Analysis of infection mechanism of muscardine disease in the silkworm, Bombyx mori, using starvation stress genes

利用饥饿应激基因分析家蚕白僵菌病感染机制

基本信息

批准号：
09460033
负责人：
SHIMIZU Susumu
金额：
$ 4.35万
依托单位：
Kyoto Institute of Technology
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

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09460033/
关键词：
Muscardine disease Starvation stress Silkworm, Bombyx mori Gene Expression

项目摘要

Synthesis of the cuticle-degrading-enzymes including protease (Pr1) by the entomopathogenic fungi occurs rapidly during carbon and nitrogen derepression in minimal media. Pr1 levels from M. anisopliae were enhanced when minimal media were supplemented wit insect cuticle. Addition of more readily utilized metabolites (e. g. glucose) repressed Pr1 production confirming that production is repressible.Nested PCR using 4 primers on DNA from strains of M. anisopliae consistently gave a strong product of about 1.2Kbp and very few non-specific product. The product is significantly larger than the predicted size of the Pr1 PCR product using cDNA as template. This is because the Pr1 gene contain at least three introns. Apart from Sau 3A1, all of the endonucleases tested cut the Pr1 product showed multiple polymorphisms. Restriction patterns generated by these endnucleases produced 5 different patterns. The results were suggested that more than 5 different Pr1s were existed in M. anisoliae. Base on differences of restriction patterns, the strains of M. anisopliae were clusterd into 4 groups at 87% similarities.To determine the relationships of the phylogeny based on Pr1 and ribosomal DNA (rDNA) from M. anisopliae, sequence data of the rDNA were aligned and analysis of the ITS and 5.8S sequences using Pasimony analysis supports Metarhizium as a monophyletic group. Ribosomal DNA has proved an extremely useful identification region for Metarhizium. It may also eventually help to construct phylogenies between species. The phylogenies based on rDNA were similar to those based on Pr1 genes from M. anisopliae.

在最小培养基中，昆虫病作用真菌在碳中蛋白酶（PR1）的合成迅速发生在碳发育性真菌中。当补充昆虫角质层的最小培养基时，来自各向异性分枝杆菌的PR1水平得到了增强。添加更容易利用的代谢物（例如葡萄糖）抑制了PR1的产生，证实了生产是可抑制的。使用4个引物在Anisopliae菌株中使用4个引物在DNA上始终如一地提供了约1.2kbp的强大产物，并且很少有非特异性产品。使用cDNA作为模板，该产物明显大于PR1 PCR产物的预测尺寸。这是因为PR1基因至少包含三个内含子。除了SAU 3A1外，所有测试的核酸内切酶都削减了PR1产品均显示出多态性的多态性。这些终止核酸酶产生的限制模式产生了5种不同的模式。结果表明，在弧菌杆菌中存在5种以上不同的PR1。基于限制模式的差异，弧菌菌株的菌株以87％的相似性聚类为4组。为了确定基于PR1和核糖体DNA（rDNA）的系统发育的关系，使用其和5.8s序列分析的RDNA序列数据对RDNA的序列数据进行了分析。核糖体DNA已被证明是metarhizium的非常有用的鉴定区。它最终也可能有助于在物种之间构建系统发育。基于rDNA的系统发育与基于来自各向异性分枝杆菌的PR1基因的系统发育相似。