- 學位論文
蟲生真菌Paecilomyces javanicus幾丁質酶基因特性及表現分析與其抑菌功能的研究
Molecular cloning, characterization, expression analysis, and fungal inhibitory effect of an endochitinase cDNA from the entomopathogenic fungus, Paecilomyces javanicus
摘要
Paecilomyces javanicus是一種蟲生真菌，在自然界裡主要感染雙翅目和鱗翅目昆蟲。利用SDS-PAGE的活性分析，發現Paecilomyces javanicus的菌絲有很強的chitinase活性，於是根據chitinase的conserved region設計一對degenerated primer然後利用touchdown PCR及RACE的技術，釣出一條Paecilomyces javanicus的endochitinase的cDNA，命名為PjCHi-1。PjCHi-1全長有1180-bp，含有1035-bp的ORF，可轉譯成345個氨基酸。利用ExPaSy資料庫分析其氨基酸序列，發現PjCHi-1含有chitinase activity conserved motif（-F82DGIDIDWE90-）且屬於Glycosyl hydrolases family18及classⅤ chitinase。Southern blot分析顯示PjCHi-1基因在單套的基因組裡只有一個拷貝。進一步的Northern blot及RT-PCR結果顯示，其在含有幾丁質（chitin）的培養基中，幾丁質能增強PjCHi-1的基因表現。因此從以上的資料大致可以說明PjCHi-1在P. javanicus生理上的功能可能跟入侵宿主有關。以大腸桿菌來表現GST-PjCHi-1的重組蛋白，經膠體中幾丁質酶螢光活性分析，發現必須把GST切除PjCHi-1才會有分解幾丁質的酵素活性；用4-Mu-（GlcNAc）3當受質進行酵素活性分析，證實PjCHi-1是屬於內切型幾丁質酶。PjCHi-1的最適反應pH值為5。PjCHi-1除了能夠抑制植物致病菌星辰花白絹病菌（Sclerotium rolfsii）的菌絲生長及菌核（slerotial body）的菌絲萌發，也能抑制其他真菌的（Colletotrichum gloeosporioides、Aspregillus sp. and Rhizoctonia solani）菌絲生長。未來可以將PjCHi-1配合甘藷sporamin基因同時轉殖進高麗菜或芥藍裡，以獲得更有效的抗蟲、抗真菌的效果。
並列摘要
Paecilomyces javanicus is an entomopathogenic fungi naturally parasitizing on Diptera and Lepidopteran. Based on the analysis of SDS-PAGE gel activity assay, a strong chitinase activity was discovered in artificial cultured mycelium. The full-length cDNA, designated PjCHi-1, was subsequently cloned from mycelium by using both degenerated primer/RT-PCR amplication and 5’-/3’-RACE extension. This cDNA gene, comprising 1,180 bp, coveres a 1035-bp open reading frame and encodes a 345-amino acid polypeptide. A conserved motif for chitinase activity ,–F82DGIDIDWE90- ,was present in deduced amino acid sequence. It implicated that it is belonged to class V, family 18 chitinase. Recombinant PjCHi-1 protein, expressed from Eschaerichia coli, was purified and its endochitinase activity toward 4-metilumbelliferilβ-D-N,N,Ntriacetilchitotrioside(4MU-(GlcNAc)3) was verified. Southern blot analysis showed that a single copy of PjCHi-1 gene was contained in haploid genome. Both of RT-PCR and Northern gel analysis revealed that the expression of chitinase gene was constitutive but in low-level, only enhanced to high-level expression in chitin-containing medium. This demonstrated that the physiological function of PjCHi-1 (family 18) in P. javanicus plays an invasive role in insect parasitism, and evolutionarily different from those chitinase genes in plants. To study the effect of pH on chitinase activity, buffers of different pH were used. The results showed that the enzyme was highly active at acidic pH with the highest activity at pH 5. An in vitro assay showed that the purified PjCHi-1 protein could inhibit mycelia growth and sclerotial body germination of Sclerotium rolfsii, a major fungal pathogen of plants.
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