刺五加内生青霉鲨烯合酶基因的克隆与序列分析

Cloning and sequence analyzing of squalene synthase gene from Eleutherococcus senticosus endogenetic Penicillium minioluteum P116-1a

摘要:

【目的】克隆刺五加内生青霉Penicillium minioluteum P116-1a的鲨烯合酶(Squalene synthase, SS)基因。【方法】采用cDNA 5?末端快速扩增(Rapid Amplification of cDNA 5? Ends, 5? RACE)技术扩增P. minioluteum P116-1a SS基因的全长cDNA序列和DNA序列; 运用生物信息学方法对该基因进行分析, 预测其编码蛋白的结构与功能; 并通过RT-PCR法和SDS-PAGE法检测SS的表达情况。【结果】P. minioluteum P116-1a的SS基因含有4个外显子和3个内含子, 开放阅读框长1 416 bp, 编码471个氨基酸, 预测蛋白含67.73%的α螺旋, 5.31%的延伸链, 2.97%的β折叠, 23.99%的无规则卷曲, 含有鲨烯合酶和八氢番茄红素合成酶的特异性识别区域, 定位于内质网膜。与P. marneffei和Talaromyces stipi-tatus中SS蛋白的氨基酸同源性达90%以上。不同温度下SS的表达情况不同。【结论】首次在刺五加内生青霉P. minioluteum P116-1a中克隆到SS基因, 为进一步研究P. minioluteum P116-1a提高刺五加皂苷含量的机制奠定基础。

Abstract:

[Objective] To clone squalene?synthase (SS) gene from Penicillium?minioluteum P116-1a isolated from Eleutherococcus senticosus. [Methods] Using rapid amplification of cDNA 5￠ ends (5￠ RACE) method, we isolated completed cDNA and DNA sequence of SS from P. minioluteum. The gene was analyzed and corresponding structure and functions were predicted by the bioinformatic method. Expression of SS was detected by RT-PCR and SDS-PAGE. [Results] The results showed that SS gene had 4 exons and 3 introns. The open reading frame was 1?416?bp encoding a protein of 471 amino acid residues. The predicted secondary structure composition for the protein contained about 67.73% α helixes, 5.31% extended strand, 2.97% β turns and 23.99% random coil. SS had conserved binding regions of squalene synthase and phytoene synthase and located in endoplasmic reticulum membrane. The SS amino acid sequence of P. minioluteum showed more than 90% homology with that of P. marneffei and Talaromyces stipitatus. Expression of P. minioluteum P116-1a SS gene varied in different tempreture. [Conclusion] The SS gene of P. minioluteum form E. senticosus was successfully cloned for the first time, providing a stable foundation for studying on mechanism of improving eleutheroside content by P. minioluteum P116-1a.