松材线虫fem-1基因的克隆与表达特性研究

 松材线虫病是世界性的检疫病害,严重威胁松林等森林资源和生态安全。有研究表明,Feminization(fem)基因家族在秀丽隐杆线虫中参与性别决定和分化。本研究克隆了松材线虫fem-1基因,并对其表达特征和调控功能进行研究。结果表明,该基因cDNA全长为856 bp,包含681 bp的开放阅读框,编码226个氨基酸,进化分析显示与秀丽隐杆线虫位于同一进化分支。荧光定量PCR以及原位杂交结果显示, fem-1基因在松材线虫各个发育阶段均有表达,其中在2龄虫期表达水平最高;在卵期至3龄虫期阶段于虫体中后部表达,而发育后期,多位于皮下细胞中表达,肠道、性腺等部位极少出现杂交反应。利用RNA干扰技术沉默fem-1基因后,线虫种群雌雄比例出现下降,说明fem-1基因可能参与松材线虫性别分化和决定过程。研究结果有助于了解松材线虫性别决定相关基因表达特性与功能,为深入研究松材线虫发育生物学提供一定的理论依据。

Cloning and expression of fem-1 gene from Bursaphelenchus xylophilus

Pine wilt disease is a worldwide quarantine disease that seriously threatens forest resources such as pine forests and ecological security. The Feminization (fem) gene family is involved in sex determination and differentiation in the model Caenorhabditis elegans. In this research, the pine wood nematode fem-1 gene was cloned and its expression characteristics and functions were studied. The full-length cDNA of fem-1 is 856 bp long,containing a 681 bp open reading frame and encodes a 226-amino-acids protein, which shares same clade with that of C. elegans in phylogenetic analysis. The results of real-time quantitative PCR(RT-qPCR)and in situ hybridization showed that fem-1 gene was expressed in all developmental stages of pine wood nematodes, with the highest expression level in the second juvenile stage. The fem-1 gene was expressed in the rear of the body from the egg stage to the third juvenile stage. In the later stage of development, it was mostly expressed in subcutaneous cells and rarely occurred in the intestinal tract, gonadal and other parts. After the fem-1 gene was silenced by RNA interference technology, the female-male ratio of the nematode population decreased, indicating that the fem-1 gene may be involved in the sex differentiation and determination of pine wood nematodes. This study is helpful for understanding the expression characteristics and function of fem-1 gene in B. xylophilus, and provides a theoretical basis for further research on the reproductive development of pine wood nematodes.