表达barstar基因及bar基因的转基因油菜的研究

Transgenic Oilseed Rape Plants Expressing barstar Gene and bar Gene

从细菌Ｂａｃｉｌｕｓａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ染色体ＤＮＡ中克隆了ｂａｒｎａｓｅ抑制剂ｂａｒｓｔａｒ的基因，构建了带有ＴＡ－２９基因５′调控区（－１３００－＋３）与ｂａｒｓｔａｒ基因编码区、ＣａＭＶ３５Ｓ启动子与除草剂抗性基因ｂａｒ两个表达框架的植物表达质粒ｐＢＢＳ。以“双低”油菜“５－４”的子叶柄为受体，通过农杆菌介导的遗传转化，获得了在含有１０ｍｇ／Ｌ卡那霉素和２０ｍｇ／ＬＰＰＴ的筛选培养基上再生的转基因植株。ＰＣＲ分析结果表明，ｂａｒｓｔａｒ基因已整合到油菜染色体上；Ｎｏｒｔｈｅｒｎｂｌｏｔ检测表明，ｂａｒｓｔａｒ基因及ｂａｒ基因在转基因植物中得到了正确的调控与表达。以转基因油菜“５－４”为父本授粉给表达ｂａｒｎａｓｅ基因的雄性不育植株，不育株能正常结实。

The barstar gene coding for the barnase inhibitor was cloned from Bacillus amyloliquefaciens genomic DNA. Chimeric genes containing the 5′ regulatory region (-1300 to +3) of TA 29 gene fused to the barstar coding region, and the CaMV 35S promoter fused to the herbicide (PPT)-resistant gene bar were constructed into pBI121.1, resulting in plasmid pBBS. Cotyledonary petioles of a “Double Low” variety of oilseed rape, “5-4”, were transformed with A. tumefaciens strain LBA4404 harboring pBBS. Plantlets regenerated on the selective medium containing 20mg/L PPT and 10mg/L Kanamycin were obtained. PCR and DNA hybridization analyses showed that PPT-and Kanamycin-resistant oilseed rape plants contained both bar and barstar genes. Northern blotting analysis indicated that the bar gene was expressed normally and the barstar gene was only transcribed in anthers of regenerated plants as expected. These male-fertile rapeseed plants with a chimeric anther-specific barstar gene could be crossed with male-sterile plants which expressed the barnase gene specifically in anthers to produce normal fruit capsules and seeds by cross-pollination.