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

• 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

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.

在基本培养基中，昆虫病原真菌在碳和氮的去阻遏过程中迅速合成包括蛋白酶（Pr1）在内的角质层降解酶。Pr1水平从M.当基本培养基中添加昆虫表皮时，对绿僵菌的生长有促进作用。添加更容易利用的代谢物（e. G.葡萄糖）抑制Pr1的产生，证实Pr1的产生是可抑制的。绿僵菌一致地产生约1.2Kbp的强产物和非常少的非特异性产物。该产物明显大于使用cDNA作为模板的Pr1 PCR产物的预测大小。这是因为Pr1基因含有至少三个内含子。除Sau 3A1外，所有切割Pr1产物的核酸内切酶都显示出多态性。由这些核酸内切酶产生的限制性内切酶图谱产生5种不同的图谱。结果表明，M.大茴香根据酶切图谱的差异，将M.在87%的相似性上，绿僵菌可聚为4个类群。绿僵菌属（Metarhizium）与金龟子绿僵菌属（Anisopliae）的rDNA序列进行比对，并利用Pasimony分析法对ITS和5.8S序列进行分析，结果支持绿僵菌属为单系类群。核糖体DNA已被证明是一个非常有用的识别区域的绿僵菌。它也可能最终有助于构建物种间的亲缘关系。基于rDNA的PCR扩增结果与基于M.绿僵菌

项目成果

UO, T. YOSHIMURA, T. SHIMIZU, S. and ESAKI, N.: "Occurrence of Pyridoxal 5'-Phosphate-Dependent Serine Racemase in Silkworm, Bombyx mori."Biochemical And Biophys. ical Research Communications. 246. 31-34 (1998)

UO, T. YOSHIMURA, T. SHIMIZU, S. 和 ESAKI, N.：“家蚕中吡哆醛 5-磷酸盐依赖性丝氨酸消旋酶的发生。”生物化学和生物物理学。

WADA, S. and SHIMIZU, S.: "Isolation of protoplasts from an entomopathogenic fungus Beauveria brongniartii"The Journal of Sericultual Science of Japan. 67巻6号. 499-502 (1998)

WADA, S. 和 SHIMIZU, S.：“从昆虫病原真菌布氏白僵菌中分离原生质体”，日本蚕业科学杂志，第 67 卷，第 6 期，499-502（1998 年）。

SHIMIZU, S.: "Chromosome length polymorphism of insect pathogenic fungi"Proceeding of VIIth International Colloquium on Invertebrate and Microbial Control Sapporo. 216-219 (1998)

SHIMIZU, S.：“昆虫病原真菌的染色体长度多态性”第七届札幌无脊椎动物和微生物控制国际研讨会论文集。

清水進、吉岡勉: "昆虫病原性糸状菌Paecilomyces fumosoroseusの細胞外プロテアーゼの産生条件"日本蚕糸学雑誌. 66巻5号. 357-359 (1997)

Susumu Shimizu，Tsutomu Yoshioka：“昆虫病原真菌 Paecilomyces fumosoroseus 中产生细胞外蛋白酶的条件”，《日本硅学杂志》，第 66 卷，第 5. 357-359 期（1997 年）。

UO, T. YOSHIMURA, T. SHIMIZU, S. and ESAKI, N.: "Occurrence of Pyridoxal 5'-Phosphate-Dependent Serine Racemase in Silkworm, Bombyx mori"Biochemical And Biophysical Research Communications. 246巻1号. 31-34 (1998)

UO, T. YOSHIMURA, T. SHIMIZU, S. 和 ESAKI, N.：“家蚕中吡哆醛 5-磷酸依赖性丝氨酸消旋酶的发生”，生物化学和生物物理研究通讯，第 246 卷。 31-34 (1998)