys?Phe?Ile?Glu?Ala?Phe?Leu?Gln
241 245 250 255
Lys?Asn?Ile?Phe?Leu?Lys?Val?Phe?Gly?Lys?Gln?Lys?Lys?Ala?Arg
256 260 265 270
Glu?MET?Phe?Asp?Thr?Gln?Ile?Ala?Asn?Ala?Ser?Asn?MET?Glu?Val
271 275 280 285
Leu?Glu?Lys?Ile?Gly?Glu?Leu?Ala?Ile?Glu?Cys?Leu?Arg?Arg?Asp
286 290 295 300
Ile?Lys?Lys?Arg?Pro?Glu?MET?Asn?His?Val?Val?Glu?Arg?Leu?Arg
301 305 310 315
MET?Leu?Gly?Lys?Asp?His?Glu?Lys?Arg?Gln?Asn?Arg?Lys?Ala?Glu
316 320 325 330
Lys?Gln?His?Gly?Val?Val?Pro?Pro?Asp?Gly?Asn?Thr?Thr?Phe?Ser
331 335 340 345
Ser?Ser?Trp?Pro?Lys?Gly?Arg?Leu?Glu?Asn?Gly?Arg?Lys?Ser?Ser
346 350 355 360
Ser?Ser?Asp?Ala?Glu?Ser?Leu?Phe?Ser?His?Arg?Glu?Val?Glu?Ala
361 365 370 375
Val?Asp?Glu?Glu?Asn?Gln?His?Pro?Leu?Leu?Arg?Arg?Lys?Ser?Ile
376 380 385 390
Gly?Asn?Gly?Pro?Pro?Glu?Ser?Phe?His?Asp?Trp?Thr?Tyr?Gly?Asn
391 395 400 405
Asp?Ile?Gly?Gly?Pro?Asp?Glu?Val?Phe?Ser?Arg?Gly?Tyr?Trp?His
406 410 415 420
Leu?Leu?Gly?Cys?Gln?Asn?Gly?Leu?His?Ile?Phe?Glu?Pro?Leu?Glu
421 425 430 435
Tyr?Gly?Asp?Tyr?Leu?Val?Arg?Ala?Val?Gly?Lys?Val?MET?Lys?Ala
436 440 445 450
Val?Gly?Val?Ile?Glu?Ala?Pro?Cys?Glu?Ala?Ile?Phe?Gln?Leu?Leu
451 455 460 465
MET?Ser?Ile?Asp?Ala?Ser?Arg?Tyr?Glu?Trp?Asp?Cys?Ser?Phe?MET
466 470 475 480
Tyr?Gly?Ser?Leu?Val?Glu?Glu?Leu?Asp?Gly?His?Thr?Ala?Ile?Leu
481 485 490 495
Tyr?His?Arg?Leu?His?Leu?Asp?Trp?Phe?Leu?Thr?Phe?Val?Trp?Pro
496 500 505 510
Arg?Asp?Leu?Cys?Tyr?Val?Arg?Tyr?Trp?Gln?Arg?Asn?Asp?Asp?Gly
511 515 520 525
Gly?Tyr?Val?Val?Leu?Phe?Gln?Ser?Arg?Glu?His?Pro?Asn?Cys?Gly
526 530 535 540
Pro?Gln?Pro?Gly?Phe?Val?Arg?Ala?His?Ile?Glu?Ile?Gly?Gly?Phe
541 545 550 555
Lys?Ile?Ser?Pro?Leu?Lys?Thr?Arg?Asn?Gly?Arg?Thr?Arg?Thr?Gln
556 560 565 570
Val?Gln?Tyr?Leu?MET?Lys?MET?Asp?Leu?Lys?Gly?Trp?Gly?Val?Gly
571 575 580 585
Tyr?Leu?Ser?Ser?Phe?Gln?Gln?His?Cys?Val?Leu?Arg?MET?Leu?Asn
586 590 595 600
Thr?Ile?Ala?Gly?Leu?Arg?Glu?Trp?Phe?Ser?Arg?Ser?Asp?Glu?Ile
601 605 610 615
Pro?Thr?Ser?MET?Asp?Gln?Ser?Arg?Tyr?Ser?Thr?MET?Leu?Glu?Glu
616 620 625 630
Glu?Ser?Asp?Glu?Asp?Glu?Leu?Ser?Ser?Gly?Ser?Asp?Glu?Ser
631 635 640 644
＜110〉Shandong Agricultural University
＜120〉clone, transformation and the application of long fringe couchgrass TeWKS1 gene
<210>3
<211>1935
<212>DNA
＜213〉long fringe couchgrass (Thinopyrum elongatum, Syn:Agropyron elongatum)
<400>3
1 atggagctcc?cacgaaacaa?acttgcagat?ttgaaccaag?gaaatgaaaa?tttaaaggca 60
61 aaggcaaagt?ggacacccaa?tgccaggagg?ttcacagaac?atcagattaa?aagaattact 120
121 aagaactata?gaactcatgt?tggcaaaggt?gcctttggtg?aggttttccg?aggttttctt 180
181 gacgatggca?gtccagttgc?agtgaagaaa?tacatcaacc?agaatatgaa?agaagggttt 240
