应用抑制性消减杂交技术从枳中筛选根特异表达基因

Identification of Root-specific Genes with Subtractive Suppression Hybridization from Poncirus trifoliate

为筛选分离植物根组织特异表达基因,以枳根RNA为试验组,叶RNA为驱动组,构建了枳根消减文库。从该消减文库中共获得有效序列1 177个,其片段长度主要分布在200~500 bp;序列拼接后得到455个非重复序列,其中245个与已知基因匹配,具有结合功能、催化活性和转运活性等生物学功能,可参与植物的代谢、细胞生长、个体发育、应激反应等生物学过程。实时定量PCR检测结果显示这些基因中的主要乳液蛋白、早期结瘤素样蛋白和贝壳杉烯酸氧化酶等基因在根中高表达。

A suppression subtractive hybridization(SSH)library of Poncirus trifoliata was successfully constructed for screening the citrus root-specific genes by using the root RNA as Driver and the leaf one as Tester. Of 1 362 sequenced clones,1 177 ESTs(expressed sequence tags)in good quality were acquired from the SSH library and assembled into 455 Unigenes. Among them,245 Unigenes were homologous with the genes in Gen Bank database,which have biology function such as catalytic activity,transporter activity,carrier activity and so on. They play the important roles in biology process such as metabolic process,cellular process,developmental process,response to stimulus and so on. The 17 genes were further analyzed by real-time PCR and confirmed that they were highly expressed in the roots,but very low in the leaves. Bioinformatic analysis indicated that they are the major latex protein gene,the early nodulin-like protein gene,the ent-kaurenoic acid oxidase gene and etc.