Bar基因的转化及抗草丁膦除草剂转基因玉米植株的获得

Transformation of Bar Gene and Regeneration ofPhosphinothricin-tolerant Plants in Maize

杂草危害已成为玉米(Zea mays L.)栽培中的一大难题,采用常规人工拔草的方式去除杂草存在操作麻烦、成本高等问题,而培育抗除草剂转基因玉米是解决这一问题的有效途径。为了获得生产中可用的抗草丁膦(phosphinothricin,PPT)除草剂的优良转基因玉米,本研究通过基因枪法将p35SIH3X质粒上的bar基因导入了优良玉米自交系18-599红幼胚诱导的愈伤组织。转化后的愈伤组织经过在含Bialaphos浓度(以有效成分PPT计算)为6,10,15mg.L-1的选择培养基中筛选3次以后进行分化,长出的小苗再经炼苗后移栽至大田,最后得到长大成活植株75株,结实收获8株。通过PCR(polymerase chain reaction)及Southern杂交等技术对抗性再生植株进行了鉴定,结果显示bar基因已整合到玉米基因组中,且已遗传至下一代,再对T1代转基因植株通过人工涂抹0.5mg.L-1的PPT进一步做表型鉴定,结果对照植株全部死亡,而转基因植株呈现出不同程度的抗性。

At present, the works of weeding are tedious, time consuming and labour intensive in maize （Zea rnays L.） planting by popular artificial manner. However, it is an effective approach to overcome the problem by breeding herbicide-resistant varieties. In order to obtain phosphinothricin-tolerant transgenic elite maize materials, in this study, the bar gene was introduced into embryonic calli derived from immature embryos of maize elite inbred line 18-599hong via mieroprojectile bombardment. Bombarded calli were selected on selection medium containing 6, 10, 15 mg-L^-1 Bialaphos （Phosphinothricin, PPT）. After that, the generated young plants from resistant calli were first exercised in laboratory, then, the live plants were transplanted to field. At last, 75 plantlets were regenerated and grown, and 8 of 75 were harvested. The results of PCR （polymerase chain reaction）and Southern blotting analysis demonstrated that bar gene has been integrated into the genome of transgenic plants. PCR-positive progeny plants were artificially daubed with herbicide, and the damage of each plant was investigated after 10 days. The results showed that the non-transgenic plants were dead, while, the presence of the bar gene provided resistance to the herbicide.