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作物学报 ›› 2012, Vol. 38 ›› Issue (01): 62-70.doi: 10.3724/SP.J.1006.2012.00062

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

TMV侵染烟草基因差异表达的cDNA-AFLP分析

陈荣平1,2，刘磊3，万秀清2，邱恩建2，王春军2，宋宝刚2，颜培强2，杨铁钊1,*   

  1. 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
  • 收稿日期:2011-05-10 修回日期:2011-09-12 出版日期:2012-01-12 网络出版日期:2011-11-07
  • 通讯作者: 杨铁钊, E-mail: yangtiezhao@126.com, Tel: 0371-63358030
  • 基金资助:

    本研究是由中国烟草总公司面上项目(HN200903)资助。

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

CHEN Rong-Ping1,2,LIU Lie3,WAN Xiu-Qing2,QIU En-Jian2,WANG Chun-Jun2,SONG Bao-Gang2,YAN Pei-Qiang2,YANG Tie-Zhao1,*   

  1. 1 College of Tobacco Science, Henan Agricultural University, Zhengzhou 450002, China; 2 Mudanjiang Tobacco Science and Research Institute of Heilongjiang Province Tobacco Company, Mudanjiang 157011, China; 3 Centers for Disease Control and Prevention in Mudanjiang, Mudanjiang 157022, China
  • Received:2011-05-10 Revised:2011-09-12 Published:2012-01-12 Published online:2011-11-07
  • Contact: 杨铁钊, E-mail: yangtiezhao@126.com, Tel: 0371-63358030

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2411

被引次数

16

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 9 500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, 并利用荧光定量PCR分析一个诱导表达片段TIF2在0~72 h之间5个时间点(0、12、24、48和72 h)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857 bp, 预测的编码区在101~613 bp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。

关键词: 烟草, TMV, 差异基因表达, cDNA-AFLP

Abstract: 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 functions 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 indicated that the genes isolated were 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.

Key words: Tobacco, TMV, Differentially Expressed Genes, cDNA-AFLP

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