桉树焦枯病菌CpSit1基因的鉴定与表达

利用Blast及TCDB数据库对桉树焦枯病菌(Calonectria pseudoreteaudii)的Cpsit1基因进行鉴定;再利用SMART数据库和Prot Param、TMHMM、PHD、Pro Comp 9.0在线分析工具对Cpsit1基因编码蛋白的理化性质、跨膜螺旋、蛋白质二级结构和亚细胞定位进行预测分析;最后采用qRT-PCR方法对CpSit1基因在焦枯病菌侵染桉树过程中的表达情况进行分析.结果表明:CpSit1基因长度为1 780 bp,其编码的蛋白序列共有592个氨基酸残基组成,并将其鉴定为铁载体-铁:H+同向转运蛋白.其保守结构域为MFS_1;共含有14个跨膜螺旋;在二级结构中α螺旋占45.10%,延伸链占26.01%,无规则卷曲占28.89%.通过qRT-PCR相对定量的方法分析焦枯病菌侵染桉树24、48和72 h后CpSit1基因的表达情况,结果显示,CpSit1基因在这3个时段均发生上调表达,但24 h的表达量明显大于48和72 h.说明桉树焦枯病菌CpSit1基因在病原菌侵染寄主的过程中通过调控铁载体-铁化合物的转运来完成铁元素的摄入,协助其在寄主中的定植.

Identification and expression analysis of CpSit1 gene in Calonectria pseudoreteaudii

Cpsit1 was identified by Blast and TCDB database, and then the physicochemical property, transmembrane helix, sec-ondary structure and subcellular localization of CpSit1 were analyzed and predicted by SMART database, ProtParam, TMHMM, PHD and ProComp 9.0 online service. Finally expression of CpSit1 gene was detected by qRT-PCR within 24, 48 and 72 h Calonec-tria pseudoreteaudii infection of Eucalyptus. Results showed that Cpsit1 was identified as proton-coupled symporter for siderophore, with CpSit1 gene being 1780 bp and encoding 592 amino acids. And the conserved domain of CpSit1 is MFS 1. α-helix accounted for 45.10% of the secondary structure, with extension chain and random coil reaching 26.01% and 28.89%, respectively. QRT-PCR results revealed that expressionsof CpSit1 gene were all upregulated within 24, 48 and 72 h C. pseudoreteaudii infection, and the expression level in 24 h was significantly higher than the other 2 stages. It proved that CpSit1 gene could mediate the iron metabolism pathway of C. pseudoreteaudii by regulating siderophore and assist C. pseudoreteaudii in the process of colonization.