piggyBac转座子AgoPLE1.1在黑腹果蝇种系转化中的应用

Application of piggyBac transposon AgoPLE1.1 in germline transformation of Drosophila melanogaster

【目的】通过检测黑腹果蝇Drosophila melanogaster中piggyBac(PB)转座子AgoPLE1.1的转化活性,明确AgoPLE1.1开发为昆虫转基因载体的潜力。【方法】构建AgoPLE1.1转座酶辅助质粒pAgoHsp和带有红色荧光标记的供体质粒pXLAgo-PUbDsRed,辅助质粒和供体质粒以170 ng/μL∶400 ng/μL,90 ng/μL∶200 ng/μL和90 ng/μL∶100 ng/μL 3种不同的比例混合后分别注射新鲜的W1118黑腹果蝇胚胎,筛选注射后代中的转基因黑腹果蝇个体;利用Southern杂交验证转基因黑腹果蝇中AgoPLE1.1转座子的插入拷贝数;利用染色体步移技术克隆AgoPLE1.1插入位点旁侧序列,明确AgoPLE1.1转座子的转座特征。【结果】AgoPLE1.1转座子在黑腹果蝇中具有转化活性,转基因频率为1.32%~1.94%。Southern杂交结果显示,AgoPLE1.1转座子在黑腹果蝇中至少有6个插入位点。染色体步移法克隆了其中4个位点,分别位于黑腹果蝇的3R,3L,2L和X染色体,并且AgoPLE1.1转座子在黑腹果蝇染色体中的整合带有供体质粒的骨架。【结论】PB转座子AgoPLE1.1仅可以在黑腹果蝇中以较低的频率进行非精确的剪切和转座,不具有开发为新型昆虫转基因载体的潜力。

【Aim】To clarify the potential of a piggyBac(PB)transposon AgoPLE1.1 to be developed as an insect transgenic vector by detecting the transformation activity of AgoPLE1.1 in Drosophila melanogaster.【Methods】AgoPLE1.1 transposase helper plasmid(pAgoHsp)and red fluorescent labeled donor plasmid(pXLAgo-PUbDsRed)were constructed.The helper and donor plasmids were mixed at three different ratios(170 ng/μL∶400 ng/μL,90 ng/μL∶200 ng/μL and 90 ng/μL∶100 ng/μL)and microinjected into fresh W1118D.melanogaster embryos,respectively.The transgenic offsprings were screened out.Southern blot was used to verify the insertion copy number of AgoPLE1.1 in the transgenic D.melanogaster,and the flanking sequences of AgoPLE1.1 insertion sites were cloned by genome walking to identify the transposition characteristics of AgoPLE1.1.【Results】AgoPLE1.1 transposon showed transformation activity in D.melanogaster,and the transgenic frequencies were 1.32%-1.94%.Southern blot analysis showed that there were at least six insertion sites of AgoPLE1.1 transposon in the transgenic flies.The genome walking PCR revealed that four of the insertion sites were located in D.melanogaster 3R,3L,2L and X chromosomes,respectively,and the integration of AgoPLE1.1 in D.melanogaster chromosomes contained the skeleton of donor plasmid.【Conclusion】The PB transposon AgoPLE1.1 can only perform imprecise excision and transposition in D.melanogaster with a low transformation frequency.Therefore,AgoPLE1.1 transposon has no potential to be developed as a new insect transgenic vector.