Hayes C K, Klemsdal S, Lorito M, Di Pietro A, Peterbauer C, Nakas J P, Tronsmo A, Harman G E
Department of Horticultural Sciences, Cornell University, Geneva, NY 14456.
Gene. 1994 Jan 28;138(1-2):143-8. doi: 10.1016/0378-1119(94)90797-8.
There are no reports of gene sequences coding for extracellular chitinolytic enzymes from filamentous fungi, even though these enzymes are considered critical to the biological control of plant pathogenic fungi. The purpose of this paper was to report the isolation of a gene (ThEn-42) encoding endochitinase (Ech) from Trichoderma harzianum strain P1, describe its sequence, and to determine whether it was related to genes coding for enzymes with similar functions from prokaryotic or other eukaryotic sources. A clone containing a 1096-bp foreign cDNA fragment was isolated from thalli grown under induced conditions. This cDNA molecule was sequenced and found to lack a portion of the 5' terminus. Polymerase chain reaction (PCR) was used to isolate a fragment from the lambda gt11 library which contained the 5' terminus plus an overlap region with the 1096-bp cDNA clone. The full-length cDNA sequence, consisting of 1554 bp, contained an open reading frame (ORF) expressing a protein of 424 amino acids (aa). Southern analysis of genomic DNA indicated that there is only a single gene in strain P1 with sequence identity to the sequence described in this report. One region within the protein, thought to be required for catalytic activity of the enzyme, was highly conserved between genes coding for Ech from Th, Serratia marcescens, Bacillus circulans, Streptomyces plicatus, Vibrio parahemolyticus and Kluyveromyces lactis.
尽管胞外几丁质分解酶被认为对植物病原真菌的生物防治至关重要，但目前尚无关于丝状真菌编码此类酶的基因序列的报道。本文旨在报道从哈茨木霉P1菌株中分离出一个编码内切几丁质酶（Ech）的基因（ThEn - 42），描述其序列，并确定它是否与原核生物或其他真核生物来源的编码具有相似功能酶的基因相关。从诱导条件下生长的菌体中分离出一个含有1096 bp外源cDNA片段的克隆。对该cDNA分子进行测序后发现其5'端缺失一部分。利用聚合酶链反应（PCR）从λgt11文库中分离出一个片段，该片段包含5'端以及与1096 bp cDNA克隆的重叠区域。全长cDNA序列由1554 bp组成，包含一个开放阅读框（ORF），可表达一个由424个氨基酸（aa）组成的蛋白质。对基因组DNA的Southern分析表明，P1菌株中只有一个与本报告中描述的序列具有序列同一性的基因。该蛋白质中一个被认为是酶催化活性所必需的区域，在哈茨木霉、粘质沙雷氏菌、环状芽孢杆菌、褶皱链霉菌、副溶血性弧菌和乳酸克鲁维酵母编码Ech的基因之间高度保守。
