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Cloning and Expression of Chitinase Gene from Bacillus subtilis CHU26 in Saccharomyces cerevisiae
幾丁質（chitin）是一種由 N-乙醯葡萄糖胺（N-acetylglucosamine）以 β-1,4 碳鍵結而成之不可溶的線狀多醣類聚合物，構成地球上甲殼類動物之外骨骼；是自然界中含量最多之多醣類之一。能分解幾丁質的幾丁質酵素（chitinase）在細菌、植物、動物都可以發現，而細胞壁含有幾丁質成分的真菌細胞；其本身也有幾丁質酵素基因；可以產生幾丁質酵素，所以幾丁質酵素基因在原核及真核生物中都可以發現，只是基因的表現目的及調控機制不盡相同。
幾丁質酵素可以抑制大部份致病性真菌的生長，其作用原理可能是經由幾丁質酵素破壞部份真菌細胞壁之結構而使其致病力降低。在膠狀幾丁質培養基測試幾丁質酵素活性表現時發現，本實驗室從土壤中所分離出來的野生種枯草桿菌（Bacillus subtilis CHU26），其幾丁質酵素的活性比另一已被研究討論的環狀芽孢桿菌（Bacillus circulans WL-12）還強，因此利用 B. circulans WL-12 的 chiA 當作探針，在 B. subtilis CHU26 染色體基因庫中找到一段 4 kb 的同源片段，此 DNA 片段中；由 PstI 切割的 1.8 kb 片段經 DNA 序列分析及 GenBank 序列比對之後可以確定是為幾丁質酵素基因，繼續以此段 DNA 當作探針可以將此菌之幾丁質酵素基因完整的從基因庫中選殖出來並將完整的序列解讀出來。此一完整的開放解讀架構（ORF）由 1791 個核?酸轉錄成 596 個氨基酸的序列所構成，將此段 DNA 構築在載體 pGEM3Zf(+)；可以在大腸桿菌中表現幾丁質酵素的活性，經由另一載體 pAAH5 也可以在酵母菌 Saccharomyces cerevisiae 中表現出幾丁質酵素活性。在蘿蔔幼苗對 Rhizoctonia solani CHU341的感病測驗中發現；由酵母菌所產生的胞外幾丁質酵素的確可以抑制 R. solani 的致病力、減少幼苗的倒枯，因此利用酵母菌的載體和發酵系統，應用到生物防治系統方面；降低致病性真菌對植株的危害；應該是可行的一個方法。
Chitin, a polymer of N-acetylglucosamine (GlcNAc), is an important element of insect exo-skeletons, shells of crustaceans, and fungal cell walls, apart from cellulose, it constitutes the second most abundant polysaccharide in the nature. Chitinase (E. C. 3. 2. 1. 14) can degrade chitin into chitosan, not only found in fungi, which contains chitin in the cell wall but also found in the other organisms, including bacteria, yeast and plants. Most chitinase genes have the same conserved regions, but they do not have all of the same expression systems or degradation mechanisms.
It has been proved that expression of chitinase genes in plants would enhance resistance activity to pathogenic fungi by the degradation of the fungal cell wall. To evaluate whether chitinases could play a role in enhancing disease tolerance of plants, transgenic plant is another method to do but it seems to be not efficiently, so another more effective enzymes and systems have to be developed.
Bacillus spp. are gram-positive soil organism, secrete antifungal substances into the culture medium. A native isolate, Bacillus subtilis CHU26 which can secrete chitinase into the culture medium, with a more strong extracellular chitinase activity than Bacillus circulans WL-12. During to the same genus, using the B. circulans WL-12 chiA as a probe, a conserved 4 kb fragment of B. subtilis CHU26 could be cloned from the genomic DNA library by the southern hybridization method. After continuous screening of the library, a complete open reading frame which had 1791 nucleotides and encoded a protein of 596 amino acids was found, and the deduced amino acid sequence has a high identity to the other chitinases. Subcloning the B. subtilis chi into pGEM3Zf(+), the chitinase gene could be expressed in Escherichia coli on colloidal chitin plate. When the gene was subcloned into the shuttle vector pAAH5, it could be expressed in the Saccharomyces cerevisiae. In the infection test of radish seedling with Rhizoctonia solani CH341, the five days culture broth of S. cerevisiae which harberd the pAAHCHI could inhibit the activity of R. solani on radish seedling. By the yeast fermentation system, the chitinase maybe produced abundantly and inhibit the pathogen, directly, but in the biocontrol analysis their still have many works to evaluate.
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