两个棉花几丁质酶基因的克隆与表达分析

 从棉花黄萎病缩减杂交库中得到了分属Ⅲ型和Ⅳ型几丁质酶的两个片段，它们都具有完整的3’末端。通过RACE与RT-PCR结合获得了这2个基因完整的读码框序列。其中Ⅳ型几丁质酶的开放读码框为678 bp，编码长为226个氨基酸的蛋白，命名为Ghachi4；Ⅲ型几丁质酶的全长为1390 bp，编码长为298个氨基酸的蛋白，命名为Ghachi3。它们与拟南芥同源基因的相似性分别达到65%和71%。利用网上分析软件发现这2个基因都有信号肽序列，并预测编码的蛋白位于胞质体外。半定量RT PCR发现Ghachi3在花、蕾和韧皮部中的表达量较高，而Ghachi4在韧皮部表达量最高。Ghachi4仅在抗病品种受黄萎病和枯萎病的诱导后表达上调，而Ghachi3还在感病品种中受黄萎病的诱导表达。同时，在常抗棉中2个基因的表达都受到ABA的强烈诱导。推测这2个基因可能参与生物胁迫或非生物胁迫的防御反应。

Molecular Cloning and Expression Analysis of Two Chitinase in Upland Cotton

ESTs encoding class Ⅲ and classⅣ chitinase respectively were isolated from the SSH library inoculated with Verticillium wilt. Both of them contained full 3’ end and their full open reading frame were obtained by RACE and RT-PCR. As a result, the entire coding region of class Ⅲ and classⅣ chitinase is 1390 bp and 678 bp respectively and encodes a polypeptide of 298 and 226 amino acids. The polypeptide encoded by these two genes shared 71% and 65% homology to their homologous proteins of Arabidopsis. They were designated as Ghachi3 and Ghachi4. All of these two genes have signal peptide and were predicted to be extracellular chitinase. Ghachi3 were abundant in petals, buds and phloem, whereas Ghachi4 only showed an extremely high level in phloem by semi quantitative RT PCR. The accumulation of Ghachi4 increased remarkably after treatment of Verticillium wilt and Fusarium wilt in the disease resistant cultivar exclusively. But Ghachi3 was also induced by Verticillium wilt in the susceptible cultivar. In addition, the expression of both the two genes increased remarkably after treatment of ABA. All the results indicated that these two genes might play roles in response to biotic and abiotic stresses.