大豆在暗诱导条件下差异表达cDNA文库的构建及分析

CONSTRUCTION AND ANALYSIS OF DIFFERENTIALLY EXPRESSED CDNA LIBRARY FROM SOYBEANS INDUCED BY DARKNESS

大豆属于短日照植物,其生长发育对光周期反应非常敏感,这一特性严重阻碍大豆品种的适应性,是制约大豆单产提高的关键因素。本研究的目的是在细胞分子水平上从基因差异表达的角度研究短日照光周期诱导开花和衰老过程中基因转录物丰度的变化,从而探索大豆叶片感受日长变化诱导开花和衰老的复杂机制。利用抑制性消减杂交技术(Suppression Subtractive Hybridiza-tion,SSH)成功构建了短日照诱导的大豆光周期敏感品种‘东农L13’叶片中差异表达的cDNA消减文库,从文库中一共筛选到738个克隆,通过反向Northern杂交,从中得到148个克隆并测序,它们代表76个不同基因的非重复序列ESTs。利用BLAST在GenBank数据库进行序列相似性比对,功能分析表明这些基因与植物的转录、信号转导和细胞凋亡的调控,解毒和防卫,大分子降解细胞壁修饰的合成代谢等多种植物的生理生化功能相关。本研究为揭示短日照诱导大豆开花和加速衰老的作用机理提供依据。

Soybean was a shortday plant,and its growth and development was very sensitive to photoperiod response which seriously limited the adaptation of soybean variety and improvement in yield of per unit area.The aim of this study reported in this paper was to break the limitation in soybean growth areas,determining what transcripts were increased in abundance in short day treated plants compared to long day treated plants during the photoperiod inductive flowering and senescent process on the cellular and molecular level,and further to discover the complicated mechanisms of flowering induction and senescence in the SD soybean leaves perceiving daylength.A cDNA subtractive library enriched for mRNAs encoding ESTs that increased in abundance during short days was constructed by suppression subtractive hybridization(SSH) from leaf tissues of soybean cultivar Dongnong L13′,a photoperiod sensitive cultivar,and 738 clones were selected from the library.A total of 148 clones were obtained by Reverse Northern blotting and these were sequenced that encoded 76 unique ESTs.The ESTs were annotated by similarity to orthologs and paralogs detected with Blastn and Blastx.The proteins predicted to be encoded were inferred to be involved in diverse physiological and biochemistrical functions such as transcription,signal transduction,programmed cell death,protein,macromolecule degradation,detoxification and defense,cell wall modification.This research established foundation to reveal the mechanisms of short day inducing flowering and accelerating senescence.