利用激光微束穿刺法获得抗黄萎病转基因棉花的研究

Studies on Verticillium and Fusarium Wilt Resistant Cotton Plants with Trangenosis by Laser Microbeam Puncture

通过激光微束穿刺法 ,将 β 1,3 葡聚糖酶及几丁质酶基因双价植物表达载体 pBLGC导入棉花幼胚。转化一代棉花幼苗用蘸根法进行抗黄萎病筛选 ,将存活的苗移入病圃进行了卡那霉素(Kan 1% )抗性测定。结果表明 ,在移栽的 2 9株幼苗中 ,有 9株表现出明显的Kan抗性 ,对 9株抗Kan植株进行了PCR检测 ,结果显示 ,其中 7株表现为阳性。病圃中的T1代转基因植株在经历了黄萎病发病高峰期后 ,7株PCR阳性植株表现明显抗病 ,并已正常开花结铃 ,其他T1代植株及对照植株全部因后期发病死亡。这初步证明外源基因已整合到棉花基因组中 ,且使转基因植株对黄萎病表现出一定的抗性。

By the method of laser microbeam puncture, the maturing embryoes of cotton were transformed with plant bivalent expression vector pBLGC including β 1,3 glucanase and chitinase genes. Fungal challenge of T1 generation with Verticillium albo atrum were carried out by dipping their roots into hyphal and spores solution. The survived T1 seedings were subjected to Kan resistant assay, and the results showed that 9 out of 29 T1 plants showed obvious Kanamycin resistance (Kan 1%). Upon PCR analysis, 7 out of 9 Kan resistant plants produced a PCR fragment with expected size, indicating that they are probably transgenic. After T1 plants in disease inducing plot were subjected to the period, in which the Veritcillium wilt serious took place, 7 PCR positive plants showed obvious resistance to this fungus, at last, flowered and bore cotton bolls normally, while the other T1 plants and the non transformants all died of infection. This result primarily suggested that the foreign genes had integreted in cotton genome, and the transgenic plants showed high resistance to Verticillium wilt.