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Stable transformation of phaC2 gene in tobacco chloroplast genome

Published online by Cambridge University Press:  20 March 2007

Wang Yu-Hua ,
Wu Zhong-Yi ,
Zhang Xiu-Hai ,
Wang Yong-Qin ,
Huang Cong-Lin  and
Yang Qing
Wang Yu-Hua
Affiliation:
College of Life Science, Northwest University, Xian 710069, China Beijing Agro-biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100089, China
Wu Zhong-Yi
Affiliation:
Beijing Agro-biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100089, China
Zhang Xiu-Hai
Affiliation:
Beijing Agro-biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100089, China
Wang Yong-Qin
Affiliation:
Beijing Agro-biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100089, China
Huang Cong-Lin*
Affiliation:
Beijing Agro-biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100089, China
Yang Qing*
Affiliation:
College of Life Science, Nanjing Agricultural University, Nanjing 210095, China
*
*Corresponding authors. E-mail: huangconglin@hotmail.com; qyang19@njau.edu.cn
*Corresponding authors. E-mail: huangconglin@hotmail.com; qyang19@njau.edu.cn
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Abstract

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The key enzyme for mcl-PHA biosynthesis is type II PHA synthase. The gene phaC2 encoding type II PHA synthase was placed under the control of psbA-pro and psbA-ter of rice (Oryza sativa) to construct a phaC2 cassette, which was ligated with the screening marker gene aadA cassette (prrn–aadA–TpsbA-ter). These recombined fragments were cloned between the plastid rbcL and accD genes for targeting to the large single copy region of the chloroplast genome. A chloroplast transformation vector, pTC2, was constructed and introduced into the tobacco (Nicotiana tobacum) chloroplast genome by particle bombardment. PCR and Southern blot analysis confirmed stable integration of phaC2 into the chloroplast genomes of T0 and T1 transgenic plants, and T1 transgenic plants exhibited homoplasmy. The expression of phaC2 at transcription level was detected by reverse transcriptase–polymerase chain reaction (RT-PCR). Recombinant transgenes in the tobacco chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. To our knowledge, this is the first report on the stable transformation of phaC2 encoding type II PHA synthase in tobacco via chloroplast genetic engineering.

Keywords

chloroplast genetic transformationmaternal inheritancemedium-chain-length polyhydroxyalkanoatestobaccotransgenic safety
Type
Research Article
Information
Chinese Journal of Agricultural Biotechnology , Volume 3 , Issue 3 , December 2006 , pp. 201 - 207
Copyright
China Agricultural University and Cambridge University Press 2006

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