药用猪苓菌核9种主要协助转运蛋白超家族基因的克隆与表达分析

Cloning and Expression Analysis of Nine Major Facilitator Superfamily Encoding Genes in Polyporus umbellatus

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 facilitator superfamily（MFS） genes of Polyporus umbellatus and carry out bioinformatic analysis. METHODS Nine major facilitator superfamily （MFS）genes were cloned from Polyporus umbellatus selerotia by RT-PCR and the expression analysis of the nine genes in different parts of Polyporus umbellatus sclerotia was carried out using quan- titative Real-time PCR. RESULTS The full open reading frame eDNA sequence of these nine genes was between 1 321 and 1 860 bp, the putative encoding proteins were between 441 and 620 amino acids, the molecular weight was between 48. 45 x 103 and 64. 79 ~ 103 and the theoretical pI was between 6. 59 and 9. 56. The amino acids of these nine genes possessed 11 to 14 membrane-spanning do- mains. Phylogenetie tree analysis indicated that Comp34750, Comp34832, Comp29252, Comp42895, Cornp32579 and Comp27555 had the highest similarity with MFS general substrate transporter, Comp28872 and Comp26306 had the highest similarity with MFS monosac- eharide transporter, and Comp33117 had the highest similarity with MFS sugar transporter. Quantitative real-time PCR showed that these nine genes were expressed in both the symbiotic part and non-symbiotic part. Meanwhile, the expressions of seven genes were sig- nificantly up-regulated in the symbiotic part except Comp34382 and Comp32579. CONCLUSION The investigated nine genes might play an important role during the defense response and nutrient absorption of P. umbellatus.