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【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 ofcDNA 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 stipitatus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。
【Objective】 Cloning Squalene synthase (SS) gene of Penicillium minioluteum P116-1a. 【Method】 The full-length cDNA and DNA sequences of P. minioluteum P116-1a SS gene were amplified by Rapid Amplification of cDNA 5 Ends (5 RACE). The gene was analyzed by bioinformatics , Predicting the structure and function of the encoded protein; and detecting the expression of SS by RT-PCR and SDS-PAGE. 【Result】 The SS gene of P.minioluteum P116-1a contained four exons and three introns. The open reading frame (ORF) length of the SS gene was 1 416 bp, encoding 471 amino acids. The predicted protein contained 67.73% alpha helix and 5.31% Stretching chain, 2.97% beta sheet, 23.99% random curl, a specific recognition region containing squalene synthase and phytoene synthase localized to the endoplasmic reticulum membrane. Amino acid homology of more than 90% with SS protein in P.marneffei and Talaromyces stipitatus. The expression of SS in different temperature is different. 【Conclusion】 The SS gene was cloned from P. minioluteum P116-1a for the first time, which laid the foundation for further study on the mechanism of P. minioluteum P116-1a in increasing the content of acanthopanax senticosus.