基于甾体皂苷的襄麦冬块根抑制性差减杂交文库的构建

摘要

[Objective] Steroidal saponins is one of the main active compounds of Liriope spicata var.prolifera Y.T.Ma (LSVP).For improving its content in tuber by gene expression regulation,it was very necessary to screen the proteins or enzymes involved in steroidal saponins synthesis.[Method] In the present paper,the suppression subtractive hybridization (SSH) libraries were constructed using the cDNA of LSVP tuber from different growth period:the beginning growth stage tuber (SJQ) and the harvest stage tuber (CSQ).[Result]308 effective EST sequences,which 586.99 bp average length and mainly distributed in 250-1000 bp,were obtained from SJQ and CSQ SSH cDNA library.In addition,the sequences that could match the genes in the database of NR,SWISS PROT,TREMBL,CDD,PFAM were 65.26 ％,34.42 ％,63.96 ％,65.26 ％ and 14.94 ％,respectively.And the 23 and 103 Unigenes were found that could be classified into 12 COG classifications and 19 KOG classifications respectively as well.The KEGG analysis showed that.there were 81 unigenes which could be noted into 48 enzymes and mapped into 92 KEGG pathways.Gene Ontology analysis was also conducted and the unigene' s distribution in different functional category was found that in biological process,mainly focused on cellular process and metabiolic process,and in cellular component,mainly related to cell,cell part and organelle,and in molecular function,were binding and catalytic activity.[Conclusion] In this experiment,the suppression subtractive hybridization (SSH) libraries were successfully constructed based on steroidal saponins,and several differentially expressed genes were screened.%[目的]甾体皂苷是襄麦冬块根主要活性成分之一,为了能在分子水平上实现对相关基因的表达调控,从而使甾体皂苷含量的提高成为可能,筛选参与甾体皂苷合成的蛋白或酶就非常必要.[方法]本实验以襄麦冬始见期块根和采收期块根作为实验样品,构建了不同生育期襄麦冬块根抑制性差减杂交(SSH)文库.[结果]构建的始见期和采收期块根差减文库的外源片段,有效基因序列308条,长度主要分布在250～1000 bp左右,片段平均长度586.99 bp;文库样本序列注释上NR、SWISS-PROT、TREMBL、CDD、PFAM库的基因分别有65.26％、34.42％、63.96％、26.30％、14.94％;样本中分别有23和103个Unigene可注释上12个COG分类与19个KOG分类中;样本中有81个预测基因,可以注释到48种酶与映射到92个代谢通路上;样本基因功能在Biological Process分类中聚集于cellular process和metabiolie process,在Cellular Component主要聚集于cell、cell part和intracellular,在Molecular Function分类中主要聚集于catalytic activity,binding和transferase activity.[结论]本实验成功构建了基于甾体皂苷的襄麦冬块根抑制性差减杂交文库,筛选出一批差异表达目的基因.