241 gacaaagaga?taactatcca?ttgccaagtc?aaccacaaga?acatagtcaa?gcttttgggt 300
301 tattgttcag?aggaaaatgc?cttgacgatg?gtcactgagt?acattcctag?aggaaacctc 360
361 aaagacctcc?ttcatggaag?tgatgatccc?atttcttttg?aggcaagatt?gtgtattgct 420
421 atagattgtg?cagaggcatt?agctttcatg?cattcaaagg?atccgccaat?cattcacggt 480
481 gacatcaaac?ctgacaatat?actcttggat?gataacttgg?gtgcaaaatt?atctgacttt 540
541 ggaatatcaa?ggttgctttc?tatggagaat?agttatttta?ctaataatgt?aataggaagc 600
601 agaggttaca?tggatccaga?acacattcag?actggccgcg?ttgatcctaa?gaatgatgtt 660
661 tacagttttg?gggttgtttt?ggtagaacta?gttaccagag?ctatggcagc?tcagaatggg 720
721 acatgtagcg?accttgcaaa?aaaattcatt?gaagctttcc?tccaaaagaa?tatttttttg 780
781 aaagtttttg?gaaagcaaaa?aaaggcaaga?gagatgtttg?atacccagat?agcaaatgcg 840
841 agcaacatgg?aggttctaga?aaaaattgga?gagctggcaa?ttgagtgtct?tagaagggat 900
901 atcaagaaac?gtcctgaaat?gaatcatgtt?gtagaacgtc?ttcgaatgct?tggtaaagat 960
961 catgaaaaaa?gacaaaatag?aaaagcagaa?aagcaacatg?gagtggtgcc?tcctgatggt 1020
1021 aatacaacct?ttagctcttc?gtggccaaag?ggtagattag?aaaatggaag?gaaatcttct 1080
1081 tcctctgatg?ccgagagtct?gttcagtcat?agggaggtgg?aagcagtaga?cgaagaaaat 1140
1141 caacatccat?tattgcgaag?aaaatcaatt?ggaaatggtc?ctccggaatc?atttcatgat 1200
1201 tggacctatg?ggaacgacat?aggaggccct?gacgaagtat?tctctagagg?atactggcat 1260
1261 cttctcggat?gccagaatgg?cctccacatt?tttgagcccc?tcgagtatgg?tgattacctt 1320
1321 gtaagagccg?ttggcaaagt?gatgaaggcc?gttggtgtga?ttgaggcacc?ttgtgaggct 1380
1381 atatttcagc?ttctcatgag?catggacgct?agccgttatg?agtgggactg?cagcttcatg 1440
1441 tatggtagtc?tagtggagga?gctagatggc?cacactgcaa?tactatacca?taggctacac 1500
1501 ctagattggt?tcttgacgtt?tgtttggcct?cgtgatcttt?gttatgtacg?atattggcag 1560
1561 cgtaatgacg?acggaggtta?cgtggcgttg?ttccaatcca?gagagcaccc?aaactgtggt 1620
1621 ccacagccag?gatttgtgag?ggcacacatt?gagattggcg?ggttcaaaat?ttctccactg 1680
1681 aaaatccgta?atggaagaac?tcgaacacaa?gtacaatacc?ttatgaagat?ggatttgaag 1740
1741 ggttggggcg?ttggttactt?atcctcgttt?cagcagcatt?gcgtcctccg?catgctgaac 1800
1801 actattgctg?ggctcaggga?atggttttca?cgaagtgatg?aaattccaac?ttcgatggat 1860
1861 caaagtagat?attctacaat?gcttgaagag?gagtcagatg?aagacgagtt?gtcttcagga 1920
1921 agtgatgaaa?gttga 1935
＜110〉Shandong Agricultural University
＜120〉clone, transformation and the application of long fringe couchgrass TeWKS1 gene
<210>4
<211>644
<212>PRT
＜213〉long fringe couchgrass (Thinopyrum elongatum, Syn:Agropyron elongatum)
<400>4
MET?Glu?Leu?Pro?Arg?Asn?Lys?Leu?Ala?Asp?Leu?Asn?Gln?Gly?Asn
1 5 10 15
Glu?Asn?Leu?Lys?Ala?Lys?Ala?Lys?Trp?Thr?Pro?Asn?Ala?Arg?Arg
16 20 25 30
Phe?Thr?Glu?His?Gln?Ile?Lys?Arg?Ile?Thr?Lys?Asn?Tyr?Arg?Thr
