松材线虫Bx-Daf-22基因鉴定及表达

目的]为探究松材线虫(Bursaphelenchus xylophilus)抗逆态幼虫DL3(Dauer larvae3)形成的分子机理,对其鸟苷酸环化酶路径(cyclic guanosine monophosphate,cGMP)中的抗逆态基因daf-22进行了鉴定和表达研究。方法]根据松材线虫基因组数据设计引物克隆松材线虫Bx-Daf-22基因。进一步对Bx-Daf-22基因编码的蛋白进行系统发育分析、保守结构域分析等生物信息学分析。应用BLAST比对技术在松材线虫基因组数据中鉴定得到固醇载体蛋白SCP2同源基因,命名为Bx-Daf-22。应用PCR技术扩增Bx-Daf-22完整蛋白编码区,分析同源序列系统进化和三维结构。结果]结果表明,Daf-22作为路径的起点是cGMP路径中一个受体蛋白,可将起始信号传递到cGMP路径的膜结合受体上,再通过胰岛素相似信号途径(insulin like signaling,Insulin/IGF)中的β-胰岛素(β-insulin,Daf-28)以及转化生长因子β路径(transforming growth factor β,TGFβ-like)中的热激蛋白90(HSP90/Daf-21)连接从而调控线虫进入抗逆态。结论]通过结构比对及基因表达量分析表明Bx-Daf-22与秀丽线虫Daf-22具有相近的功能。Bx-Daf-22编码的蛋白质具有固醇载体蛋白SCP2构域。因此松材线虫的cGMP路径与DL3形成具有直接相关。

Bx-Daf-22 in Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae): Cloning and Expression

To explore the molecular mechanism ofBursaphelenchus xylophilus dauer larvae 3 formation, the cGMP pathway geneBx-Daf-22 was identified. Based on theB. xylophilus genome, a SCP2 homologous gene was identified by BLAST, and named asBx-Daf-22 .Bx-Daf-22 was cloned by the method of PCR. And some bioinformatics analysis, such as phylogenetic and conserved domain analysis were conducted based on the amino acid sequence of Bx-Daf-22. Then, the orthologous alignment and 3D structure of the Bx-Daf-22 were constructed. The expression levels ofBx-Daf-22 in different larvae and adults were tasted by Q-PCR. The results indicated that: the Bx-Daf-22 consisted of 4α helices, 1 310 helix at the C-terminal, and 5β -pleated sheets among the middle of theα helix. Compared with the expression level in L2, the expression of DL3 were significantly up- regulated, the expression of Bx- Daf- 22 in male adult was significantly suppressed. The structure of Bx-Daf-22 and the expression ofBx-Daf-22 indicate that the gene is involved in cGMP pathway and directly related to DL3 formation.