摘要
ω3-脂肪酸脱氢酶是生物体内催化多不饱和脂肪酸合成的关键酶之一,其表达受生物胁迫和非生物胁迫因素的影响。从家蚕蛹中克隆了类ω3-脂肪酸脱氢酶基因(Bm FAD3-like),该基因具有一个长度为1 083 bp的开放阅读框,编码由360个氨基酸残基组成的分子质量为41.5 k D的蛋白质。将该基因克隆到p YES2.0载体,构建重组表达载体p YBm FAD3-like及重组酿酒酵母工程菌株YBm FAD3-like后进行诱导表达,收集菌体提取脂肪酸,经气相色谱检测表明异源表达的重组Bm FAD3-like能够将诱导表达体系中加入的外源底物亚油酸(LA)转化为α-亚麻酸(ALA)。半定量RT-PCR检测Bm FAD3-like基因mRNA几乎在家蚕5龄第3天幼虫的所有组织中都有转录,在5龄幼虫期、蛹期和蛾期的转录水平较高,并且其转录水平受低温(0℃)和真菌(球孢白僵菌Beauveria bassiana)侵染(6~36 h)诱导显著上调。对家蚕蛹体注射siRNA沉默目的基因12~72 h后,Bm FAD3-like mRNA转录水平显著下调。研究结果表明,Bm FAD3-like的表达产物能催化LA在ω3位脱氢生成ALA,并且该基因在蚕体受到低温诱导和真菌侵染等胁迫时显著上调表达,外源siRNA能够介导该基因的沉默,显著下调其转录水平。
Omega3-fatty acid desaturase is one of the key enzymes catalyzing the biosynthesis of polyunsaturated fatty acids. Its expression is affected by both biotic and abiotic stresses. In this study,a novel omega3-deaturase like gene（Bm FAD3-like） was cloned from Bombyx mori pupae. Sequence analysis showed that Bm FAD3-like had an open reading frame of 1 083 bp and encoded 360 amino acids with predicted molecular weight of 41. 5 k D. This gene was cloned into p YES2. 0 vector to construct recombinant expression vector p YBm FAD3-like and recombinant Saccharomyces cerevisiae strain YBm FAD3-like for induced expression. Gas chromatographic analysis to fatty acids isolated from the recombinant yeast indicated that Bm FAD3-like protein from heterologous expression could synthesize α-linolenic acid（ ALA） using exogenous substrate linoleic acid（ LA） added into the expression system. Semi-quantitative RT-PCR assay revealed that Bm FAD3-like transcription occurred in almost all tissues of day 3 Bombyx mori larvae of the 5th instar,and was at high level during the 5th larval instar, the pupa and moth stages. Moreover,its expression was significantly upregulated under low temperature（0 ℃） stress and fungus infection（6 h to 36 h by Beauveria bassiana）. At 12 h to 72 h after Bombyx mori pupae were injected with siRNA to silence the target gene,Bm FAD3-like mRNA transcriptional levels were significantly down-regulated. Our findingssuggested that the expression product of Bm FAD3-like gene could catalyze dehydrogenation of LA at ω3 position to form ALA. Furthermore,its expression was significantly up-regulated under low temperature stress and fungus infection,and exogenous siRNA could mediate silencing of it and significantly down-regulate its transcriptional level.
出处
《蚕业科学》
CAS
CSCD
北大核心
2017年第4期577-586,共10页
ACTA SERICOLOGICA SINICA
基金
公益性行业(农业)科研专项(No.201403064)
关键词
家蚕
ω3-脂肪酸脱氢酶
基因克隆
酿酒酵母表达系统
表达谱
多不饱和脂肪酸合成
诱导表达
Bombyx mori
Omega3-fatty acid desaturase
Gene cloning
Saccharomyces cerevisiae expression system
Expression profile
Polyunsaturated fatty acid synthesis
Induced expression
