【作者】 龙松华；

【导师】 黎定军； 邱德文；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2005， 硕士

【摘要】 新型微生物蛋白质农药最近几年才出现的一种新农药,是对多种农作物具有很强生物活性的一类蛋白质药物,它正逐步成为生物农药发展的一个趋势。过敏蛋白Harpin是一种从病原细菌分离出,分子量约44KD、能激发HR的蛋白类激发子总称。2001年美国Cornell大学和EDEN生物科技公司基于过敏蛋白中共同开发和研制成功了具有抗病防虫功能的广谱性无公害微生物蛋白农药Messenger,开创了新型微生物蛋白农药的兴起。Elicitins是一类从主要从疫霉属和腐霉属分离出、能在烟草上HR和SAR的小分子耐热胞外蛋白质的总称。通过把编码Harpin和Elicitins蛋白基因导入植物,使植物获得广谱抗病性。邱德文博士从交链孢菌纯化出一类新型的蛋白质,分子量从35-62KD。氨基酸和核酸序列分析表明其氨基酸和基因的序列完全不同于Harpin蛋白和Elicitins蛋白,与蛋白质数据库中已知的蛋白序列均无同源性,属于一类全新的蛋白,并命名为激活蛋白。目前还没有关克隆编码激活蛋白基因的成功报道。本实验采用国际上目前最新的Gateway技术构建起交链孢属JH505菌株cDNA文库,以克隆编码激活蛋白的基因。经检测,入门文库的滴度达到1×10~7,文库总容量为9×10~7cfu,其阳性克性率为100%,插入片段大小大部分在1kb以上,平均插入片段大小大约为1510bp左右。通过LR重组把入门文库转换为表达文库,从而能够用激活蛋白抗血清筛选文库克隆其编码基因。表达文库的滴度为1.58×10~6,文库总容量为6.32×10~6cfu,其阳性克性率为100%,插入片段大小大部分在1kb以上,平均插入片段大小大约为1680bp左右。用免疫的方法筛选cDNA表达文库,共得4个阳性克隆,通过检测确定为三种片段,测序后,确定基编码区分别为1095bp、993bp、510bp,进Gene Bank比对后,发现除510bp与与脉孢菌一种假想蛋白同源性为67%,其它同源性都不高。更多还原

【Abstract】 Micro protein pesticide is a kind of new pattern pesticide coming into being lately, which is a group of proteins that play great biological reaction on most plants. Micro protein pesticide may be next wave within pesticide. Either of Harpin protein and Elicitins is a set of protein elicitors which induce HR and SAR of plants. But Harpin protein are separated from plants pathogeny bacterias which has a molecular weight of about 44 KD, while Elicitins mainly derive from Phytophthora and Pythium, which are two kinds of fungies, and their molecular weight are mostly about 10 KD. Neither of Harpin protein and Elicitins is hypersensitive to temperature. The plants can get broad-spectrum resistance to plants pathogeny, which the genes coding Harpin proteins and Elicitins are transformed to. Activator protein is new type protein elictiors differing from Harpin protein and Elicitins by analyzing their sequence of AA(amino acid), and have not induce HR of tobacco. It has a molecular weight from 35-62 KD and is not hypersensitive to temperature and has less identity with the proteins in GenBank. There were not reports about genes coding Activator proteins. Gateway system is a new method of the gene engineering, which mainly includes BXP recombination action and LXR recombination action. cDNA library construction based on Gateway system singnificantly reduces the major drawbacks of conventional cDNA cloning methods, because it eliminated restriction digestion to cDNA. In our experiment, Gateway system was firstly used to construct cDNA library of Alternaria, one kind of epiphytes. As a result, the entry library had a high titer of 1X107 and a total clones of 9X107 cfu, in更多还原