甜菜(Beta vulgaris L.)叶绿体转化体系建立及抗虫和抗除草剂植株的获得

Establishment of an Efficient Chloroplast Gene Transformation System in Sugar Beet(Beta vulgaris L.)and Obtainment of Insect and Herbicide Resistant Sugar Beet Plants

为建立外源基因甜菜叶绿体转化体系,利用分子生物学方法构建了包含有编码苏云金芽孢杆菌晶体蛋白基因Btcry1Ac和编码膦丝菌素乙酰转移酶基因bar的甜菜叶绿体转化载体pSKARBt/bar,以甜菜叶绿体基因组中atpB/rbcL做同源片段,以甜菜叶绿体16S启动子和终止子为调控基因,以bar基因为筛选标记基因.基因枪法转化甜菜叶柄,经筛选获得抗性转基因植株.对转基因植株进行外源基因Btcry1Ac和bar的PCR检测、DNA印迹分析,结果表明:外源基因Btcry1Ac和bar确已导入到甜菜叶绿体基因组中.转基因植株除草剂抗性鉴定及其离体叶片虫试鉴定结果表明:转基因植株具有较强的杀虫活性和抗除草剂特性,表达了相应的蛋白质.研究结果还表明:bar基因在植物叶绿体转化中,既可以用作抗性基因,又可用作转化体筛选的标记基因.建立了甜菜叶绿体转化体系.

Insects pests and weeds are the main factors that reduce the yield of sugar beet. Genetic engineering breeding is an effective method to breed insect-resisitant and herbicide-resisitant sugar beet. A transformation system for foreign genes in sugar beet chloroplast was established. The expression of the foreign genes can confers resistance in transgenic sugar beet plants to insects pests and weeds. The chloroplast transformation vector pSKARBt/bar, which carries Bt crylAc gene and bar gene expression cassettes, was constructed by using molecular method. The Bt gene expression cassette contained the 3.5 kb Bt crylAc gene under the control of psbA promoter and terminator cloned from sugar beet chloroplast genome. The bar gene expression cassette contained the bar gene, 16S promoter and terminator cloned from sugar beet chloroplast genome, The atpB and rbcL gene cloned from sugar beet chloroplast genome were used as homologous fragment, the bar gene was the selective marker. Plasmid pSKARBt/bar were transformed into the petioles of sugar beet with particle bombardment method. The petioles were planced onto the shoot-inducing selection medium which contained spectinomycin （20 mg/L）, 6-BA （1.5 mg/L） and NAA （0.2 mg/L） at first. And when the green shoots regenerated, the green shoots were transfered into the shoot-propagation medium for optimal shoot development which contained spectinomycin （20 mg/L） and 6-BA （0.5 mg/L） and NAA （1.0 mg/L） one subculture at 20-day intervals, and then the shoots were transfered into the shoot-propagation medium for optimal shoot development with herbicide （PPT 10 mg/L） several subcultures. The shoots were transfered into the root-induction medium with herbicide （PPT 10 mg/L） and the transgenic plants were obtained at last. The transgenic sugar beet plants were testsed by PCR and Southern blot. The results showed that the Bt gene and bar gene had been transferred into the chloroplast genome of sugar beet. The transgenic plants had tolerance to both PPT and bioassays testsed. The insecticidal activity （the mortality of larvaes was 33%-80%） and herbicide resistance of the transgenic plants indicated that the relevant protein had been expressed already in sugar beet. The study showed that the bar gene can also be used as an efficient selective marker gene besides antibiotic resistant markers in plant transformation. Efficient transformation system in sugar beet chloroplast had been established.