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formation of transqenic plant resistant to bacterial infection containing insect antibacterial protein genes

含有昆虫抗菌蛋白基因的抗细菌感染转基因植物的形成

基本信息

批准号：
01890004
负责人：
NATORI Shunji
金额：
$ 14.78万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01890004/
关键词：
transgenic plant sarcotoxin I sarcotoxin II resistant plant Sarcophage peregrina antifungal protein tobacco cDNA ザルコトキシンI.II 抗菌性蛋白 cDNAクロ-ニング抗菌蛋白耐病性ノザンブロット分析

项目摘要

We have been isolating antibacterial proteins from the hemolymph of injured larvae of Sarcophaga peregrina (flesh fly). So far we have purified three protein named sarcotoxin I, II and sapecin. Of these, sarcotoxin I is effective to Gram-negative bacteria and sapecin is effective to Gram-positive bacteria. cDNAs for these proteins have been isolated and complete nucleotide sequences were determined. We have also purified an anti-fungal protein from the same hemolymph. This protein was toxic to Candida albicans and various yeast strains.The purpose of this project was to integrate the genes (cDNAs) for these antimicrobial proteins into the clomosomes of tobacco and to test if the plant resistant to bacterial infection is established.We first tried this experiment using cDNA for sarcotoxin I. This is a small protein consisting of 39 amino acid residues. We integrated this cDNA to a plasmid named pGA643 having multi-cloning sites, and then introduced this plasmid to tobacco leaves using A … More grobacterium LAB 4404. As this plasmid contained 35S RNA promoter, it is possible to express the sarcotoxin I gene in the plant. We isolated cells from tobacco leaves and established callus. From callus we developed transgenic plants. We extracted RNA from the leaves of transgenic plants, and tested if mRNA for sarocotoxin I was present or not by Northern blot hybridization. Clearly, we detected a positive signal, indicating that integrated sarcotoxin I gene was expressed. However, we could not detect neither antibacterial activity nor sarcotoxin I itself in the crude extract of the leaves. It is likely that plant cells cannot translate sarcotoxin I mRNA. We tried similar experiment using sarcotoxin II cDNA, but the results were the same as those of sarcotoxin I cDNA. Namely, we could not detect protein although we could detect mRNA.It seems to be important to investigate why plant cells cannot translate mRNA of insect protein. We are going to test this using in vitro translation system. Less

从肉蝇（Sarcophaga peregrina）受伤幼虫的血淋巴中分离出抗菌蛋白。目前已纯化出三种蛋白，分别命名为肌毒素I、肌毒素II和皂苷。其中，肌毒素I对革兰氏阴性菌有效，皂甙对革兰氏阳性菌有效。这些蛋白的cdna已被分离出来并确定了完整的核苷酸序列。我们还从相同的血淋巴中纯化了一种抗真菌蛋白。该蛋白对白色念珠菌和多种酵母菌株均有毒性。本项目的目的是将这些抗菌蛋白的基因（cdna）整合到烟草的染色体中，并测试烟草是否建立了对细菌感染的抗性。我们首先用肉毒素i的cDNA进行实验，这是一种由39个氨基酸残基组成的小蛋白质。我们将该cDNA整合到具有多克隆位点的质粒pGA643上，然后利用a…More grobacterium LAB 4404将该质粒导入烟叶。由于该质粒含有35S RNA启动子，因此可以在植物中表达肌毒素I基因。从烟叶中分离细胞，建立愈伤组织。我们从愈伤组织中培育出转基因植株。我们从转基因植株的叶片中提取RNA，通过Northern blot杂交检测是否存在sarocotoxin I的mRNA。显然，我们检测到阳性信号，表明整合的肌毒素I基因表达。然而，我们既没有检测到抗菌活性，也没有检测到肉毒素I本身。很可能植物细胞不能翻译肌毒素I mRNA。我们用肌毒素II cDNA进行了类似的实验，但结果与肌毒素I cDNA相同。即，我们不能检测到蛋白质，但我们可以检测到mRNA。研究植物细胞不能翻译昆虫蛋白mRNA的原因似乎很重要。我们将使用体外翻译系统进行测试。少