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

1)甜菜叶绿体转基因体系的建立甜菜叶绿体转基因体系的研究已取得进展，该体系将cry基因导入到甜菜叶绿体基因组中，在叶绿体中产生高效的杀虫晶体蛋白（ICP）。用微弹轰击法将携带选择因子（aadA）、标记因子（GUS）、启动子（rrn）和终止子（rps 16）的质粒导入甜菜茎盘组织。在组织培养中还没有实现植株的再生。多次构建质粒，并将其导入甜菜中。甜菜细胞器上蛋白浓缩系统的建立甜菜细胞器上毒性蛋白浓缩系统的建立取得了进展。从烟草植株中克隆到叶绿体传递肽的碱基序列，并与GUS基因N端位置相连。利用农杆菌法构建了该质粒，并将其导入甜菜中。在农杆菌法再生植株的一半植株中，通过PCR southern、ICP分析和PCR southern blotting检测到多个携带cryIA(b)的转化子。这些转化体的几片叶片在第三幼虫龄提供给卷心菜粘虫。白菜粘虫幼虫能在无哭基因的叶片上正常生长，不能在多片转化叶片上生长。4)产杀虫蛋白的高效cry基因筛选白菜粘虫产杀虫蛋白的高效cry基因筛选。CryIC基因对甘蓝粘虫幼虫的杀虫能力优于cryIA(b)。

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.