[发明专利]抗柑橘溃疡病pthA－nls基因及其构建方法与应用

摘要

抗柑橘溃疡病pthA－nls基因及其构建方法与应用。本发明利用柑橘溃疡病的致病基因pthA介导广谱抗性原理，构建了抗柑橘溃疡病的新基因pthA－nls。其构建方法包括以下步骤：(1)引物设计；(2)NLS序列的克隆；(3)PCR产物回收及酶切；(4)植物表达载体双酶切；(5)将pBI121酶切产物用T4 DNA连接酶与相应的NLS序列的PCR酶切产物连接；(6)将连接产物转化大肠杆菌DH5α，获得阳性克隆基因pthA－nls。基因pthA－nls可进一步对柑橘的感病品种进行遗传转化，获得抗柑橘溃疡病的新种质。本发明之抗柑橘溃疡病pthA－nls基因为柑橘抗溃疡病育种提供了有效工具。

主权项

1.一种抗柑橘溃疡病pthA-nls基因，其特征在于，具有如下序列：tgagcgtcgc aaaggcgctc ggtcttgcct tgctcgtcgg tgatgtactt caccagctccgcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcaccccccggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcatgaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggcatcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccaggcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcccgtgattttg tagccctggc cgacggccag caggtaggcc gacaggctca tgccggccgccgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgggctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgccggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaataagggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccggctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtatatcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggttatgcagcggaa aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcggcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatctttatagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcaggggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggccttttgctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgtattaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagtcagtgagcga ggaagcggaa gagcgccaga aggccgccag agaggccgag cgcggccgtgaggcttggac gctagggcag ggcatgaaaa agcccgtagc gggctgctac gggcgtctgacgcggtggaa agggggaggg gatgttgtct acatggctct gctgtagtga gtgggttgcgctccggcagc ggtcctgatc aatcgtcacc ctttctcggt ccttcaacgt tcctgacaacgagcctcctt ttcgccaatc catcgacaat caccgcgagt ccctgctcga acgctgcgtccggaccggct tcgtcgaagg cgtctatcgc ggcccgcaac agcggcgaga gcggagcctgttcaacggtg ccgccgcgct cgccggcatc gctgtcgccg gcctgctcct caagcacggccccaacagtg aagtagctga ttgtcatcag cgcattgacg gcgtccccgg ccgaaaaacccgcctcgcag aggaagcgaa gctgcgcgtc ggccgtttcc atctgcggtg cgcccggtcgcgtgccggca tggatgcgcg cgccatcgcg gtaggcgagc agcgcctgcc tgaagctgcgggcattcccg atcagaaatg agcgccagtc gtcgtcggct ctcggcaccg aatgcgtatgattctccgcc agcatggctt cggccagtgc gtcgagcagc gcccgcttgt tcctgaagtgccagtaaagc gccggctgct gaacccccaa ccgttccgcc agtttgcgtg tcgtcagaccgtctacgccg acctcgttca acaggtccag ggcggcacgg atcactgtat tcggctgcaactttgtcatg cttgacactt tatcactgat aaacataata tgtccaccaa cttatcagtgataaagaatc cgcgcgttca atcggaccag cggaggctgg tccggaggcc agacgtgaaacccaacatac ccctgatcgt aattctgagc actgtcgcgc tcgacgctgt cggcatcggcctgattatgc cggtgctgcc gggcctcctg cgcgatctgg ttcactcgaa cgacgtcaccgcccactatg gcattctgct ggcgctgtat gcgttggtgc aatttgcctg cgcacctgtgctgggcgcgc tgtcggatcg tttcgggcgg cggccaatct tgctcgtctc gctggccggcgccagatctg gggaaccctg tggttggcat gcacatacaa atggacgaac ggataaaccttttcacgccc ttttaaatat ccgattattc taataaacgc tcttttctct taggtttacccgccaatata tcctgtcaaa cactgatagt ttaaactgaa ggcgggaaac gacaatctgatcatgagcgg agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagccgttttacgtt tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttctggagtttaa tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcctaaggtcact atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataaattcccctcg gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccggatctggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgcttgggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccgccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccggtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcgttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgggcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatccatcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgaccaccaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatcaggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctcaaggcgcgcat gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc tgcttgccgaatatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtggcggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcgaatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcgccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccgaccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaaggttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatctcatgctggag ttcttcgccc acgggatctc tgcggaacag gcggtcgaag gtgccgatatcattacgaca gcaacggccg acaagcacaa cgccacgatc ctgagcgaca atatgatcgggcccggcgtc cacatcaacg gcgtcggcgg cgactgccca ggcaagaccg agatgcaccgcgatatcttg ctgcgttcgg atattttcgt ggagttcccg ccacagaccc ggatgatccccgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgcgatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatgcatgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaatacgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatctatgttacta gatcgggcct cctgtcaatg ctggcggcgg ctctggtggt ggttctggtggcggctctga gggtggtggc tctgagggtg gcggttctga gggtggcggc tctgagggaggcggttccgg tggtggctct ggttccggtg attttgatta tgaaaagatg gcaaacgctaataagggggc tatgaccgaa aatgccgatg aaaacgcgct acagtctgac gctaaaggcaaacttgattc tgtcgctact gattacggtg ctgctatcga tggtttcatt ggtgacgtttccggccttgc taatggtaat ggtgctactg gtgattttgc tggctctaat tcccaaatggctcaagtcgg tgacggtgat aattcacctt taatgaataa tttccgtcaa tatttaccttccctccctca atcggttgaa tgtcgccctt ttgtctttgg cccaatacgc aaaccgcctctccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaagcgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctttacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcacacaggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc cccagattagccttttcaat ttcagaaaga atgctaaccc acagatggtt agagaggctt acgcagcaggtctcatcaag acgatctacc cgagcaataa tctccaggaa atcaaatacc ttcccaagaaggttaaagat gcagtcaaaa gattcaggac taactgcatc aagaacacag agaaagatatatttctcaag atcagaagta ctattccagt atggacgatt caaggcttgc ttcacaaaccaaggcaagta atagagattg gagtctctaa aaaggtagtt cccactgaat caaaggccatggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg gcgaacagttcatacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca tggtggagcacgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaattgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctatctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattgcgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacccccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagtggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgcaagacccttcc tctatataag gaagttcatt tcatttggag agaacacggg ggactctagagttgcccagt tatctcgccc tgatccggcg ttggccgcgt tgaccaacga ccacctcgtcgccttggcct gcctcggcgg acgtcctgcg ctggatgcag tgaaaaaggg attgccgcacgcgccggcct tgatcaaaag aaccaatcgc cgtattcccg aacgcacatc ccatcgcgttgccgaccacg cgcaagtggt tcgcgtgctg ggttttttcc agtgccactc ccacccagcgcaagcatttg atgacgccat gacgcagttc gggatgagca ggcacgggtt gttacagctctttcgcagag tgggcgtcac cgaactcgaa gcccgcagtg gaacgctccc cccagcctcgcagcgttggg accgtatcct ccaggcatca gggatgaaaa gggccaaacc gtcccctacttcaactcaaa cgccggacca ggcgtctttg catgcattcg ccgattcgct ggagcgtgaccttgatgcgc ccagcccaac gcacgaggga gatcagaggc gggcaagcag ccgtaaacggtcccgatcgg atcgtgctgt caccggtccc tccgcacagc aatcgttcga ggtgcgcgttcccgaacagc gcgatgcgct gcatttgccc ctcagttgga gggtaaaacg cccgcgtaccagtatcgggg gcggcctccc ggatcctggt acgcccacgg ctgccgacct ggcagcgtccagcaccgtga tgcgggaaca agatgaggac cccttcgcag gggcagcgga tgatttcccggcattcaacg aagaggagct cgcatggttg gagctcgaat ttccccgatc gttcaaacatttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatataatttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttatgagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaaaatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcgggaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaacttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgcaccgatcgccc ttcccaacag ttgcgcagcc tgaatggcgc ccgctccttt cgctttcttcccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccctttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgatggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtccacgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggctattcttttg atttataagg gattttgccg atttcggaac caccatcaaa caggattttcgcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtgaagggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccacccca gtacattaaaaacgtccgca atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatcctgccaccag ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgatacaggcagcc catcagtccg ggacggcgtc agcgggagag ccgttgtaag gcggcagactttgctcatgt taccgatgct attcggaaga acggcaacta agctgccggg tttgaaacacggatgatctc gcggagggta gcatgttgat tgtaacgatg acagagcgtt gctgcctgtgatcaaatatc atctccctcg cagagatccg aattatcagc cttcttattc atttctcgcttaaccgtgac aggctgtcga tcttgagaac tatgccgaca taataggaaa tcgctggataaagccgctga ggaagctgag tggcgctatt tctttagaag tgaacgttga cgatatcaactcccctatcc attgctcacc gaatggtaca ggtcggggac ccgaagttcc gactgtcggcctgatgcatc cccggctgat cgaccccaga tctggggctg agaaagccca gtaaggaaacaactgtaggt tcgagtcgcg agatcccccg gaaccaaagg aagtaggtta aacccgctccgatcaggccg agccacgcca ggccgagaac attggttcct gtaggcatcg ggattggcggatcaaacact aaagctactg gaacgagcag aagtcctccg gccgccagtt gccaggcggtaaaggtgagc agaggcacgg gaggttgcca cttgcgggtc agcacggttc cgaacgccatggaaaccgcc cccgccaggc ccgctgcgac gccgacagga tctagcgctg cgtttggtgtcaacaccaac agcgccacgc ccgcagttcc gcaaatagcc cccaggaccg ccatcaatcgtatcgggcta cctagcagag cggcagagat gaacacgacc atcagcggct gcacagcgcctaccgtcgcc gcgaccccgc ccggcaggcg gtagaccgaa ataaacaaca agctccagaatagcgaaata ttaagtgcgc cgaggatgaa gatgcgcatc caccagattc ccgttggaatctgtcggacg atcatcacga gcaataaacc cgccggcaac gcccgcagca gcataccggcgacccctcgg cctcgctgtt cgggctccac gaaaacgccg gacagatgcg ccttgtgagcgtccttgggg ccgtcctcct gtttgaagac cgacagccca atgatctcgc cgtcgatgtaggcgccgaat gccacggcat ctcgcaaccg ttcagcgaac gcctccatgg gctttttctcctcgtgctcg taaacggacc cgaacatctc tggagctttc ttcagggccg acaatcggatctcgcggaaa tcctgcacgt cggccgctcc aagccgtcga atctgagcct taatcacaattgtcaatttt aatcctctgt ttatcggcag ttcgtagagc gcgccgtgcg tcccgagcgatactgagcga agcaagtgcg tcgagcagtg cccgcttgtt cctgaaatgc cagtaaagcgctggctgctg aacccccagc cggaactgac cccacaaggc cctagcgttt gcaatgcaccaggtcatcat tgacccaggc gtgttccacc aggccgctgc ctcgcaactc ttcgcaggcttcgccgacct gctcgcgcca cttcttcacg cgggtggaat ccgatccgca catgaggcggaaggtttcca gcttgagcgg gtacggctcc cggtgcgagc tgaaatagtc gaacatccgtcgggccgtcg gcgacagctt gcggtacttc tcccatatga atttcgtgta gtggtcgccagcaaacagca cgacgatttc ctcgtcgatc aggacctggc aacgggacgt tttcttgccacggtccagga cgcggaagcg gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcggacgtgaagc ccatcgccgt cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaataccggccat tgatcgacca gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatcggctcgccga taggggtgcg cttcgcgtac tccaacacct gctgccacac cagttcgtcatcgtcggccc gcagctcgac gccggtgtag gtgatcttca cgtccttgtt gacgtggaaaatgaccttgt tttgcagcgc ctcgcgcggg attttcttgt tgcgcgtggt gaacagggcagagcgggccg tgtcgtttgg catcgctcgc atcgtgtccg gccacggcgc aatatcgaacaaggaaagct gcatttcctt gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttggcctcgctga cctgttttgc caggtcctcg ccggcggttt ttcgcttctt ggtcgtcatagttcctcgcg tgtcgatggt catcgacttc gccaaacctg ccgcctcctg ttcgagacgacgcgaacgct ccacggcggc cgatggcgcg ggcagggcag ggggagccag ttgcacgctgtcgcgctcga tcttggccgt agcttgctgg accatcgagc cgacggactg gaaggtttcgcggggcgcac gcatgacggt gcggcttgcg atggtttcgg catcctcggc