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

Longjiang 925 is a flue-cured tobacco variety with high resistance to Tobacco mosaic virus (TMV). In this study, the cDNA extracted from the TMV-inoculated leaves of Longjiang 925 was amplified with 240 pairs of primers. Approximately 9500 gene transcript fragments were obtained, in which 12 inducible expressed gene fragments were selected by cloning and sequencing. The inducible fragments functions involved in the nucleic acid metabolism, protein synthesis and modulation, energy metabolism, stress responding, intracellular transport, and metabolism of carbohydrates. The function of a differentially expressed gene sequence, TIF2, was validated using real-time PCR with the samples collected at 0, 12, 24, 48, and 72 h post inoculation. The result indicated that TIF2 was related to TMV-resistance. Both 5′- and 3′-rapid amplification of cDNA ends (RACE) were preformed using TIF2 sequence, and the full-length cDNA sequence was 875 bp in length, containing a conjecturable coding region from 101 bp to 613 bp and encoding 170 amino acids. Analyses of Blastn and Blastp showed that this gene was probably a novel gene related to TMV resistance in tobacco.

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。利用荧光定量PCR分析诱导表达片段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。