31 35 40 45
His?Val?Gly?Lys?Gly?Ala?Phe?Gly?Glu?Val?Phe?Arg?Gly?Phe?Leu
46 50 55 60
Asp?Asp?Gly?Ser?Pro?Val?Ala?Val?Lys?Lys?Tyr?Ile?Asn?Gln?Asn
61 65 70 75
MET?Lys?Glu?Gly?Phe?Asp?Lys?Glu?Ile?Thr?Ile?His?Cys?Gln?Val
76 80 85 90
Asn?His?Lys?Asn?Ile?Val?Lys?Leu?Leu?Gly?Tyr?Cys?Ser?Glu?Glu
91 95 100 105
Asn?Ala?Leu?Thr?MET?Val?Thr?Glu?Tyr?Ile?Pro?Arg?Gly?Asn?Leu
106 110 115 120
Lys?Asp?Leu?Leu?His?Gly?Ser?Asp?Asp?Pro?Ile?Ser?Phe?Glu?Ala
121 125 130 135
Arg?Leu?Cys?Ile?Ala?Ile?Asp?Cys?Ala?Glu?Ala?Leu?Ala?Phe?MET
136 140 145 150
His?Ser?Lys?Asp?Pro?Pro?Ile?Ile?His?Gly?Asp?Ile?Lys?Pro?Asp
151 155 160 165
Asn?Ile?Leu?Leu?Asp?Asp?Asn?Leu?Gly?Ala?Lys?Leu?Ser?Asp?Phe
166 170 175 180
Gly?Ile?Ser?Arg?Leu?Leu?Ser?MET?Glu?Asn?Ser?Tyr?Phe?Thr?Asn
181 185 190 195
Asn?Val?Ile?Gly?Ser?Arg?Gly?Tyr?MET?Asp?Pro?Glu?His?Ile?Gln
196 200 205 210
Thr?Gly?Arg?Val?Asp?Pro?Lys?Asn?Asp?Val?Tyr?Ser?Phe?Gly?Val
211 215 220 225
Val?Leu?Val?Glu?Leu?Val?Thr?Arg?Ala?MET?Ala?Ala?Gln?Asn?Gly
226 230 235 240
Thr?Cys?Ser?Asp?Leu?Ala?Lys?Lys?Phe?Ile?Glu?Ala?Phe?Leu?Gln
241 245 250 255
Lys?Asn?Ile?Phe?Leu?Lys?Val?Phe?Gly?Lys?Gln?Lys?Lys?Ala?Arg
256 260 265 270
Glu?MET?Phe?Asp?Thr?Gln?Ile?Ala?Asn?Ala?Ser?Asn?MET?Glu?Val
271 275 280 285
Leu?Glu?Lys?Ile?Gly?Glu?Leu?Ala?Ile?Glu?Cys?Leu?Arg?Arg?Asp
286 290 295 300
Ile?Lys?Lys?Arg?Pro?Glu?MET?Asn?His?Val?Val?Glu?Arg?Leu?Arg
301 305 310 315
MET?Leu?Gly?Lys?Asp?His?Glu?Lys?Arg?Gln?Asn?Arg?Lys?Ala?Glu
316 320 325 330
Lys?Gln?His?Gly?Val?Val?Pro?Pro?Asp?Gly?Asn?Thr?Thr?Phe?Ser
331 335 340 345
Ser?Ser?Trp?Pro?Lys?Gly?Arg?Leu?Glu?Asn?Gly?Arg?Lys?Ser?Ser
346 350 355 360
Ser?Ser?Asp?Ala?Glu?Ser?Leu?Phe?Ser?His?Arg?Glu?Val?Glu?Ala
361 365 370 375
Val?Asp?Glu?Glu?Asn?Gln?His?Pro?Leu?Leu?Arg?Arg?Lys?Ser?Ile
376 380 385 390
Gly?Asn?Gly?Pro?Pro?Glu?Ser?Phe?His?Asp?Trp?Thr?Tyr?Gly?Asn
391 395 400 405
Asp?Ile?Gly?Gly?Pro?Asp?Glu?Val?Phe?Ser?Arg?Gly?Tyr?Trp?His
406 410 415 420
Leu?Leu?Gly?Cys?Gln?Asn?Gly?Leu?His?Ile?Phe?Glu?Pro?Leu?Glu
421 425 430 435
Tyr?Gly?Asp?Tyr?Leu?Val?Arg?Ala?Val?Gly?Lys?Val?MET?Lys?Ala
436 440 445 450
Val?Gly?Val?Ile?Glu?Ala?Pro?Cys?Glu?Ala?Ile?Phe?Gln?Leu?Leu
451 455 460 465
MET?Ser?MET?Asp?Ala?Ser?Arg?Tyr?Glu?Trp?Asp?Cys?Ser?Phe?MET
466 470 475 480
Tyr?Gly?Ser?Leu?Val?Glu?Glu?Leu?Asp?Gly?His?Thr?Ala?Ile?Leu
481 485 490 495
Tyr?His?Arg?Leu?His?Leu?Asp?Trp?Phe?Leu?Thr?Phe?Val?Trp?Pro
496 500 505 510
Arg?Asp?Leu?Cys?Tyr?Val?Arg?Tyr?Trp?Gln?Arg?Asn?Asp?Asp?Gly
511 515 520 525
Gly?Tyr?Val?Ala?Leu?Phe?Gln?Ser?Arg?Glu?His?Pro?Asn?Cys?Gly
526 530 535 540
