CAJ | 학술논문

于葡萄黑痘病发病盛期,用病叶压片法对高抗黑痘病的中国野生毛葡萄'商-24'接种黑痘病病原菌,采用mRNA差异显示技术进行抗黑痘病基因表达差异的研究.结果显示:(1)获得了T11GG/B0304-400、T11CC/S428-350、T11GG/S424-700、T11AG/S432-350、T11GG/S433-250、T11GG/S433-300、T11AG/S432-300、T11GG/S438-353和T11AG/S424-300等9个基因表达差异cDNA片段.其中T11GG/S438-353 mRNA片段表达在接种后3 d被诱导显著降低,并在之后2 d几乎检测不到.(2)采用RACE技术克隆了T11GG/S438-353 mRNA片段的cDNA全长序列;序列分析表明,该cDNA包含一个607 bp完整的开放阅读框架,编码149个氨基酸;其编码氨基酸序列与拟南芥、欧洲葡萄、柳杉、党参、无梗花栎、欧洲栗及寄生草钙调蛋白的一致性分别为99%、97%、94%、91%、90%、88%和77%.(3)本实验克隆到了负向调控中国野生毛葡萄抗黑痘病的钙调蛋白基因,并命名为VqCaM,其GenBank登录号为EU694099;实时荧光定量PCR结果再次验证VqCaM表达受葡萄黑痘病侵染下调,
우포도흑두병발병성기,용병협압편법대고항흑두병적중국야생모포도'상-24'접충흑두병병원균,채용mRNA차이현시기술진행항흑두병기인표체차이적연구.결과현시:(1)획득료T11GG/B0304-400、T11CC/S428-350、T11GG/S424-700、T11AG/S432-350、T11GG/S433-250、T11GG/S433-300、T11AG/S432-300、T11GG/S438-353화T11AG/S424-300등9개기인표체차이cDNA편단.기중T11GG/S438-353 mRNA편단표체재접충후3 d피유도현저강저,병재지후2 d궤호검측불도.(2)채용RACE기술극륭료T11GG/S438-353 mRNA편단적cDNA전장서렬;서렬분석표명,해cDNA포함일개607 bp완정적개방열독광가,편마149개안기산;기편마안기산서렬여의남개、구주포도、류삼、당삼、무경화력、구주률급기생초개조단백적일치성분별위99%、97%、94%、91%、90%、88%화77%.(3)본실험극륭도료부향조공중국야생모포도항흑두병적개조단백기인,병명명위VqCaM,기GenBank등록호위EU694099;실시형광정량PCR결과재차험증VqCaM표체수포도흑두병침염하조,
mRNA differential display was employed to study the differential expression genes of anthracnose disease resistance with E.ampelina inoculated leaves in Chinese wild V.quinquangularis clone 'Shang-24'.Nine differential expressed cDNA fragments T11GG/B0304-400,T11CC/S428-350,T11GG/S424-700,T11AG/S432-350,T11GG/S433-250,T11GG/S433-300,T11AG/S432-300,T11GG/S438-353 and T11AG/S424-300 were obtained,in which a cDNA fragment,T11GG/S438-353,was obtained which expression levels were down-regulated significantly in inoculated leaves during first three days post-inoculation and were nearly undetectable within additional two days.Rapid Amplification of cDNA Ends (RACE) was used to obtain the full-length cDNA sequence of T11GG/S438-353.Full size of 607 bp is obtained and predicted to encode a polypeptide of 149 residues.The deduced amino acid (aa) sequence shared fine identity of 99%,97%,94%,91%,90%,88% and 77% with calmodulin (CaM) proteins from Arabidopsis thaliana,V.vinifera,Cryptomeria japonica,Codonopsis lanceolata,Quercus petraea,Castanea sativa and Striga asiatica respectively.We designated it as VqCaM (GenBank accession number is EU694099).qRT-PCR result also confirmed the expression pattern of VqCaM post E.ampelina infection in V.quinquangularis 'Shang-24'.These results suggest that VqCaM is most likely a specific CaM gene function negatively in message transfer in disease resistance of V.quinquangularis 'Shang-24' against E.ampelina.