Yun D J, D'Urzo M P, Abad L, Takeda S, Salzman R, Chen Z, Lee H, Hasegawa P M, Bressan R A
Center for Plant Environmental Stress Physiology, Purdue University, West Lafayette, Indiana 47905-1165, USA.
Plant Physiol. 1996 Aug;111(4):1219-25. doi: 10.1104/pp.111.4.1219.
NaCl (428 mM)-adapted tobacco (Nicotiana tabacum L. var Wisconsin 38) cells accumulate and secrete several antifungal chitinases. The predominant protein secreted to the culture medium was a 29-kD peptide that, based on internal amino acid sequence, was determined to be a class II acidic chitinase with similarity to PR-Q. The four predominant chitinases (T1, T2, T3, and T4) that accumulated intracellularly in 428 mM NaCl-adapted cells were purified. Based on N-terminal sequence analyses, two of these were identified as class I chitinase isoforms, one similar to the N. tomentosiformis (H. Shinshi, J.M. Neuhaus, J. Ryals, F. Meins [1990] Plant Mol Biol 14:357-368) protein (T1) and the other homologous to the N. sylvestris (Y. Fukuda, M. Ohme, H. Shinshi [1991] Plant Mol Biol 16:1-10) protein (T2). The other two proteins (T3 and T4) were determined to be novel chitinases that have sequence similarity with class I chitinases, but each lacks a chitin-binding domain. All four chitinases inhibited Fusarium oxysporum f. sp. lycopersici and Trichoderma longibrachiatum hyphal growth in vitro, although the isoforms containing a chitin-binding domain were somewhat more active. Conditions were established for the successful expression of soluble and active bacterial recombinant T2. Expression of soluble recombinant T2 was achieved when isopropyl beta-D-thiogalactopyranoside induction occurred at 18 degrees C but not at 25 or 37 degrees C. The purified recombinant protein exhibited antifungal activity comparable to a class I chitinase purified from NaCl-adapted tobacco cells.
适应428 mM氯化钠的烟草（烟草品种Wisconsin 38）细胞积累并分泌几种抗真菌几丁质酶。分泌到培养基中的主要蛋白质是一种29 kDa的肽，根据内部氨基酸序列，它被确定为与PR-Q相似的II类酸性几丁质酶。在适应428 mM氯化钠的细胞中细胞内积累的四种主要几丁质酶（T1、T2、T3和T4）被纯化。基于N端序列分析，其中两种被鉴定为I类几丁质酶同工型，一种与绒毛状烟草（H. Shinshi、J.M. Neuhaus、J. Ryals、F. Meins [1990] Plant Mol Biol 14:357 - 368）的蛋白质相似（T1），另一种与野生烟草（Y. Fukuda、M. Ohme、H. Shinshi [1991] Plant Mol Biol 16:1 - 10）的蛋白质同源（T2）。另外两种蛋白质（T3和T4）被确定为新型几丁质酶它们与I类几丁质酶有序列相似性，但每种都缺乏几丁质结合结构域。所有四种几丁质酶在体外均抑制尖孢镰刀菌番茄专化型和长枝木霉的菌丝生长，尽管含有几丁质结合结构域的同工型活性稍高一些。建立了成功表达可溶性和活性细菌重组T2的条件。当在18℃而不是25℃或37℃进行异丙基β - D - 硫代半乳糖苷诱导时，实现了可溶性重组T2的表达。纯化的重组蛋白表现出与从适应氯化钠的烟草细胞中纯化的I类几丁质酶相当的抗真菌活性。
