phaG和phaC基因在烟草叶绿体中的转化及其遗传分析  

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

作　　者：王玉华[1] 吴忠义[2] 张秀海[2] 王永勤[2] 黄丛林[2] 贾敬芬[1] 

机构地区：[1]西北大学生命科学学院/陕西省生物技术重点实验室/西部资源生物与现代生物技术教育部重点实验室,陕西西安710069 [2]北京市农林科学院北京农业生物技术研究中心,北京100097

出　　处：《作物学报》2009年第11期1949-1957,共9页Acta Agronomica Sinica

基　　金：国家高技术研究发展计划(863计划)(2002AA213051);国家自然科学基金项目(30900914);北京市自然科学基金项目(5062012);陕西省教育厅自然科学专项(09JK777)资助

摘　　要：中长链羟基脂肪酸聚酯(medium-chain-length-PHAs,mcl-PHAs)属于微生物聚酯。羟酰-CoA-ACP-转移酶和II型PHA合酶是mcl-PHAs生物合成途径中的两个关键酶。将编码羟酰-CoA-ACP-转移酶的基因phaG与水稻叶绿体psbA基因的启动子和终止子连接构建表达盒RpsbA-pro-phaG-RpsbA-ter,将II型PHA合酶的基因phaC与水稻叶绿体16SrRNA基因的强启动子Prrn及rbcL基因的终止子连接构建表达盒prrn-phaC-RrbcL-ter,连同壮观霉素抗性基因aadA表达盒prrn-aadA-TpsbA-ter一起克隆进烟草叶绿体基因组同源片段rbcL和accD之间,得到烟草叶绿体表达载体pTGC。用包裹有质粒pTGC的金粉子弹轰击烟草无菌苗叶片,经壮观霉素筛选后获得6株叶绿体型转基因植株。对T0代和T1代转基因植株进行PCR检测和Southern blot分析表明,外源基因已整合进烟草叶绿体基因组中,且T1代转基因植株已同质化。RT-PCR分析结果证实外源基因已在转录水平上表达。转基因植株的自交及正反交结果表明,外源基因在转基因后代中能够稳定遗传,遗传方式遵循母性遗传规律,不存在转基因的花粉漂移现象。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 genes into chloroplast genome of T0 and T1 transgenic plants, and T1 transgenic plants exhibited homogenization.The expression of phaC and phaG at transcription l

关 键 词：烟草 叶绿体转化 phaG和phaC 遗传分析 

分 类 号：S572[农业科学—烟草工业]