【摘要】 丙酮酸脱氢酶(PDH)是丙酮酸脱氢酶复合体(PDC)中的前件酶,参与生成柠檬酸循环(TCA)的起始物乙酰辅酶A,决定生物体内营养成分的分配。利用电子克隆、RT-PCR和cDNA末端快速扩增(RACE)等方法克隆了与果蝇lethal(1)G0334基因类似的具有丙酮酸脱氢酶功能的家蚕Bm-l(1)基因。Bm-l(1)基因的cDNA全长为1 630 bp,由1 200 bp的完整ORF序列、186 bp的5’-UTR和207 bp的3’-UTR组成,含8个外显子和7个内含子,编码蛋白为399个氨基酸残基,分子质量43.93kD,pI 8.07。Bm-l(1)基因编码蛋白的69-365氨基酸残基为E1-dh结构域,该结构域为硫胺素焦磷酸依赖性脱氢酶所特有。蛋白质二级结构预测结果表明α螺旋占28.8%,β折叠占12.0%。采用Clustal W进行多序列比对发现,Bm-l(1)基因编码蛋白与赤拟谷盗等昆虫的PDH具有63%以上的序列相似性,且保守区域高度一致。Bm-l(1)基因mRNA在家蚕整个卵期、幼虫期、蛹期和刚羽化的成虫中,以及5龄3 d幼虫的头部、丝腺、生殖腺、脂肪体、中肠和血液6种组织中都有较高的转录水平,且存在较小的组织差异性。更多还原

【Abstract】 Pyruvate dehydrogenase (PDH) is the former enzyme in pyruvate dehydrogenase complex and participates in generating acetyl coenzyme A,the initiator of tricarboxylic acid (TCA) cycle.It also plays a decisive role in the distribution of nutrition composition.By means of in silico cloning,RT-PCR and rapid amplification of cDNA ends (RACE) technology,a full-length cDNA which was similar to Drosophila melanogaster lethal(1) G0344 gene with pyruvate dehydrogenase function was cloned from silkworm (Bombyx mori) and designated as Bm-l(1) .It is 1 630 bp long,contains a complete ORF of 1 200 bp,186 bp of 5’-UTR and 207 bp of 3’-UTR.Bm-l(1) gene contains 8 exons and 7 introns.It encodes 399 amino acids,with predicted molecular mass of 43.93 kD and isoelectric point of 8.07.The deduced amino acids showed that an E1-dh domain was located between the 69th and the 365th amino acid residues.This structural domain is uniquely owned by thiamine pyrophosphate dependent dehydrogenases.Protein seconda-ry structure prediction showed that 28.8% of the protein is composed of α helix and 12.0% of β sheet.Multiple sequence alignment with Clustal W program revealed that Bm-l(1) coded protein has over 63% sequence identity with PDHs from Tribolium castaneum and other insect species.Nevertheless,their conservative regions are in high consensus.Bm-l(1) mRNA had high transcriptional levels during the whole egg,larval,and pupal stages,and in emerging adults,head,silk gland,gonad,fat body,midgut and hemolymph of 3-day-old larvae of the 5th instar,and the difference between tissues was relatively low.更多还原