家蚕Bmugt 3基因cDNA序列的克隆、表达与序列分析

Cloning,expression and sequence analysis of cDNA encoding the silkworm Bmugt 3 gene

【目的】克隆分析家蚕Bmugt 3基因cDNA序列,并对其在家蚕不同组织中的表达进行检测。【方法】以家蚕ak1和873为供试材料,采用生物信息学方法和RT-PCR技术,对家蚕Bmugt3基因cDNA序列进行克隆和组织表达谱研究,并通过XbaⅠ和HindⅢ双酶切重组质粒,将Bmugt 3亚克隆至具有2个反向T7启动子,用于体外诱导合成双链RNA(dsRNA)的L4440载体。【结果】克隆得到了家蚕Bmugt 3基因1675bp的cDNA序列,编码462个氨基酸,编码蛋白的分子质量为52.3ku,等电点6.5;多序列比对显示,Bmugt 3基因缺失了C末端的跨膜结构域;聚类分析结果显示,其与家蚕phenol-UGT基因聚合在同一分支上。Bmugt 3基因主要在家蚕丝腺中表达。【结论】Bmugt 3基因是组织特异性表达基因,可能在家蚕类黄酮素的代谢中起作用。

[Objective] The study was to clone and analyze the cDNA sequence of silkworm Brnugt 3 gene and the expression in 5 tissues. [Method] Using silkworm strain,akl and 873 ,as materials,the cDNA sequence of Bmugt 3 gene was cloned by RT-PCR and analyzed by bioinformaties. The gene was subcloned to L4440 vector, which had two T7 polymerase promoter in opposite orientation to synthesize dsRNA in vitro,through double-digestion with Xba Ⅰ and Hind Ⅲ. [Result] Bmugt 3 gene of silkworm with 1 675 bp in length was cloned. 426 amino acids were coded and the MW and pI of the predicted protein was 52.3 ku and 6.5 respectively. Bmugt 3 had no C-end transmembrane domain by the multiple sequence alignment and it clustered in the same branch with silkworm phenol-UGT in the clustering analysis. The Brnugt 3 gene expressed mostly in the silk glands in all the tested silkworm tissues. [Conclusion] The Bmugt 3 gene is a tissue-specific gene and may function in the metabolism of silkworm flavonoids.