ggaaaaccccgcgtcgatca gttcttgcct gtatgccttc cggtcaaacg tccgattcat tcaccctccttgcgggattg ccccgactca cgccggggca atgtgccctt attcctgatt tgacccgcctggtgccttgg tgtccagata atccacctta tcggcaatga agtcggtccc gtagaccgtctggccgtcct tctcgtactt ggtattccga atcttgccct gcacgaatac cagcgaccccttgcccaaat acttgccgtg ggcctcggcc tgagagccaa aacacttgat gcggaagaagtcggtgcgct cctgcttgtc gccggcatcg ttgcgccaca tctaggtact aaaacaattcatccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca gtaagtcaaaaaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga cgcagaaggcaatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc cacttactttgccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga caagttcctcttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa atggagtgtcttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt aatccaattcggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga tggagtgaaagagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt catcttcatactcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc agccatcatgccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata gcatcatgtccttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca tttttaaatataggttttca ttttctccca ccagcttata taccttagca ggagacattc cttccgtatcttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc tcattttagccatttattat ttccttcctc ttttctacag tatttaaaga taccccaaga agctaattataacaagacga actccaattc actgttcctt gcattctaaa accttaaata ccagaaaacagctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga gccgattttgaaaccacaat tatgggtgat gctgccaact tactgattta gtgtatgatg gtgtttttgaggtgctccag tggcttctgt gtctatcagc tgtccctcct gttcagctac tgacggggtggtgcgtaacg gcaaaagcac cgccggacat cagcgctatc tctgctctca ctgccgtaaaacatggcaac tgcagttcac ttacaccgct tctcaacccg gtacgcacca gaaaatcattgatatggcca tgaatggcgt tggatgccgg gcaacagccc gcattatggg cgttggcctcaacacgattt tacgtcactt aaaaaactca ggccgcagtc ggtaacctcg cgcatacagccgggcagtga cgtcatcgtc tgcgcggaaa tggacgaaca gtggggctat gtcggggctaaatcgcgcca gcgctggctg ttttacgcgt atgacagtct ccggaagacg gttgttgcgcacgtattcgg tgaacgcact atggcgacgc tggggcgtct tatgagcctg ctgtcaccctttgacgtggt gatatggatg acggatggct ggccgctgta tgaatcccgc ctgaagggaaagctgcacgt aatcagcaag cgatatacgc agcgaattga gcggcataac ctgaatctgaggcagcacct ggcacggctg ggacggaagt cgctgtcgtt ctcaaaatcg gtggagctgcatgacaaagt catcgggcat tatctgaaca taaaacacta tcaataagtt ggagtcattacccaattatg atagaattta caagctataa ggttattgtc ctgggtttca agcattagtccatgcaagtt tttatgcttt gcccattcta tagatatatt gataagcgcg ctgcctatgccttgccccct gaaatcctta catacggcga tatcttctat ataaaagata tattatcttatcagtattgt caatatattc aaggcaatct gcctcctcat cctcttcatc ctcttcgtcttggtagcttt ttaaatatgg cgcttcatag agtaattctg taaaggtcca attctcgttttcatacctcg gtataatctt acctatcacc tcaaatggtt cgctgggttt atcgcacccccgaacacgag cacggcaccc gcgaccacta tgccaagaat gcccaaggta aaaattgccggccccgccat gaagtccgtg aatgccccga cggccgaagt gaagggcagg ccgccacccaggccgccgcc ctcactgccc ggcacctggt cgctgaatgt cgatgccagc acctgcggcacgtcaatgct tccgggcgtc gcgctcgggc tgatcgccca tcccgttact gccccgatcccggcaatggc aaggactgcc agcgctgcca tttttggggt gaggccgttc gcggccgaggggcgcagccc ctggggggat gggaggcccg cgttagcggg ccgggagggt tcgagaagggggggcacccc ccttcggcgt gcgcggtcac gcgcacaggg cgcagccctg gttaaaaacaaggtttataa atattggttt aaaagcaggt taaaagacag gttagcggtg gccgaaaaacgggcggaaac ccttgcaaat gctggatttt ctgcctgtgg acagcccctc aaatgtcaataggtgcgccc ctcatctgtc agcactctgc ccctcaagtg tcaaggatcg cgcccctcatctgtcagtag tcgcgcccct caagtgtcaa taccgcaggg cacttatccc caggcttgtccacatcatct gtgggaaact cgcgtaaaat caggcgtttt cgccgatttg cgaggctggccagctccacg tcgccggccg aaatcgagcc tgcccctcat ctgtcaacgc cgcgccgggtgagtcggccc ctcaagtgtc aacgtccgcc cctcatctgt cagtgagggc caagttttccgcgaggtatc cacaacgccg gcggccgcgg tgtctcgcac acggcttcga cggcgtttctggcgcgtttg cagggccata gacggccgcc agcccagcgg cgagggcaac cagcccgg。