Studies on Development of Efficient Transgenic System for Alien Gene of Insecticidal Protein in Sugarbeet

1)甜菜叶绿体转基因系统的建立我们已经建立了一个甜菜叶绿体转基因系统，将cry基因导入叶绿体基因组中，并在叶绿体中高效地产生杀虫晶体蛋白（ICP）。用基因枪法将携带选择因子（aadA）、标记因子（GUS）、启动子（rrn）和终止子（rps16）的质粒导入甜菜茎盘组织。在组织培养中还没有实现再生植株。2）甜菜细胞器蛋白质富集系统的建立我们在甜菜细胞器蛋白质富集系统的建立方面取得了进展。从烟草植株中克隆了叶绿体转运肽的碱基序列，并将其与GUS基因的N端位置连接。3）cry基因在甜菜中的表达通过PCR southern、ICP分析和PCR southern印迹分析，对农杆菌介导法转化的植株进行了检测，结果表明，在转化植株中，有一半的植株带有cryIA（B）基因。将这些转化子的几片叶片提供给第三龄幼虫的甘蓝夜蛾。甘蓝夜蛾幼虫能在非cry基因的叶片上正常生长，而不能在转化子的多片叶片上生长。4）筛选高效产杀虫蛋白的cry基因对甘蓝夜蛾进行了高效产杀虫蛋白的cry基因筛选。CryIC基因对菜青虫幼虫的杀虫活性高于cryIA（B）基因。

1)Development of chloroplast transgenic system in sugarbeetWe have progressed to develop a chloroplast transgenic system in sugarbeet in which is introduced cry genes to chloroplast genome and produce efficiently insecticidal crystal protein (ICP) in chloroplast. Plasmid carrying selective factor (aadA), marker factor (GUS), promoter (rrn) and terminator (rps 16) was incorporated into disc tissue of shoot of sugarbeet by microprojectile bombardment. In the tissue culture regeneration plant has not achieved. Repeatedly plasmid was constructed and incorporated into the sugarbeet.2)Development of concentration system of protein upon organelle in sugarbeetWe have progressed to develop a concentration system of toxic protein upon organelle in sugarbeet. Base sequence coding transit peptide to chloroplast was cloned from tobacco plant and was linked with position of N terminal in GUS gene. Thus the plasmid was constructed and introduced into sugarbeet by Agrobacterium method.3)Expression of cry gene in transformant sugarbeetSeveral transformants carrying cryIA(b) were detected by PCR southern, ICP analysis and PCR southern blotting in half of plants regenerating after procedure of Agrobacterium method. Several leaves of these transformants were supplied for cabbage armyworm with the third larva age. Larva of cabbage armyworm was able to grow normally on leaves with non-cry gene and not to grow on several leaves of transformant.4)Screening for efficient cry gene to produce insecticidal proteinScreening for efficient cry gene to produce insecticidal protein was conducted for cabbage armyworm. CryIC gene has more insecticidal ability than cryIA(b) in larva of cabbage armyworm.