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中长链羟基脂肪酸聚酯(medium-chain-length-PHAs,mcl-PHAs)属于微生物聚酯.PHA合酶(PHAsynthase,由phaC基因编码)和3-羟酰基-酰基转移蛋白-辅酶A转移酶(3-hydroxyacyl-acylcarrierprotein-coenzymeAtransferase,由phaG基因编码)是mcl-PHAs生物合成途径中的关键酶.以aadA(氨基糖苷-3′-磷酸转移酶)基因做筛选标记,分别构建了含phaC2基因的单价叶绿体转化表达载体pTC2以及同时嵌合phaC和phaG基因的双价叶绿体转化表达载体pTGC.通过PDS1000/He基因枪转化法转化烟草,获得具有壮观霉素抗性的叶绿体型转基因烟草植株,PCR和SouthernBlot结果表明,外源基因确已整合进烟草叶绿体基因组中且基本达到同质化.气相色谱法检测转基因株系中的mcl-PHAs含量最高达4.8mg/g干重,合成的PHAs单体主要成分为3-羟基辛酸(3-hydroxyoctanoate,3-HO)和3-羟基癸酸(3-dydroxydecanoate,3-HD).透射电子显微镜观察到转基因烟草叶片叶绿体中确有PHAs颗粒累积.
Medium-chain-length-PHAs (mcl-PHAs) belong to microbial polyesters, and PHA synthase (encoded by the phaC gene) and 3-hydroxyacyl-acyltransferase-coenzyme A transferase (3-hydroxyacyl-acylcarrier protein-coenzyme Atransferase, encoded by the phaG gene) is a key enzyme in the mcl-PHAs biosynthesis pathway.The phaC2 gene was constructed using aadA (aminoglycoside-3’-phosphotransferase) Of monovalent chloroplast transformation vector pTC2 and chimeric phaC and phaG bivalent chloroplast transformation vector pTGC.The tobacco plants were transformed with PDS1000 / He by gene gun method to obtain spectinomycin-resistant chloroplast-type transgenic tobacco plants, and PCR And SouthernBlot results showed that the exogenous gene was indeed integrated into the tobacco chloroplast genome and basically reached homogeneity.Gc chromatography detected the content of mcl-PHAs in transgenic lines up to 4.8mg / g dry weight, the synthesized PHAs monomer The main constituents were 3-hydroxyoctanoate (3-HO) and 3-hydroxydecanoate (3-HD) .There were PHAs in the chloroplasts of transgenic tobacco leaves by transmission electron microscopy Particle accumulation.