Acta Entomologica Sinica ›› 2021, Vol. 64 ›› Issue (6): 676-681.doi: 10.16380/j.kcxb.2021.06.003

• RESEARCH PAPERS • Previous Articles     Next Articles

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

 ZHANG Hao-Miao1, WANG Xiao-Fang1, LUO Guang-Hua2, HAN Xiang-Yu1, WANG Qiu-Xia1, HAN Zhao-Jun1, WU Min1,*   

  1.  (1. College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; 2. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)
  • Online:2021-06-20 Published:2021-06-15

Abstract: 【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.

Key words: Drosophila melanogaster, piggyBac transposon, piggyBac-like elements, germline transformation, excision, transposition