暴马桑黄细胞色素P450家族CYP5150AW6基因克隆及表达分析

Cloning and expression analysis of CYP5150AW6 gene of cytochrome P450 family in Sanghuangporus baumii

目的为深入了解细胞色素P450家族CYP5150AW6基因在暴马桑黄Sanghuangporus baumii生长发育调控中的功能。方法采用PCR技术获得目的基因的cDNA序列全长,通过生物信息学在线软件分析其编码的氨基酸序列;采用同源重组的方法构建原核表达载体;采用荧光定量PCR技术分析其不同发育阶段的表达特性。结果暴马桑黄中克隆得到的基因开放阅读框(open reading frame,ORF)序列全长1758 bp,编码585个氨基酸,相对分子质量为66080,经国际P450命名委员会命名为CYP5150AW6;跨膜区预测表明CYP5150AW6蛋白不具有跨膜结构;亚细胞定位显示CYP5150AW6蛋白在细胞质和线粒体中修饰合成;系统进化分析结果表明,暴马桑黄CYP5150AW6蛋白与灵芝CYP5150L8蛋白亲缘关系最近,符合物种分类原则;SDS-PAGE结果证实在75000~100000出现目的蛋白条带(包含20000的标签蛋白),说明所获得的基因能够成功表达出蛋白;从荧光定量分析结果中可以看出CYP5150AW6基因转录水平从菌丝体阶段到子实体阶段生长逐渐升高,并且在子实体阶段达到最高,说明该基因可能与暴马桑黄的生长发育相关。结论通过对CYP5150AW6基因的鉴定与分析,为进一步研究该基因在暴马桑黄生长发育调控过程的功能提供了理论依据。

Objective To explore the function of CYP5150AW6 gene of cytochrome P450 family in the regulation of growth and development of Sanghuangporus baumii.Methods The full-length cDNA sequence of target gene was obtained by PCR,and the amino acid sequence was analyzed by bioinformatics online software.Prokaryotic expression vectors were constructed by homologous recombination.The qRT-PCR technology was used to analyze the expression characteristics in different developmental stages.Results The total length of the P450 gene(ORF)was 1758 bp,encoding 585 amino acids with a relative molecular weight of 66080,and it was named CYP5150AW6 by P450 international naming committee.The prediction of the transmembrane region showed that CYP5150AW6 protein did not have a transmembrane structure.Subcellular localization showed that CYP5150AW6 was modified and synthesized in cytoplasm and mitochondria.Phylogenetic analysis showed that CYP5150AW6 protein was closely related to CYP5150L8 protein of Ganoderma lucidum,which was in accord with the principle of species classification.SDS-PAGE results confirmed the presence of target protein bands between 75000—100000(containing 20000 of the labeled protein),indicating that the obtained gene could successfully express the protein.According to the results of qRT-PCR analysis,the transcript level of CYP5150AW6 gene increased gradually from mycelium stage to fruiting body stage,and reached the highest level in fruiting body stage,indicating that this gene may be related to the growth and development of S.baumii.Conclusion Through the identification and analysis of CYP5150AW6 gene,it provides a theoretical basis for further study of the function of CYP5150AW6 gene in the regulation of growth and development of S.baumii.