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目的克隆分离药用真菌猪苓菌核的主要协助转运蛋白超家族基因并进行生物信息学分析。方法采用RT-PCR技术从中国野生猪苓菌核(Polyporus umbellatus)中克隆得到9个主要协助转运蛋白超家族(major facilitator superfamily,MFS)基因,利用生物信息学方法分析这9个基因的特点,并利用qPCR验证这9个基因在不同猪苓菌核部分的表达情况。结果该9个基因cDNA包含的完整开放阅读框在1 321~1 860 bp之间,编码的氨基酸的数量在441~620之间,相对分子质量在(48.45~64.79)×10~3之间,理论等电点在6.59~9.56之间。氨基酸序列分析表明,这9个基因编码的主要协助转运蛋白的跨膜区在11~14之间。氨基酸序列多重比对及系统发育树结果显示,Comp34750、Comp34832、Comp29252、Comp42895、Comp32579和Comp27555与MFS general substrate transporter同源性比较高,Comp28872和Comp26306与MFS单糖转运蛋白(MFS monosaccharide transporter)同源性比较高,Comp33117与MFS糖转运蛋白(MFS sugar transporter)同源性比较高。实时荧光定量RT-PCR分析表明,这9个基因在不同的菌核部分都有表达,除基因Comp34382和Comp32579在共生部位显著下调外,其他7个基因都是表达显著上调。结论提示这些基因可能以不同方式参与了猪苓的防御反应及吸收外界营养元素的过程。
Objective To clone and isolate the major gene assisting transporter superfamily of Polyporus umbellatus and make bioinformatic analysis. Methods Nine major facilitator superfamily (MFS) genes were cloned by RT-PCR from Chinese wild Polyporus umbellatus. The characteristics of these nine genes were analyzed by bioinformatics methods. The qPCR was used to verify the expression of these nine genes in different parts of Polyporus umbellatus. Results The complete open reading frame (ORF) of the 9 cDNAs was between 1 321 and 1 860 bp, the number of amino acids encoded was between 441 and 620, and the relative molecular mass was between (48.45 and 64.79) × 10 3. The theoretical isoelectric point is between 6.59 ~ 9.56. Amino acid sequence analysis showed that the nine genes encoding the major facilitator transporter transmembrane region between 11 to 14. Amino acid sequence multiple alignment and phylogenetic tree analysis showed that Comp34750, Comp34832, Comp29252, Comp42895, Comp32579 and Comp27555 had higher homology with MFS general substrate transporter. Comp28872 and Comp26306 were homologous with MFS monosaccharide transporter Comp33117 has high homology with MFS sugar transporter. Real-time quantitative RT-PCR analysis showed that these nine genes were expressed in different sclerotia, except for genes Comp34382 and Comp32579, which were significantly down-regulated in the symbiotic sites. The other seven genes were significantly up-regulated. Conclusions suggest that these genes may be involved in different ways Polyporus defense response and absorption of external nutrients process.