利用cDNA-AFLP分析内生解淀粉芽胞杆菌 Fy11诱导辣椒差异表达基因

doi:10.3864/j.issn.0578-1752.2013.12.006

摘要/Abstract

摘要： 【目的】分析内生解淀粉芽胞杆菌（Bacillus amyloliquefaciens）Fy11诱导辣椒（Capsicum annuum）幼苗差异表达基因，了解辣椒与内生芽胞杆菌互作的分子机制。【方法】利用cDNA-AFLP技术，采用256对引物，分析辣椒幼苗接种内生解淀粉芽胞杆菌Fy11后5个时间点的基因表达谱；利用qRT-PCR分析差异基因表达模式，验证cDNA-AFLP表达谱。【结果】256对引物共产生18 620个转录本（TDF），筛选获得353条差异表达条带，占扩增条带总数的1.89%。经克隆、测序分析，最终获得257个差异TDF，聚类分析得到229个独立基因（EST，unigenes），其中144个基因上调表达，85个基因下调表达。经Blastx比对和功能分类分析，其中65条EST（28.38%）未找到同源性匹配，8条（3.49%）与未知功能蛋白同源性较高。其余156条EST主要涉及基础代谢基因（10.92%）；与能量和抗病与防御类相关基因，各占8.73%；信号转导基因占7.42%；转运子或转座子基因占6.99%；与细胞结构相关基因占6.55%；参与转录调控基因占5.68%；细胞生长类基因占3.93%；参与蛋白质运输和储存有8个基因，占3.49%；蛋白质合成类基因占2.18%；次生代谢类和胞间运输类基因，其数量相对较少，各占1.75%。选取与抗病防御、转录调控及信号转导类等相关的10个差异基因，qRT-PCR分析结果显示其表达模式符合cDNA-AFLP表达谱。【结论】内生细菌与植物互作的分子机制涉及植物多方面生理生化反应，包括抗病防御、转录调控、蛋白质代谢、信号转导、以及非生物胁迫等多种途径相关基因的协同控制。

关键词: 辣椒 , 内生解淀粉芽胞杆菌 , 互作 , 基因表达 , cDNA-AFLP , qRT-PCR

Abstract: 【Objective】The objective of this study is to analyze extensive transcription profiling of pepper seedlings inoculated with an endophytic bacterial strain, Bacillus amyloliquefaciens Fy11, and elucidate the molecular mechanism of endophytic Bacillus and plant interaction.【Method】cDNA-AFLP technique was used to conduct transcription profiling of pepper seedlings across five sampling time points after inoculation with Fy11. The validation of cDNA-AFLP expression patterns was analyzed by qRT-PCR.【Result】 A total of 18 620 transcript derived fragments (TDFs) were obtained using cDNA-AFLP with 256 primer pairs, 353 (1.89%) displayed altered expression patterns after inoculation. Two-hundred and fifty seven differentially expressed TDFs produced reliable sequences after cloning and sequencing. Two-hundred and twenty nine expressed sequence tags (ESTs) of unigenes were obtained after assembling, of which 144 showed up-regulated and 85 down-regulated. Blastx analysis and functional annotations were then performed and the results revealed that the 156 ESTs had predicted gene products mainly implicated in energy (10.92%), metabolism and disease/defense (each accounted for 8.73%), signal transduction (7.42%), transporter (6.99%), cell structure (6.55%), transcription (5.68%), cell growth (3.93%), protein destination and storage (3.49%), protein synthesis (2.18%), secondary metabolism and intracellur traffic (each accounted for 1.75%). The 65 ESTs (28.38% of the sequenced total 229 ESTs) had no match to known genes, and the 8 ESTs (3.49% of the sequenced total 229 ESTs) were highly homologous to unknown functional proteins. Ten differential genes related to disease/defense, transcription, and signal transduction were chosen for further qRT-PCR expression patterns, which confirmed the cDNA-AFLP profiles. 【Conclusion】 Endophytic bacteria and plant interaction involved in multifaceted biochemical and physiological reactions, including concerted regulation of the genes involved in different pathways, like disease/defense, transcription, protein metabolism, signal transduction, as well as abiotic stresses. These results provide information for further elucidation of molecular mechanism of endophytic bacteria and plant interaction.

Key words: Capsicum annuum , Bacillus amyloliquefaciens , interaction , gene expression , cDNA-AFLP , qRT-PCR

杨瑞先, 范晓静, 邱思鑫, 蔡学清, 胡方平. 利用cDNA-AFLP分析内生解淀粉芽胞杆菌 Fy11诱导辣椒差异表达基因[J]. 中国农业科学, 2013, 46(12): 2449-2458.

YANG Rui-Xian, FAN Xiao-Jing, QIU Si-Xin, CAI Xue-Qing, HU Fang-Ping. cDNA-AFLP Analysis of Differential Gene Expression in Pepper Inoculated with Endophytic Bacillus amyloliquefaciens Fy11[J]. Scientia Agricultura Sinica, 2013, 46(12): 2449-2458.

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