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Acta Agron Sin ›› 2009, Vol. 35 ›› Issue (11): 1949-1957.doi: 10.3724/SP.J.1006.2009.01949

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Transformation of phaG and phaC Genesis into Tobacco Chloroplast Genome and Genetic Analysis

WANG Yu-Hua1,WU Zhong-Yi2,ZHANG Xiu-Hai2,WANG Yong-Qin2,HUANG Cong-Lin2,*,JIA JIng-Fen1   

  1. 1College of Life Sciences,Northwest University/Shaanxi Provincial Key Laboratory of Biotechnology/Key Laboratory of Resource Biology and Biotechnology in Westem China,Xi'an 710069,China;2Beijing Agro-Biotechnology Research Center,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,China
  • Received:2009-04-14 Revised:2009-07-25 Online:2009-11-12 Published:2009-09-07

Abstract:

Polyhydroxyalkanoates (PHAs) belong to the group of microbial polyesters, which are a class of polymers produced by various species of bacteria as source of carbon and energy reserve. Due to their properties of biodegradable thermoplastics and elastomers, PHAs have been regarded as ideal alternatives to traditional petroleum-derived plastics in medical areas and many other areas of high technology and high-value. Compared with short-chain-length-PHAs (scl-PHAs), such as polyhydroxybutyrate (PHB), medium-chain-length-PHAs (mcl-PHAs) copolymers are less crystalline, and are more flexible polymers with low melting points, and generally regarded as elastomers. PHAs were produced commercially by bacterial fermentation method, but the process was not economically competitive with petrochemical-based polymers. At present, novel efforts are focused on using the transgenic plants as bioreactors to produce PHAs.Both 3-hydroxyacyl-CoA-ACP-transferase and type II PHA synthase are the key enzymes for mcl-PHAs biosynthesis. The gene phaG encoding 3-hydroxyacyl-CoA-ACP-transferase was placed under the control of psbA-pro and psbA-ter of rice to construct phaG expression cassette, and the gene phaC encoding type II PHA synthase was placed under the control of prrn and rbcL-ter of rice to construct phaC expression cassette, which were ligated with the screening marker gene aadA expression cassette prrn-aadA-TpsbA-ter. These recombined fragments were cloned between the plastid rbcL and accD genes of tobacco for targeting to the large single copy region of chloroplast genome. Chloroplast expression vector of pTGC was constructed and then transformed into tobacco chloroplast genome through particle bombardment. Six transplastomic tobacco plants were obtained by spectinomycin screening. PCR and Southern blot analysis confirmed integration of phaG and phaC genesinto chloroplast genome of T0 and T1 transgenic plants, and T1 transgenic plants exhibited homogenization. The expression of phaC and phaG 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.

Key words: Tobacco, Chlorophlst genetic transformation, phaG and phaC, Maternally inheritance, Genetic analysis

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