陆地棉开花后20d纤维抑制性消减文库的构建及分析

本研究以高比强度纤维材料0-153和转基因抗虫棉sGK9708为亲本构建的高代重组自交系群体(F6:9)中选育出的高比强度纤维品系(69307)作为材料,利用抑制性消减杂交技术,以15DPA纤维为driver、20DPA纤维为tester,成功构建出陆地棉开花后20d纤维的cDNA消减文库。通过蓝白斑筛选、菌落PCR及反向Northern技术最终筛选出差异表达的阳性克隆340个。通过对阳性克隆测序及序列分析,共得到115个单一序列,其中35个重叠群,80个单拷贝。利用Blast2GO等对差异表达基因进行生物信息学分析,结果表明这些差异表达基因广泛参与糖类、脂类、氨基酸等物质的代谢,以及纤维素生物合成、细胞壁合成与修饰、氧化还原、细胞信号转导等生物学过程。

Construction and Analysis of SSH cDNA Library of Fiber in 20 Days Post Anthesis of Upland Cotton

In this study,one of excellent recombinant inbred line(69307) with high fiber strength was chosen as material from an F6:9 generation of upland cotton(Gossypium Hirsutum L.) derived from the combination between sGK9708 and 0-153.sGk9708 is a commercial transgenic cultivar with resistant to budworm,and 0-153 is a line with high fiber strength.An SSH cDNA library of fiber in 20 days post-anthesis of upland cotton had been successfully constructed via the approach of Suppressive Subtractive Hybridization(SSH),in which the mRNAs isolated from the fibers in 15 days post anthesis(dpa) were used as driver,and the mRNAs from the fibers in 20 days post anthesis(dpa) as tester.Finally,the 340 positive clones expressed differentially had been chosen by Blue-white bolting,colony PCR and Reverse Northern Dot-Blot.115 unigenes were obtained based on sequencing of the positive clones and analysis of the sequences,which included 35 contigs and 80 singlets.Bioinformatic analysis with Blast2GO software revealed that these differentially expressed genes extensively involved in metabolism of carbohydrate,lipid,amino acid and other substances,and in biological process,such as cellulose biosynthesis,cell wall formation and modification,oxidation and reduction,signal transduction,etc.