软枣猕猴桃L-半乳糖内酯脱氢酶基因的克隆及原核表达分析

Cloning and Prokaryotic Expression Analysis of L-Galactono-1, 4-Lactone Dehydrogenase in Actinidia arguta

为更好了解L-半乳糖内酯脱氢酶及编码基因在软枣猕猴桃中的生理功能及作用,以‘魁绿’软枣猕猴桃果实cDNA为模板,用RT-PCR及RACE技术,获得L-半乳糖酸-1,4-内酯脱氢酶基因AaGalLDH(GenBank:KP145007.1)。序列分析表明该基因全长为2394 bp,开放阅读框为1833 bp,编码610个氨基酸,与其它植物GalLDH氨基酸序列具有78%~95%的同源性。编码的蛋白序列具有GalLDH蛋白家族典型的保守结构域。进一步将该基因连接到原核表达载体并转化大肠杆菌进行诱导表达,经Western Blotting验证,目的基因在pET-28a表达系统中有表达,但基本为不溶性表达。这为进一步研究该酶蛋白的体外表达和功能以及选育高品质软枣猕猴桃品种奠定了基础。

In order to better understand the physiological function of L-galactono-1,4-lactone dehydrogenase and coding gene in Actinidia arguta,2394 bp cDNA encoding L-galactono-1,4-lactone dehydrogenase(GalLDH)fragment was cloned from fruit of A.arguta Planch by the method of RT-PCR and RACE,named AaGalLDH(GenBank:KP145007.1).Sequence analysis showed that the open reading frame was 1833 bp,encoding 610 amino acids,and had 78%to 95%homology with other plants’GalLDH amino acid sequences.The encoded protein sequence had a typical conservative domain of the GalLDH protein family.The gene was constructed to the prokaryotic expression vector and transformed into E.coli for induction expression.The target gene was expressed in the pET-28 a expression system,but it was basically insoluble after Western Blotting verified.This had laid the foundation for further research on the expression and function of the enzyme protein in vitro and the selection of high-quality varieties.