柳树几丁质酶基因SlChi的克隆和功能验证

Cloning and Functional Identification of Chitinase Gene SlChi in Salix

本研究以灌木柳‘苏柳2345’(Salix Jiangsuensis 2345)为材料,克隆了柳树几丁质酶基因,将该基因命名为SlChi基因。SlChi基因包含一个编码289个氨基酸序列的开放阅读框,含有信号肽并定位于胞外。生物信息学分析显示SlChi编码蛋白是典型的第Ⅲ类几丁质酶,具有几丁质酶特征性催化结构,其特征序列为LDGVDFDIE,其中谷氨酸(E)为质子供体,属于糖基水解酶第18家族(GH18);系统进化树分析表明SlChi基因与毛果杨几丁质酶基因的亲缘关系最为亲近。构建过表达载体将其转入拟南芥,获得T3代阳性转基因株系纯合子。对转基因植株进行100 mmol/L Na Cl胁迫处理,SlChi基因的过量表达能够促进转基因植株在高盐条件下的种子萌发和幼苗生长。在盐胁迫条件下,转基因种子的萌发率>92%,远高于野生型植株(<50%);转基因植株幼苗的生长势旺盛,叶绿素含量下降幅度较小,低于野生型植株。结果表明SlChi基因在提高植物耐盐能力方面具有重要作用。本研究为几丁质酶基因的新功能开辟了新的思路,同时也为培育柳树抗病、耐盐兼具的新品种提供了坚实的依据。

In this study, the Salix saposhnikovii cultivar ’Salix Jiangsuensis 2345’ was used as the material, and the chitinase gene was cloned which was named as SlChi. This gene contained an open reading frame encoding289 amino acids sequence, owned signal peptide and was located outside the cell. Bioinformatics analysis revealed that SlChi encoded protein was a typical class Ⅲ chitinase with the characteristic catalytic structure of chitinase. Its signature sequence sequence was LDGVDFDIE, in which glutamic acid(E) was the proton donor, belonging to the family 18 of glycosylhydrolase(GH18). Phylogenetic tree analysis indicated that SlChi had the closest relationship with chitinase in Populus trichocarpa. The overexpression vector was constructed and introduced into Arabidopsis thaliana, then the T3 homozygote generation transgenic lines were obtained. When the transgenic plants were treated with 100 mmol/L Na Cl, the overexpression of SlChi gene could promote the seed germination and seedling growth of transgenic plants under high salt conditions. Under salt stress, the survival rate of transgenic lines was more than 92%, which was much higher than that of wild type plants(<50%). Meanwhile, the growth potential of transgenic young seedlings was vigorous and the decrease of chlorophyll content in leaves was smaller than that of wild type plants. This result suggested that SlChi played an important role in improving salt tolerance of plants.This study gave deep insight into the new function of chitinase and could provide fundamental basis for cultivating willow varieties with salt and disease resistance as well.