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

甜菜杀虫蛋白外源基因高效转基因体系的开发研究

基本信息

批准号：
09460001
负责人：
SHIMAMOTO Yoshiya
金额：
$ 8.7万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

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.

1)甜菜叶绿体转基因系统的建立甜菜叶绿体转基因系统的研究进展：将杀虫晶体蛋白基因导入甜菜叶绿体基因组，在叶绿体中高效表达杀虫晶体蛋白。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。在组织培养再生植株方面还没有实现。2)甜菜细胞器上蛋白质浓缩体系的建立我们已经建立了甜菜细胞器上有毒蛋白质的浓缩体系。从烟草中克隆了编码叶绿体转运肽的碱基序列，并将其与GUS基因中N端的位置连锁。3)转化甜菜后，对经农杆菌法处理后再生植株的一半植株进行了PCR Southern、ICP分析和PCR Southern blotting检测。这些转化子的几片叶子被提供给三龄甘蓝粘虫。甘蓝粘虫幼虫能在不含杀虫基因的叶片上正常生长，不能在转化子的多片叶片上正常生长。4)筛选高效的杀虫蛋白基因。CryIC基因在甘蓝粘虫幼虫体内的杀虫能力强于CryIA(B)基因。