TMV侵染烟草基因差异表达的cDNA-AFLP分析  

cDNA-AFLP Analysis of Differentially Expressed Genes in Tobacco Infected by TMV

作　　者：陈荣平[1,2] 刘磊 万秀清 邱恩建 王春军 宋宝刚 颜培强 杨铁钊[1] 

机构地区：[1]河南农业大学烟草学院,河南郑州450002 [2]黑龙江省烟草公司牡丹江烟草科学研究所,黑龙江牡丹江157011 [3]牡丹江市疾病防控中心,黑龙江牡丹江157022

出　　处：《作物学报》2012年第1期62-70,共9页Acta Agronomica Sinica

基　　金：中国烟草总公司面上项目(HN200903)资助

摘　　要：以烤烟品种龙江925[高抗烟草普通花叶病(TMV)]接种TMV的叶片为材料,选用240对引物组合的扩增,获得约9500个转录基因片段,经过克隆测序最终获得了12个诱导表达片段,它们与核酸代谢、蛋白质合成与修饰、能量代谢、胁迫响应、细胞内运输、糖代谢等相关。利用荧光定量PCR分析诱导表达片段TIF2在0～72h之间5个时间点(0、12、24、48和72h)的基因差异表达,证实了所获得的基因序列为真实诱导的表达序列。对此序列进行RACE,最终获得全长cDNA序列,全序列857bp,预测的编码区在101～613bp之间,共编码170个氨基酸。经Blastn、Blastp比对分析,在烟草中没有获得其同源序列,确定该基因是在烟草中发现的与抗TMV相关的新基因。Flue-cured tobacco variety Longjiang 925 with high resistance to Tobacco mosaic virus（TMV） was used as the tested material,and we selected 240 pairs of primers and amplified cDNA from the tobacco leaf inoculated by TMV.The results showed that approximately 9500 gene transcript fragments were obtained,and twelve inducible expressed gene fragments were selected out by cloning and sequencing.The inducible fragments functions,involve in the nucleic acid metabolism,protein synthesis and modulation,energy metabolism,stress responding,intracellular transport,metabolism of carbohydrates.To validate the func-tions of these differentially expressed gene sequences obtained,we analyzed TIF2 fragment by real-time PCR with the samples collected at 0,12,24,48,and 72 hours after inoculation,and the mRNAs samples were isolated.The result of real-time PCR in-dicated that the gene isolated was related to TMV-resistance.Then,both 5＇-and 3＇-rapid amplification of cDNA ends（RACE） were preformed.A full length cDNA sequence of TIF2 contained 875 bp,with a coding zone of 101 bp to 613 bp conjecturably,encoding 170 amino acids.Analyses of Blastn and Blastp showed that the gene was not homologous to tobacco,and probably was the TMV-resistance related novel gene.The results will be helpful for the future research on tobacco resistant-breeding to TMV in molecule level and so on.

关 键 词：烟草 TMV 差异基因表达 CDNA-AFLP 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]