家蚕尿苷二磷酸-葡萄糖基转移酶基因(BmUGT004965)的克隆和表达谱分析  

Molecular Cloning and Expression Analysis of UDP-glucosyltransferase Gene BmUGT004965 in Bombyx mori

作　　者：黄飞飞[1] 刘增虎[1] 陈颜虹[1] 马艳[1] 鲁成[1] 

机构地区：[1]西南大学,农业部蚕桑学重点开放实验室,重庆400716

出　　处：《蚕业科学》2009年第1期50-57,共8页ACTA SERICOLOGICA SINICA

基　　金：国家重点基础研究发展计划“973”项目(编号2005CB12-1004);科技基础性工作专项项目(编号2007FY210401-0);高等学校学科创新引智计划(编号B07045);重庆市自然科学基金项目(编号CSTC,2007BB1351)

摘　　要：糖基化作用在抑制和排除生物体一系列内生和外生有毒化合物的过程中非常重要,尿苷二磷酸-葡萄糖基转移酶(UGTs)参与了这一过程。在分析家蚕基因组中存在的UGT基因时选取其中的一个基因进行生物信息学分析与分子生物学实验,经分子克隆和表达谱分析,将该基因命名为BmUGT004965,其开放读码框(ORF)长1 578 bp,编码525个氨基酸,预测分子质量60.3 kD,等电点9.13。该基因编码蛋白的N端具有一段由19个氨基酸组成的信号肽序列,而且N端和C端各有一段疏水的跨膜区。将该基因cDNA与家蚕基因组序列进行比对表明其具有4个外显子,外显子/内含子边界处均符合GT-AG规则。将家蚕BmUGT004965基因与人类、果蝇、昆虫杆状病毒、植物及已报道的家蚕UGT基因进行氨基酸水平的比对,显示氨基酸序列一致性为20%～30%;多重序列比对结果表明C端序列的保守性高于N端区域,可能与UGT具有2个主要的功能域有关。实时定量RT-PCR检测表明,在5龄第3天家蚕幼虫的9种组织中,BmUGT004965基因只在丝腺和脂肪体中有表达,且丝腺中的表达丰度明显高于脂肪体,这与基因芯片的结果一致,推测这种特异的表达方式可能与其特异的功能有关。Abstract UDP-glycosyltransferase is involved in glycosylation which plays a major role in the inactivation and excretion of a great variety of both endogenous and exogenous toxic compounds. In an analysis to UDP-glycosyltransferase family in the silkworm genome, we cloned and analyzed one of these genes named BmUG-fO04965 through bioinformatic analysis and molecular biological experiment. The ORF （Open Read Frame） of BmUGTO04965 is 1 578 bp long and encodes 525 amino acids with a predicted molecular weight of 60.3 kD and an isoelectric point of 9.13. It has a 19 amino acid signal peptide sequence on the N-terminus and a putative hydrophobic transmembrane domain located on both N-terminus and C-terminus. An alignment of the cDNA sequence with silkworm genomic sequence revealed that there are 4 exons in BmUG-rO04965, and all exon/intron boundaries are consistent with the GT-AG rule. A comparison of BmUG7004965 with UGT genes from other sources, such as Drosophila, human, baculovirous, plant and the reported silkworm UGT genes, revealed that their amino acids share 20% to 30% identity. It was also found that its C-terminal half has higher conservatism than its N-terminal half. This was thought to reflect the fact that the protein comprises two major functional domains. Realtime quantitative RT-PCR results indicated that BmUGTO04965 was expressed only in silk gland and fat body among 9 tissues detected on day 3 of the fifth instar larvae, and the expression level in silk gland was much higher than that in fat body, which is consistent with the microarray analyzing results, The specific expression pattern may relate to the special function of this qene.

关 键 词：糖基化作用 尿苷二磷酸-葡萄糖基转移酶基因 家蚕 基因克隆 组织表达 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]