小菜蛾NanosO基因启动子的克隆及功能验证

Cloning and functional characterization of the NanosO gene promoter in Plutella xylostella (Lepidoptera: Noctuidae)

【目的】克隆小菜蛾Plutella xylostella NanosO基因PxnosO启动子,并验证其具有生殖腺特异性活性,以期应用于基因功能研究或转基因昆虫的构建,为小菜蛾等农业害虫的综合治理提供新的研究思路。【方法】根据小菜蛾基因组序列信息,利用PCR技术克隆NanosO的启动子并进行序列分析。构建PxnosO-EGFP表达质粒,利用脂质体细胞转染技术将PxnosO-EGFP和IE1-EGFP表达质粒转入到小菜蛾胚胎细胞系(Px-6)和草地贪夜蛾Spodoptera frugiperda卵巢细胞系(Sf9)中,通过激光共聚焦荧光显微镜观察和qRT-PCR技术分别定性和定量分析EGFP基因的表达,验证小菜蛾NanosO启动子的活性。【结果】克隆获得小菜蛾PxnosO(Px004767)启动子区序列,长1 743 bp。对启动子序列进行分析,发现该序列不仅包含启动子共有核心元件TATA box以及上游启动子成分CAAT box和GC box等,还包含有数十个转录因子结合位点。利用细胞转染技术,在PxnosO启动子驱动下成功地在Px-6和Sf9细胞系中表达外源基因EGFP。【结论】克隆了小菜蛾NanosO基因PxnosO启动子,在细胞水平上验证其能驱动外源EGFP基因的表达,为分析PxnosO在小菜蛾不同发育时期的表达模式和PxnosO启动子在体内的功能验证奠定基础。

【Aim】To clone the NanosO gene(PxnosO)promoter from the diamondback moth,Plutella xylostella,and to verify its gonad-specific activity,so as to further study the gene functions and develop transgenic insects for integrated management of P.xylostella and other agricultural pests.【Methods】According to the sequence information of NanosO gene in the P.xylostella genome,the NanosO gene promoter was cloned by PCR and subjected to sequence analysis.The PxnosO-EGFP expression plasmid was constructed,and then the PxnosO-EGFP and IE1-EGFP expression plasmids were transfected into P.xylostella embryonic cell line(Px-6)and Spodoptera frugiperda ovary cell line(Sf9)by lipofection.The activity of the NanosO promoter in P.xylostella was confirmed by the qualitative and quantitative analysis of EGFP expression by laser confocal fluorescence observation and qRT-PCR,respectively.【Results】We cloned the PxnosO(Px004767)promoter(with the length of 1 743 bp)of P.xylostella.Sequence analysis showed that this promoter not only contains common core promoter elements like TATA box,and upstream promoter domains TATA box and GC box,but also contains dozens of transcription factor binding sites.Using cell transfection technology,we successfully expressed exogenous gene EGFP driven by this promoter in the Px-6 and Sf9 cell lines.【Conclusion】We cloned the PxnosO promoter of P.xylostella and verified that it can drive the expression of EGFP gene at the cellular level.This study provides a foundation for analyzing the expression pattern of PxnosO in different developmental stages of P.xylostella and the potential functions of the PxnosO promoter in vivo.