小麦几丁质酶基因Wch2的克隆与表达分析

Cloning and Expression Analysis of a Wheat Chitinase Gene Wch2

利用小麦几丁质酶基因PCR特异片段为探针 ,分离克隆了一个小麦Chid1几丁质酶基因Wch2。该基因编码 311个氨基酸 ,不含内含子 ,具有一个信号肽、一个富含半胱氨酸的几丁质结合区域、两个变异区、两个酶活性区域。Southern分析表明 ,在小麦基因组中Wch2有多个拷贝。秆锈菌接种诱导Wch2在一对小麦近等基因系中差异表达 ;在抗病系中国春Sr11中 ,接种 3d后Wch2开始表达 ,6d后表达量更高 ;而在感病等基因系中国春sr11中 ,在所有取样分析的时间内均未检测到Wch2表达。将Wch2克隆到细菌表达载体 pET2 2b ,在细菌中表达的重组Wch2具有几丁质酶活性。这些结果说明 ,分离的Wch2基因在小麦秆锈菌诱导的抗性反应中具有重要作用。

Using a wheat chitinase gene fragment derived from PCR as probe, a Chid1 chitinase gene, Wch2 , was isolated from a wheat genomic library. Sequence analysis indicated that Wch2 encodes a peptide of 311 amino acids with no intron, which contains a signal peptide, a cysteine-rich chitin-binding domain, two variable regions and two catalytic domains (Fig.1). Southern blot hybridization showed that there are multiple copies of Wch2 in wheat genome (Fig.2). Infection with stem rust fungus Puccinia graminis induced a differential expression of Wch2 in a pair of near-isogenic wheat lines (Fig.3). In resistant isogenic line Chinese Spring Sr11(CS-Sr11), an induction of Wch2 transcript was detected 3 days after inoculation and a massive increase of the expression was observed 6 days after incolation. In contrast, in susceptible line Chinese Spring sr11(CS-sr11), expression of the chitinase gene was not detected during the time courses assayed. Crude bacterial extracts of Wch2 expressed in E.coli showed a clear chitinase activity towards chitin(Fig.4). These results suggest that Wch2 may play an important role in the induced resistance responses to the stem rust fungus.