苏云金芽孢杆菌(Bacillus thuringiensis)杀虫晶体蛋白基因导入棉花获得转基因植株

苏云金芽孢杆菌的两个变种中B.t.aizawai 7-29和B.t.kurstaki HD-1是毒杀棉铃虫、红铃虫等鳞翅目害虫的优良菌株.本文通过花粉管途径将B.t.aizawai 7-29和 B.t.kurstaki HD-1的杀虫基因分别导入我国棉花品种(无毒棉、中棉12, 3118,3414)中,经Dot blotting证明,有3株棉花(A1,A2, A5)带有B.t.aizawai 7-29的杀虫基因,18株棉花(B1-4,B6, B7,B9-15,B17—21)带有B.t.kurstaki HD-1的杀虫基因.取2株点杂交呈阳性的植株(A1,A2,)进行Southern blotting,结果表明,A1和A2植物DNA经Hind Ⅲ酶切后均出现了杀虫晶体蛋白基因片段,没有酶切的A1植株DNA在50kb处具有杂交性,表明杀虫基因均已整合到棉花基因组中.组织化学法检测的结果证明,与aizawai 7-29杀虫基因融合的GUS基因(二者共用1个CaMV 35S启动子和1个终止子.)在棉花(A1,A2)中得到了表达,为杀虫基因在转基因棉花中的表达提供了证据.     

INTRODUCING EXOGENOUS DNA INTO PLANTS AFTER POLLINATION——DNA TRAVERSE PATHWAY OF POLLEN TUBE TO REACH EMBRYONIC SAC

The technique of introducing exogenous DNA into plants through the pathway of pollen tube after pollination has been applied since 1978 for transformation of the egg, zygote or carly embryonic cells. By introducing the erogenous DNA containing the goal gene(s) into cotton and rice, several new varieties with economic value and genetic stability are obtained. Thus molecular breeding of crops can be realized by this biotechnique.The pollen tube pathway is demonstrated by tritium-labelled cotton DNA (> 50kb). The labelled DNA is injected into cotton ovaries from the center of the top 24 h after selfing. 30 min to 8 h later, the ovaries are removed. It is clearly shown that the exogenous DNA can only pass from the micrnphyle to the opened nucellus, and then reach the embryonic sac. ~3H-DNA can be found in the embryonic sacs 30 min after injection. In more than 80％ of the embryonic sacs ~3H-DNA can be seen between 2 and 4 h after injection. No ~3H-DNA is found inside the pollen tubes. Therefore it is not th