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Production of insect resistant, transgenic turfgrass plants with an insecticidal protein gene.

生产具有杀虫蛋白基因的抗虫转基因草坪植物。

基本信息

批准号：
07456002
负责人：
ASANO Yoshito
金额：
$ 3.65万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

In our project, we obtained the following research results.1.A higher frequency of embryogenic callus induction from seeds was obtained using a revised callus induction medium, indicating the establishment of an efficient cell-to-plant regeneration system of turfgrass plants in vitro. 2.In electroporation, modification of buffer components and electric conditions improved gene transfer efficiency to protoplasts. 3.In particle bombardment, adjustment of several mechanical conditions improved gene transfer efficiency to intact culture cells. 4.Transgenic plants with a marker gene were obtained by electroporation, and the transformation was confirmed by PCR-southrn analysis. Difference in gene expression level between transformants with different types of promoters of transgenes was analyzed. 5.Among various types of bt genes, we found that the CryIA(c) produced the protein which gave the strongest insecticidal activity to Jananese lawn grass cutworm and Bluegrass webworm, the severest harmful insects of turfgrass. 6.The CryIA(c) gene (wild-type) was introduced into turgrass plants. Insect resistance of the transgenic plants was examined by bioassay with Bluegrass webworm. However, none of the transgenic plants showed resistance to the insect. 7.To enhance the insecticidal activity of the CryIA(c) gene, we intend 1)to produce a CryIA(c) gene with a stronger promoter, and 2)to modify the sequence of the coding region of the gene. We hope that the use of the gene with a stronger promoter or the modified gene may lead to the production of transgenic turfgrass plants with substantial insect resistance.

本课题取得了以下研究成果：1.采用改良的愈伤组织诱导培养基进行种子胚性愈伤组织的诱导，获得了较高的胚性愈伤组织诱导率，建立了一套高效的草坪草离体植株再生体系。2.在电转化中，通过改变缓冲液成分和电转化条件，提高了原生质体的转化效率。3.在基因枪法中，通过调整机械条件，提高了基因向完整培养细胞的转移效率。4.通过电转化获得了带有标记基因的转基因植株，并通过PCR-southrn分析证实了转化。分析了具有不同类型的转基因启动子的转化体之间基因表达水平的差异。5.在各种类型的bt基因中，我们发现CryIA（c）产生的蛋白质对草坪上最重要的害虫--草坪地老虎和草地螟的杀虫活性最强。6.将CryIA（c）基因（野生型）引入草坪草植物中。通过蓝草网虫生物测定来检测转基因植物的抗虫性。然而，没有转基因植物表现出对昆虫的抗性。7.为了提高CryIA（c）基因的杀虫活性，我们打算1）产生具有更强启动子的CryIA（c）基因，2）修饰该基因的编码区序列。我们希望利用该基因与一个更强的启动子或改造的基因可能导致生产的转基因草坪草具有显着的抗虫性。