家蚕类ω3-脂肪酸脱氢酶基因的克

发布时间：2018-06-08 15:09

本文选题：家蚕 + ω-脂肪酸脱氢酶　；参考：《蚕业科学》2017年04期

【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。
[Abstract]:蠅 _ 3-fatty acid dehydrogenase is one of the key enzymes for the synthesis of polyunsaturated fatty acids in vivo, and its expression is affected by biological and abiotic stress factors. The omega-3-fatty acid dehydrogenase gene (Bm FAD3-like) was cloned from silkworm pupae. The gene has an open reading frame of 1083 BP and encodes a protein with a molecular weight of 41.5 KD consisting of 360 amino acid residues. The gene was cloned into pYES2.0 vector. The recombinant expression vector pYBm FAD3-like and the recombinant S.cerevisiae engineering strain YBm FAD3-like were constructed. Gas chromatographic analysis showed that the recombinant BmFAD3-like could transform the exogenous substrate linoleic acid into 伪 -linolenic acid ALAA. The mRNA of BmFAD3-like gene was detected by semi-quantitative RT-PCR in almost all tissues of the 5th instar larvae of Bombyx mori, and the transcription level was higher in the 5th instar stage, pupa stage and moth stage. Moreover, the transcription level was significantly up-regulated by fungi (Beauveria bassiana) and fungi (Beauveria bassiana) for 6 h. The transcription level of BmFAD3-like mRNA was significantly down-regulated in silencing of silkworm pupae for 1272 hours. The results showed that the expression product of Bm FAD3-like could catalyze the dehydrogenation of LA into ALAs at 蠅 3 position, and the gene could be upregulated in silkworm under stress of low temperature induction and fungal infection. Exogenous siRNA could mediate the silencing of the gene. The transcription level was significantly down-regulated.
【作者单位】：江苏科技大学生物技术学院;中国农业科学院蚕业研究所;
【基金】：公益性行业(农业)科研专项(No.201403064)
【分类号】：Q78;S881.24

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