利用cDNA-AFLP技术分析炭疽菌危害诱导茶树的差异表达基因

cDNA-AFLP Reveals Differential Gene Expression Profiles of Tea Plant (Camellia sinensis cv. Maoxie) Induced by Colletotrichum sp.1 Infection

应用cDNA-AFLP技术筛选炭疽菌(Colletotrichum sp.1)危害诱导茶树毛蟹品种(Camellia sinensis cv. Maoxie)的差异表达基因,为探究茶树抗炭疽菌的分子机制奠定基础。利用256对选择性扩增引物组合分析病菌接种后0 h和48 h的叶片cDNA,经筛选、测序和BLAST比对获得136条差异表达片段(TDFs)。同源基因功能分析结果表明,128条TDFs与Nr数据库已有基因同源,其功能以胁迫响应、生物调控与信号转导为主;其中51条TDFs与TPIA数据库中冷害、干旱、盐碱条件下的差异表达基因高度同源。进一步对27条TDFs进行qRT-PCR验证,结果显示,有24条TDFs的表达情况与cDNA-AFLP的结果一致,WRKY转录因子、乙烯响应转录因子、过氧化物酶和超氧化物歧化酶等抗逆相关基因的表达显著上调。本试验通过筛选获得差异表达的抗逆相关基因,为后续的基因功能研究奠定基础。

Differentially expressed genes related to Camellia sinensis cv.Maoxie against Colletotrichum sp.1 infection were screened by cDNA-AFLP(cDNA-amplified fragment length polymorphism)to provide reference for further illustrating the molecular mechanism underlying tea resistance to anthracnose.A total of 256 pairs of selective primers were applied to amplify the cDNA of leaves collected at 0 h and 48 h after Colletotrichum infection,and 136 differentially expressed transcript-derived fragments(TDFs)were obtained via screening,sequencing and BLAST analysis.The analysis of the homologous genes revealed that 128 TDFs were homologous with the known genes in Nr database.Most of them were related to stress responses,biological regulation and signal transduction,and 51 TDFs were highly homologous to differential expressed genes of tea plant under cold,drought or salt conditions in TPIA database.Further qRT-PCR analysis of 27 TDFs showed that the expression profiles of 24 TDFs were consistent with those of cDNA-AFLP results,among which several resistance-related genes including WRKY transcription factor,ethylene-responsive transcription factor,peroxidase and superoxide dismutase were significantly up-regulated.The differential expressed genes found in this study would lay a foundation for further analysis of their functions.