橡胶树胶乳均一化全长cDNA文库的构建与EST测序分析

Construction and EST Analysis of the Normalized Full-Length Latex cDNA Library of Hevea 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6.0×106 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】构建了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。

【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 6.0×106 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.