松材线虫Bxpel2基因的克隆及RNA干扰载体构建

目的]为了构建松材线虫(Bursaphelenchus xylophilus)果胶酶Bxpel2基因干扰载体.方法]通过Trizol 法提取松材线虫总RNA,反转录合成cDNA,设计带T₇启动子的果胶酶Bxpel2基因引物,以cDNA为模板扩增出果胶酶Bxpel2基因片段,连接到RNA干扰载体,再以干扰载体为模板,PCR扩增出目的片段后进行测序鉴定,合成果胶酶Bxpel2基因双链RNA(dsRNA),采用RT-PCR检测松材线虫Bxpel2基因干扰后的表达情况.结果]表明:1)提取的松材线虫总RNA完整性好,无降解;2)成功克隆出松材线虫果胶酶Bxpel2基因片段(790 bp)并将其连接至pMD19-T载体;3)以RNA干扰载体为模板合成dsRNA,浓度分别为1.313 mg·mL^-1和1.152 mg·mL^-1;4)RT-PCR结果显示,松材线虫经过dsRNA干扰后,Bxpel2基因表达基本受到抑制.结论]成功构建松材线虫果胶酶Bxpel2基因干扰载体,为进一步研究Bxpel2基因在松材线虫致病过程中的作用和功能奠定了良好的基础.

Cloning of Bursaphelenchus xylophilus Pectate Lyase 2 Gene and Construction of Its RNA Interference Vector

Objective]To establish the interference vector of Bxpel2 gene in Bursaphelenchus xylophilus. Method]Total RNA was extracted and reverse transcribed into cDNA. The primer of Bxpel2 gene containing T₇ promoter was designed to amplify the fragment of Bxpel2 gene. The products of amplification were connected to RNAi vector and used to amplify the target fragment of double strand RNA (dsRNA) of Bxpel2 gene. The expression of Bxpel2 gene was tested by quantitative real-time PCR (qRT-PCR). Result](1) The total RNA extracted by Trizol method was complete and without degradation. (2) The Bxpel2 gene (790bp) of B. xylophilus was cloned and connected to pMD19-T vector. (3) dsRNA was synthesized based on RNA interference vector template, with the concentration of 1.313 and 1.152 mg·mL^-1, respectively. (4) The expression of Bxpel2 gene was inhibited by dsRNA interference. Conclusion]The RNA interference vector of Bxpel2 gene was established successfully. The constructed expression vector could provide a better understanding of the role and function of Bxpel2 gene during the pathogenic process of B. xylophilus.