کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5478529 | 1521806 | 2017 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Precise excision of a selectable marker gene in transgenic Coccomyxa strains by the piggyBac transposase
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موضوعات مرتبط
مهندسی و علوم پایه
مهندسی انرژی
انرژی های تجدید پذیر، توسعه پایدار و محیط زیست
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Precise excision of a selectable marker gene in transgenic Coccomyxa strains by the piggyBac transposase Precise excision of a selectable marker gene in transgenic Coccomyxa strains by the piggyBac transposase](/preview/png/5478529.png)
چکیده انگلیسی
The piggyBac transposon isolated from the cabbage looper moth Trichoplusia ni, is integrated into the host genome, and then excised from it without leaving a footprint. The piggyBac transposon system has been used as a genomic engineering tool in a variety of organisms. In this study, we used two improved versions of the piggyBac transposase (PBase) to create marker-free transgenic strains of the unicellular green alga Coccomyxa sp. strain KJ as follows: Uracil-auxotrophic (Uraâ) mutants of strain KJ defective in the gene for uridine monophosphate synthase (KJUMPS) were isolated on agar plates containing 5-fluoroorotic acid. Subsequently, cDNA of KJUMPS (cKJUMPS) was cloned between the promoter and terminator of the elongation factor 1 alpha gene to construct a cKJUMPS expression cassette. A DNA fragment carrying the cKJUMPS expression cassette flanked with piggyBac transposon terminal repeats was then constructed (TR_cKJUMPS) and introduced into an Uraâ mutant, and Ura+ transformants were isolated. One of the Ura+ transformants was named strain TR2-7. Hyperactive PBase (hyPBase) is a mutant PBase with increased excision and integration frequencies. Herein, we synthesized a coding sequence for hyPBase (KJhyPBase), which has optimized codons for expression in strain KJ, and its expression cassette was introduced into strain TR2-7. Fourteen transformants stably carried the KJhyPBase expression cassette, and TR_cKJUMPS was excised from seven of these. We also introduced an expression cassette of KJhyPBase_Ex, which encodes the excision-competent/integration-defective R372A/K375A/D450N mutant of KJhyPBase (KJhyPBase_Ex), into strain TR2-7, and found that the excision frequency of TR_cKJUMPS in KJhyPBase_Ex transformants was significantly higher than that in KJhyPBase transformants. In further experiments, we purified His-tagged KJhyPBase_Ex, and transfected it into strain TR2-7 using electroporation. Under these conditions, TR_cKJUMPS was precisely excised at a frequency of 8.8 Ã 10â 8 cellâ 1. The present data extend applications of the present piggyBac transposase-catalyzed excision system in green algae.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Algal Research - Volume 27, November 2017, Pages 152-161
Journal: Algal Research - Volume 27, November 2017, Pages 152-161
نویسندگان
Yuki Kasai, Kenta Matsuzaki, Fukiko Ikeda, Yuya Yoshimitsu, Shigeaki Harayama,