Pro?Gln?Pro?Gly?Phe?Val?Arg?Ala?His?Ile?Glu?Ile?Gly?Gly?Phe
541 545 550 555
Lys?Ile?Ser?Pro?Leu?Lys?Ile?Arg?Asn?Gly?Arg?Thr?Arg?Thr?Gln
556 560 565 570
Val?Gln?Tyr?Leu?MET?Lys?MET?Asp?Leu?Lys?Gly?Trp?Gly?Val?Gly
571 575 580 585
Tyr?Leu?Ser?Ser?Phe?Gln?Gln?His?Cys?Val?Leu?Arg?MET?Leu?Asn
586 590 595 600
Thr?Ile?Ala?Gly?Leu?Arg?Glu?Trp?Phe?Ser?Arg?Ser?Asp?Glu?Ile
601 605 610 615
Pro?Thr?Ser?MET?Asp?Gln?Ser?Arg?Tyr?Ser?Thr?MET?Leu?Glu?Glu
616 620 625 630
Glu?Ser?Asp?Glu?Asp?Glu?Leu?Ser?Ser?Gly?Ser?Asp?Glu?Ser
631 635 640 644
Claims (3)
1. one kind long fringe couchgrass TeWKS1 gene is characterized in that described TeWKS1 gene comprises TeWKS1-1 and two genes of TeWKS1-2M; The TeWKS1-1 gene nucleotide series is shown in SEQ ID NO 1, and aminoacid sequence is shown in SEQ ID NO2; The TeWKS1-2M gene nucleotide series is shown in SEQ ID NO 3, and aminoacid sequence is shown in SEQ ID NO 4.
2. nucleotide sequence according to claim 1 is characterized in that this gene clone is in long fringe couchgrass.
3. the application of one kind long fringe couchgrass TeWKS1 gene is characterized in that two genes of TeWKS1-1 and TeWKS1-2M in Application in Wheat, can improve the resistance of wide spectrum of wheat to the stripe rust bacterium.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010100115276A CN101955945A (en) | 2010-01-07 | 2010-01-07 | Clone, transformation and application of TeWKS1 genes of thinopyrum elongatum |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010100115276A CN101955945A (en) | 2010-01-07 | 2010-01-07 | Clone, transformation and application of TeWKS1 genes of thinopyrum elongatum |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402141A (en) * | 2015-06-04 | 2019-03-01 | 山东农业大学 | The application of wheat male sterility gene WMS and its anther specific promoter |
| CN114807187A (en) * | 2022-05-05 | 2022-07-29 | 福建农林大学 | Ural chart wheat receptor protein kinase gene TuRLK1 and application thereof |
-
2010
- 2010-01-07 CN CN2010100115276A patent/CN101955945A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402141A (en) * | 2015-06-04 | 2019-03-01 | 山东农业大学 | The application of wheat male sterility gene WMS and its anther specific promoter |
| CN114807187A (en) * | 2022-05-05 | 2022-07-29 | 福建农林大学 | Ural chart wheat receptor protein kinase gene TuRLK1 and application thereof |
| CN114807187B (en) * | 2022-05-05 | 2023-08-18 | 福建农林大学 | Ula drawing wheat receptor protein kinase gene TuRLK1 and application thereof |
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Open date: 20110126 |