家蚕超长链脂肪酸延长酶基因Bmelo424的克隆及功能分析

Cloning and function analysis of elongase of very long chain fatty acid gene Bmelo424 in silkworm

摘要:

超长链脂肪酸延长酶家族基因影响生物体的多种生理功能。文中克隆了家蚕的一个该家族成员Bmelo424基因，其ORF为558 bp。该基因的蛋白序列预测有4个跨膜结构域，并有6个丝氨酸磷酸化位点、8个苏氨酸磷酸化位点和4个酪氨酸磷酸化位点，其亚细胞定位于内质网中，二级结构分析结果显示其α-螺旋和β-折叠股分别占26.7%和20％。荧光定量PCR结果显示Bmelo424基因在家蚕各组织均有表达，尤其在头部表达量最高。通过在酿酒酵母中异源表达Bmelo424基因的方法研究其对脂肪酸延伸的作用，GC-MS结果表明，携带pYES2-Bmelo424重组质粒的酿酒酵母的C16:1n-7脂肪酸含量有显著提高，而C16:0、C18:0和C18:1n-9的含量下降。温度胁迫结果显示，Bmelo424基因能够提高酿酒酵母的低温适应能力，但却降低其高温适应能力。这为探究家蚕Bmelo424基因的功能提供了参考。

关键词:家蚕，超长链脂肪酸延长酶，表达特征，酿酒酵母，温度胁迫

Abstract:

Genes belonging to the elongases of very long chain fatty acid (ELOVL) family affect many physiological functions in organism. In this paper, Bmelo424 gene, a member of the ELOVL family in silkworm, was cloned and its ORF was 558 bp. Its protein sequence was predicted to have four transmembrane domains, six serine phosphorylation sites, eight threonine phosphorylation sites and four tyrosine phosphorylation sites, and its subcellular localization was in the endoplasmic reticulum. Secondary structure analysis showed that the percentage of alpha-helix and beta-strand was 26.7% and 20% respectively. The results of fluorescence quantitative PCR showed that Bmelo424 gene was expressed in all tissues of silkworm, especially with the highest expression in head. By heterologous expression of Bmelo424 gene in Saccharomyces cerevisiae, the effect of Bmelo424 gene on fatty acid elongation was studied. GC-MS results indicated that the fatty acid content of C16:1n-7 in S. cerevisiae with pYES2-Bmelo424 recombinant plasmid increased significantly, whereas the content of C16:0, C18:0 and C18:1n-9 decreased. The results of temperature stress revealed that Bmelo424 gene could improve the low temperature adaptability of S. cerevisiae, but its high temperature adaptability decreased. This provides a reference for exploring the function of Bmelo424 gene in silkworm.

Key words:silkworm, elongase of very long chain fatty acid, expression profile, Saccharomyces cerevisiae, temperature stress