棉铃虫蜕皮调节转录因子cDNA及其克隆方法与重组应用

摘要

棉铃虫蜕皮调节转录因子cDNA及其克隆方法与重组应用，属于生物基因工程技术领域。棉铃虫蜕皮调节转录因子具有SEQ ID NO 1和SEQ ID NO.2所示的序列。本发明的克隆方法是，从棉铃虫中提取总RNA或mRNA，反转录合成cDNA；根据蜕皮调节转录因子的保守序列设计引物，通过链式聚合酶反应扩增到一个DNA片段；将扩增产物纯化，取纯化产物克隆到pGEM－T Easy载体，转化DH5α细胞，平板过夜培养。本发明棉铃虫蜕皮调节转录因子，通过基因重组技术在昆虫核型多角体病毒中进行表达，获得有杀虫活性的基因重组病毒，用于害虫防治；或者通过基因工程方法将棉铃虫蜕皮调节转录因子专入作物，获得具有抗虫能力的作物。

主权项

1.一种克隆的棉铃虫蜕皮调节转录因子cDNA，其特征在于，具有如下序列：(1)SEQ ID NO 1的信息(a)序列特征：*长度：1668碱基对*类型：核酸*链型：双链*拓扑结构：线性(b)分子类型：cDNA(c)假设：否(d)反义：否(e)最初来源：棉铃虫(Helicoverpa armigera)(f)序列描述：SEQ ID NO.1atgaacaaca accagttcca cgatttattt gggtcccagt ggcctccgga ccagcacgga 60ggtcactcgt cggcatcgac gatgctgcac cagtcccagg gcctgcccca gggtatgcag 120ctgaagagag aacctcatac ggatgtgcag ccggtgatgc acaaccagat gggcacggac 180atcacgtcag ggtccgtggc ggacagcaca tcccccccgc ctggcagcag tgacgggatg 240ttcggctcgt ctatatctgg catgtttatg gataagaaag ccgctaattc tataagagct 300caaatcgaaa taataccatg caaggtgtgt ggggataaat catctggagt ccactatggc 360gtcatcacct gcgaggggtg caagggtttc ttcaggcgat cacagagtac agtggtgaac 420taccagtgtc cacggaataa agcctgcgtg gtcgaccggg tcaacagaaa tcgttgtcag 480tattgcagat tgcagaaatg cctcaaactt ggaatgagcc gtgatgcggt gaagtttgga 540cggatgtcga aaaagcaacg tgagaaggtg gaagatgagg tgagattcca tcgcgcacag 600atgagagctc agactgacac ggcgcccgac tcagtatacg acgctcaaca acaaacgccg 660agctcaagcg accagttcca tggtcactac aatggctacc cgggctacgg gtcgccgttg 720tcctcatatg ggtacaacaa cgcagggccg gcgctacagt ccaacatggg aggcatacag 780cctcaagcac ctcaacagca gccctatgac gtgtcggctg attacgtgga ctccaccacg 840gcttacgagc cgaaacagac cgagggattc ttggatcctg attttattag tcatgcggag 900ggtgacatca gcaaagtcct ggtgaagagt ttggcagaag cacacgcaaa taccaacccc 960aaactggagt tcattcatga gatgttcaga aagccacagg atgtctccaa gcttctatac 1020tatagttcaa tgacgtacga ggaaatgtgg ctggattgtg cagacaaatt aactgggatg 1080attcagaata ttatcgagtt cgctaaactt atacccgggt tcatgaaact gactcaggat 1140gatcagattt tgctcttgaa atctggctcg ttcgagctcg ccatagtccg gctgtcgcga 1200ctgatagacg tcaaccgtga ccaagtgcta tacggtgatg cggtgctgtc catcagagaa 1260tgtgtgcatg cacgtgaccc ccgcgacgtg gcgctggtgg ttggtatctt cgacgcggca 1320aagacgatcg ctcgactcaa actcactgag actgaactgg cgctatacca gagccttgtg 1380ctattgtggc cagagcggca cggcgtccgc ggcaaccctg agatccagtg cctgttcaac 1440atgtcgatgg cggcgatgcg gcacgagatc gagaccaacc acgcccccct caagggagac 1500gtcactgttc tggacacgct gctcgccaag atacccacct tcagagaact atcactgatg 1560cacctggaag ctttgtgccg cttcaaggcc gcccaccccc accacgtatt cccggccctc 1620tacaaggagc tgttctcact cgacagcgtc ctggactaca cccacggt 1668(2)SEQ ID NO.2的信息(a)序列特征 *长度：556氨基酸 *类型：氨基酸 *链型：单链 *拓扑结构：线性(b)分子类型：蛋白质(c)序列描述Met Asn Asn Asn Gln Phe His Asp Leu Phe Gly Ser Gln Trp Pro1 5 10 15Pro Asp Gln His Gly Gly His Ser