锌胁迫下小麦SSH文库的构建及初步分析

Construction and Analysis of Wheat SSH Libraries Induced under Zinc Stress

Zn是植物必需的微量元素,同时也是近年来造成环境污染的重金属元素之一.为了从基因表达水平揭示小麦Zn胁迫响应的分子机制,本研究利用抑制差减杂交(suppression subtractivehybridization,SSH)技术构建了Zn胁迫(0.5 mmol/L,1 mmol/L)下小麦的正反向SSH文库.从正反向文库中随机挑选阳性单克隆,并利用通用引物T7/Sp6对其进行验证.结果显示,正反库中分别获得307和821个EST序列,其片段长度在200～1 000 bp之间,它们反映了Zn胁迫下特异响应的基因.利用BLASTn和BLASTx将这些EST序列进行比对分析,在正、反库中分别筛选出221和641个uniESTs,其中751个uniESTs被注释(包括正库中193个和反库中558个).这些序列的功能主要涉及信号转导、抗氧化防御、转录与翻译、物质运输、核糖体结构、能量代谢,以及一些功能未知的基因.

Zinc is an essential trace element for plants, as well as one of heavy metals causingenvironmental pollution in recent years. In order to clarity the molecular mechamsms ot responses ot wheat （Triticum aestivum L. ） to zinc stress at the transcriptional level, we constructed forward and reverse suppression subtractive hybridization （SSH） eDNA libraries of wheat under zinc stress （0.5 retool/L, 1 mmol/L）. 307 and 821 ESTs were obtained from forward and reverse eDNA library respectively through testing positive clones randomly selected from two SSH libraries using T-/ and Sp6 primers. These cloned sequences were 200-1000 bp and represented genes specifically induced under zinc stress. Through BLASTn and BLASTx annotations, we screened 221 and 641 uniESTs from forward library and reverse library, respectively. Among these uniESTs, 751 ones were noted （including 193 uniESTs from forward library and 558 uniESTs from reverse library）. These noted uniESTs were mainly related to metabolism, signal transduction, antioxidant defense, transcription, translation, transport, ribosomal structure, energy metabolism as well as some genes with unknown functions.