巴西橡胶树顺式异戊烯基转移酶基因cDNA克隆及其序列特征分析

Cloning and Sequence Analysis of a Novel Cis-prenyltransferases Gene from Hevea brasiliensis

摘要:

以巴西橡胶树(Hevea brasiliensis)胶乳的RNA为Tester；叶片RNA为Driver，利用抑制消减杂交法(suppressive subtractive hybridization, SSH)构建了一个胶乳特异表达基因差减文库。通过反式Northern点杂交(reverse Northern dot blots)筛选到一个与顺式异戊烯基转移酶基因(橡胶生物合成的关键酶基因)高度同源的阳性克隆R363。采用RACE方法获得该克隆的全长cDNA(GenBank登陆号: AY461414)。序列分析表明，该基因长1 156 bp，含有873 bp的阅读框，编码290个氨基酸，分子量约为32.9 kD，等电点为7.2，含有N-端跨膜螺旋区。同源性分析表明R363编码的蛋白质具有异戊烯基转移酶家族的特征，含有cis-异戊烯基链转移酶的5个高度保守区，推测R363可能是一种新的顺式-异戊烯基转移酶基因。Northern blot分析显示，R363在胶乳中高度表达，在叶中不表达。乙烯处理前后表达强度一致，表明该基因表达不为乙烯所诱导。

Abstract:

Cis-prenyltransferases is the key enzyme to rubber biosynthesis. A cDNA fragment which was highly homologous to the gene encoding cis-prenyltransferases was isolated by screening of a latex subtracted cDNA library. The latex subtracted cDNA library was constructed by suppressive subtractive hybridization (SSH), in which the polyA+RNA of latex from Hevea brasiliensis served as tester, and polyA+RNA of leaves as driver. According to its sequences information, a novel full-length cDNA of 1 156 bp termed R363 (GenBank number: AY461414) was obtained by using rapid amplification of cDNA ends (RACE). R363 contained a 1 035 bp ORF, which encoded 290 amino acids with a theoretical molecular weight of 32.9 kD and an isoelectric point of 7.2. Hydropathy and transmembrane motif analysis of deduced amino acid sequences indicated that R363 possessed a signal peptide of 27 amino acids at its N-end. The result of the conserved domains analysis indicated that R363 contained five highly conserved regions as well as signature motif of undecaprenyl pyrophosphate sythetase family, which implied that R363 would be a cis-prenyltransferases gene. Furthermore, the transcripts of R363 were expressed predominantly in the latex as compared with leaves, and exerted no differences in latex with or without 2% ethylene pretreatment.