家蚕3-磷酸甘油脱氢酶基因BmGpd的结构特征与表达谱分析

Structure characteristics and expression profiles of BmGpd,a glycerol-3-phosphate dehydrogenase gene in the silkworm,Bombyx mori

3-磷酸甘油脱氢酶(GPDH)是真核细胞的烟酰胺腺嘌呤二核苷酸依赖性酶,具有能量传递等重要功能。家蚕Bombyxmori是重要的变态和能量代谢研究模式动物,为了明确GPDH在家蚕中的作用,本研究根据果蝇lethal(2)k05713的蛋白质序列,从家蚕C108品种克隆了2个完整的mRNA转录体,长度分别为3456bp和2979bp(GenBank登录号为GQ865685和GQ865686),具有相同的2166bpORF,编码721个氨基酸。克隆拼接了548bp的第9内含子后,参照大造品种的全基因组数据,推测出BmGpd基因全长27983bp(GenBank登录号为EF154335),包含16个外显子和15个内含子。编码蛋白具跨膜螺旋结构,pI为8.22,分子量为80.5kD,1～20氨基酸区域为信号肽序列。分子同源进化和蛋白质基序分析表明,BmGPD是家蚕中一种新GPDH成员,能在大肠杆菌Escherichiacoli中诱导大量表达。RT-PCR分析表明,BmGpd基因表达量在5龄中期最高,在3d的蛹中明显下调,进入滞育时表达量更低;血液中较低,中肠、丝腺、生殖腺和脂肪体中有大量表达。芯片表达谱显示,5龄第3天幼虫头和体壁中表达丰度最高,脂肪体和马氏管中最低,性别差异不显著。EST表达谱显示,4龄第2天中肠中表达丰度最高。RNAi结果显示,家蚕体内存在BmGpd基因产物代谢补偿途径。本研究为深入研究家蚕GPDH的表达调控以及与变态和能量代谢的关系创造了条件。

Glycerol-3-phosphate dehydrogenase （GPDH） is a soluble cytosolic NAD-dependent enzyme present in eukaryotic organisms and has some important functions,such as energy transfer.Bombyx mori is a central model animal to research metamorphosis and energy metabolism.In order to determine the role of GPDH in B.mori,we cloned a complete gene of BmGpd based on protein （lethal （2） k05713） sequence of Drosophila melanogaster from NCBI （GenBank accession number：NP_725495.1）,which is 27 983 bp in full length and contains 16 exons and 15 introns （GenBank accession number is EF154335）.The two complete mRNA transcripts from B.mori strain C108 are 3 456 bp and 2 979 bp in full length （GenBank accession numbers are GQ865685 and GQ865686,respectively）,both including a 2 166 bp ORF that encodes 721 amino acids （Mw 82.1 kD,pI 6.43）.BmGPD protein includes several membrane-spanning regions and a signal peptide sequence with a length of 20 aa.The results of phylogenetic analysis and motif search revealed that BmGPD belonged to glycerol-3-phosphate dehydrogenase （GPDH） family,which was not reported in B.mori and could be abundantly inducible expression in Escherichia coli.Semi-quantitative RT-PCR analysis indicated that a high-level of BmGpd mRNA occurred in tissues （organs） of the midgut,silk gland,gonad and fat body,and it occurred at the middle stage of the 5th instar larva;however,a low level in the blood,as well as on day 3 of the pupal stage,and a lower level occurred during the diapause.The result of microarray-based gene expression profile showed that the expression abundance of BmGpd was very high in head and integument,while low in fat body and Malpighian tubule on day 3 of the 5th instar larva,and there were no obvious differences between the male and female strains.Expression profile of ESTs indicated that the expression abundance was the highest in the midgut on day 2 of the 4th instar larva.RNAi result suggested that there are strong metabolic compensation pathways to make up for the silence of BmGpd gene.This study provides a basis for further investigating the expression regulation of GPDH and its relationship with metamorphosis and energy metabolism in B.mori.