Ser Ala Ser Thr Met Leu His16 20 25 30Gln Ser Gln Gly Leu Pro Gln Gly Met Gln Leu Lys Arg Glu Pro31 35 40 45His Thr Asp Val Gln Pro Val Met His Asn Gln Met Gly Thr Asp46 50 55 60Ile Thr Ser Gly Ser Val Ala Asp Ser Thr Ser Pro Pro Pro Gly61 65 70 75Ser Ser Asp Gly Met Phe Gly Ser Ser Ile Ser Gly Met Phe Met76 80 85 90Asp Lys Lys Ala Ala Asn Ser Ile Arg Ala Gln Ile Glu Ile Ile91 95 100 105Pro Cys Lys Val Cys Gly Asp Lys Ser Ser Gly Val His Tyr Gly106 110 115 120Val Ile Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Gln121 125 130 135Ser Thr Val Val Asn Tyr Gln Cys Pro Arg Asn Lys Ala Cys Val136 140 145 150Val Asp Arg Val Asn Arg Asn Arg Cys Gln Tyr Cys Arg Leu Gln151 155 160 165Lys Cys Leu Lys Leu Gly Met Ser Arg Asp Ala Val Lys Phe Gly166 170 175 180Arg Met Ser Lys Lys Gln Arg Glu Lys Val Glu Asp Glu Val Arg181 185 190 195Phe His Arg Ala Gln Met Arg Ala Gln Thr Asp Thr Ala Pro Asp196 200 205 210Ser Val Tyr Asp Ala Gln Gln Gln Thr Pro Ser Ser Ser Asp Gln211 215 220 225Phe His Gly His Tyr Asn Gly Tyr Pro Gly Tyr Gly Ser Pro Leu226 230 235 240Ser Ser Tyr Gly Tyr Asn Asn Ala Gly Pro Ala Leu Gln Ser Asn241 245 250 255Met Gly Gly Ile Gln Pro Gln Ala Pro Gln Gln Gln Pro Tyr Asp256 260 265 270Val Ser Ala Asp Tyr Val Asp Ser Thr Thr Ala Tyr Glu Pro Lys271 275 280 285Gln Thr Glu Gly Phe Leu Asp Pro Asp Phe Ile Ser His Ala Glu286 290 295 300Gly Asp Ile Ser Lys Val Leu Val Lys Ser Leu Ala Glu Ala His301 305 310 315Ala Asn Thr Asn Pro Lys Leu Glu Phe Ile His Glu Met Phe Arg316 320 325 330Lys Pro Gln Asp Val Ser Lys Leu Leu Tyr Tyr Ser Ser Met Thr331 335 340 345Tyr Glu Glu Met Trp Leu Asp Cys Ala Asp Lys Leu Thr Gly Met346 350 355 360Ile Gln Asn Ile Ile Glu Phe Ala Lys Leu Ile Pro Gly Phe Met361 365 370 375Lys Leu Thr Gln Asp Asp Gln Ile Leu Leu Leu Lys Ser Gly Ser376 380 385 390Phe Glu Leu Ala Ile Val Arg Leu Ser Arg Leu Ile Asp Val Asn391 395 400 405Arg Asp Gln Val Leu Tyr Gly Asp Ala Val Leu Ser Ile Arg Glu406 410 415 420Cys Val His Ala Arg Asp Pro Arg Asp Val Ala Leu Val Val Gly421 425 430 435Ile Phe Asp Ala Ala Lys Thr Ile Ala Arg Leu Lys Leu Thr Glu436 440 445 450Thr Glu Leu Ala Leu Tyr Gln Ser Leu Val Leu Leu Trp Pro Glu451 455 460 465Arg His Gly Val Arg Gly Asn Pro Glu Ile Gln Cys Leu Phe Asn466 470 475 480Met Ser Met Ala Ala Met Arg His Glu Ile Glu Thr Asn His Ala481 485 490 495Pro Leu Lys Gly Asp Val Thr Val Leu Asp Thr Leu Leu Ala Lys496 500 505 510Ile Pro Thr Phe Arg Glu Leu Ser Leu Met His Leu Glu Ala Leu511 515 520 525Cys Arg Phe Lys Ala Ala His Pro His His Val Phe Pro Ala Leu526 530 535 540Tyr Lys Glu Leu Phe Ser Leu Asp Ser Val Leu Asp Tyr Thr His541 545 550 555Gly556