濒危药用植物新疆阿魏cDNA文库构建与微卫星标记

cDNA Library Construction and Microsatellite Marker Analysis of the Endangered Medicinal Plant Ferula sinkiangensis

目的:测试和分析新疆阿魏转录组,挖掘基因表达谱多态性标记(EST—SSRs),为新疆阿魏属植物遗传多样性、生物学特性、功能基因鉴定和濒危机制的研究奠定基础。方法:提取新疆阿魏茎叶总RNA,反转录合成cDNA,应用454 GS FLX Titanium进行测序,并对其序列进行拼接,获得一致性序列(Unique Sequences);使用Simple Sequence Repeat Identification Tool(Perl Script)对一致性序列进行分析,检索SSRs模体,采用Primer 5及SSR Hunter软件设计引物并进行PCR扩增。结果:高通量测序获得新疆阿魏ESTs序列550200条,经拼接得到60248条Unique Sequences,从中发现1916个EST—SSRs,平均每100kb出现3.18个SSR,其中二核苷酸最多,GA重复最常见,随机挑选5条序列扩增及测序结果表明,80%的SSR位点与Sanger测序结果一致。结论:通过转录组测序首次获得新疆阿魏EST—SSRs,为新疆阿魏生物学特性及濒危机制研究奠定了基础。

This study aimed to detect and analyze of the transcriptome data of F.sinkiangensis,as well as identify its genetic polymorphism of simple sequence repeats（SSRs） of expressed sequence tags（EST-SSRs）,which laid foundations for studies on genus genetic diversity,biological characteristics,identification of functional gene and the endangered mechanism of F.sinkiangensis.Total RNA from the leaves of F.sinkiangensis was extracted and reversed to cDNA,in order to construct the 454-EST library.Then 454-EST library was sequenced using 454 GS FLX Titanium.After sequences assembly,the unique sequences of F.sinkiangensis were obtained.The SSR identification tool that named Sequence Repeat Identification Tool Simple（Script Perl）was applied to analyze the SSR motif from the obtained unique sequences.Primers were designed by Primer 5and SSR Hunter software and amplified by PCR.As a result,550 200 EST sequences of F.sinkiangensis were obtained by high-throughput sequencing,which were assembled into 60 248 unique sequences.Among these unique sequences,1 916 EST-SSRs sites were discovered with the average of 3.18 EST-SSRs sites in every 100 kb sequences.Among the EST-SSRs sites,dinucleotide SSRs was the richest with G and A nucleotide repeats mostly.In conclusion,EST-SSRs data of F.sinkiangensis was established firstly in this study,which provided the basis for further researches for biological characteristics and endangered mechanisms of F.sinkiangensis through transcriptome sequencing.