橡胶树胶乳均一化全长cDNA文库的构建与EST测序分析

摘要

[ Objective ] Construction and normalization of the latex full-length cDNA library would facilitate the identification and analysis of the laticifer-specially expressed genes so as to gain insights into the molecular and regulatory mechanisms of the latex metabolism and the rubber biosynthesis that occur in the laticifers of rubber tree. [ Method] Firstly, the primary full-length cDNA library was constructed with the latex of H. brasiliensis (clone Reyan 7-33-97) using recombination of the full-length latex cDNAs with the Gateway donor vector of pDONR222. Secondly, the plasmids of the primary cDNA library were normalized through self-subtraction with the Hevea genome DNA, and the normalized latex full-length cDNA library was constructed using the subtracted plasmids. [Result] The titer of the unamplified primary cDNA library was about 6.0×l06 cfu/mL. the average size of inserted cDNAs was 1.5 kb with a recombination of about 100％, and the ratio of full-length cDNAs was around 76％. The normalized latex full-length cDNA library had a total CFU of 1.87× 105 with a recombination ratio of 95％. Quantity RT-PCR results demonstrated that normalization produced about 1 000 fold average reduction of two high abundant latex genes, i.e., small rubber particle protein (SRPP) and rubber elongation factor (REF). [Conclusion] A high-quality latex normalized full-length cDNA library was successfully constructed, and 12 000 of random clones in this cDNA library were then sequenced. A total of 11 951 ESTs (expressed sequence tag) were attained, and could be assembled into 3 394 unigenes, including 2 061 contigs and 1 333 singlets. The latex cDNA library can be well used for Hevea's functional genomics, novel gene screening, high-through EST sequencing and the preparation of the latex cDNA microarray of H. brasiliensis.%[目的]构建橡胶树胶乳均一化全长cDNA文库,降低胶乳中存在的高丰度表达基因的拷贝数,提高胶乳表达基因的分离鉴定和EST测序分析效率.[方法]以橡胶树无性系热研7-33-97的胶乳为材料,将胶乳全长cDNA与Gateway供体载体pDONR222重组,构建了橡胶树胶乳的非剪切型全长cDNA原始文库,经检测原始文库的滴度为6.0×10(6)cfu/mL,平均插入片段长度大于1.5 kb,重组率约为100%,全长cDNA比例约为76%;利用基因组DNA饱和杂交技术对胶乳原始cDNA文库进行均一化处理,构建橡胶树胶乳的均一化全长cDNA文库.[结果]胶乳均一化全长cDNA文库的总库容量(总CFU)为1.87×105,重组率大于95%.定量RT-PCR检测表明,橡胶树胶乳2个高丰度表达基因,即小橡胶粒子蛋白(SRPP)和橡胶延长因子(REF)基因,在均一化cDNA文库中的表达量均下降了约1 000倍.[结论]构建T均一化效果良好的橡胶树胶乳全长cDNA文库,并对文库中随机的12 000个克隆进行了测序,获得高质量的有效EST(expressed sequence tag)序列为11 951条,经拼接共获得单一基因(unigene)为3 394个,其中包括片段重叠群(contig)2 061个和单一EST序列(singlet)1 333个.此cDNA文库将可用于橡胶树功能基因组研究、新基因筛选、高通量EST测序以及橡胶树胶乳cDNA芯片的制备.