松材线虫磷酸肌醇依赖性蛋白激酶1基因的克隆及表达  

Cloning and expression analysis of the phosphoinositide dependent protein kinase 1 gene from Bursaphelenchus xylophilus

作　　者：王步勇 问荣荣 马玲[2] 周天华[1] 张萌 WANG Buyong;WEN Rongrong;MA Ling;ZHOU Tianhua;ZHANG Meng(College of Agricultural and Biological Engineering,Heze University,Heze,Shangdong 274015,China;School of Forestry,Northeast Forestry University,Harbin,Heilongjiang 150040,China)

机构地区：[1]菏泽学院农业与生物工程学院,山东菏泽274015 [2]东北林业大学林学院,黑龙江哈尔滨150040

出　　处：《湖南农业大学学报（自然科学版）》2018年第5期479-486,共8页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：黑龙江省自然科学基金项目(ZD201404);中央高校基本科研业务专项(2572015AA09)

摘　　要：应用BLAST比对技术,在松材线虫基因组数据中鉴定得到模式线虫pdk–1的同源基因Bxpdk–1。结合RT–PCR技术进行Bxpdk–1基因完整蛋白编码区(CDS)扩增,得到CDS区全长2298bp。Bx PDK–1蛋白含有"STKc_PDK1""PH_PDK1"2个保守结构域,属于磷酸肌醇激酶超家族。序列比对及进化树分析显示,松材线虫Bx PDK–1蛋白与捻转血矛线虫PDK–1蛋白(CDJ88126.1)同源性为50%,进化树聚在同一小分支上,亲缘关系较近。原位杂交试验表明,Bxpdk–1基因主要在线虫的头部神经元及咽部神经处表达。q PCR结果分析显示,Bxpdk–1基因表达响应鱼藤酮药剂胁迫处理。RNAi试验显示,Bxpdk–1在dsRNA处理12 h时,基因表达量显著下降,基因被沉默。Based on the Bursaphelenchus xylophilus genome, Bxpdk–1 was identified by the method of BLAST, and cloned by RT–PCR. The results indicated that the complete CDS of Bxpdk–1 gene was 2 298 bp, which encodes a putative protein BxPDK–1. BxPDK–1 protein displayed two conserved functional domains–STKc_PDK1 and PH_PDK1, and belonged to the PKc_like superfamily. Multiple sequence alignment and phylogenetic tree showed that BxPDK–1 protein and the HcPDK–1 （accession no. CDJ88126.1） of Haemonchus contortus shared the similarity value （50%）, and they were present on the same branch of the phylogenetic tree. In situ hybridization indicated that Bxpdk–1 mRNA was mainly expressed in nematode head neurons and pharyngeal nerve. qPCR results showed that the Bxpdk–1 gene expression in response to rotenone stress. Gene silencing showed that the expression of Bxpdk–1 gene decreased under the treatment of dsRNA.

关 键 词：松材线虫 磷酸肌醇依赖性蛋白激酶1基因 基因克隆 原位杂交 表达分析 RNA干扰 

分 类 号：S763.18[农业科学—森林保护学]