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

昆虫病原真菌在微量碳氮解阻抑过程中迅速合成包括蛋白水解酶(PR1)在内的角质层降解酶。当微量培养液中添加昆虫角质层时，绿僵菌的PR1水平有所提高。添加更易利用的代谢物(如葡萄糖)抑制了PR1的产生，证实了该产物是可抑制的。用4个引物对来自绿僵菌菌株的DNA进行套式聚合酶链式反应，始终得到约1.2Kbp的强产物和极少的非特异性产物。该产物的大小明显大于以cDNA为模板的PR1PCR产物的预测大小。这是因为PR1基因至少包含三个内含子。除Sau3A1外，所有检测的切割PR1产物的内切酶均表现出多重多态性。这些核酸内切酶产生的限制性内切酶产生了5种不同的模式。结果表明，松材线虫中存在5种以上不同的Pr1。根据限制性内切酶图谱的差异，将金龟子绿僵菌聚为4个群，相似性为87%。为了确定基于PR1的系统发育与金龟子绿僵菌核糖体DNA(RDNA)的关系，对rDNA的序列数据进行了比对，并用Pasimony分析对ITS和5.8S序列进行分析，支持绿僵菌作为单系群。核糖体DNA已被证明是绿僵菌一个非常有用的鉴定区域。它最终也可能有助于构建物种之间的系统发育图。基于rDNA的系统发育与基于绿僵菌Pr1基因的系统发育相似。

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.