哈密瓜中几丁质酶基因的克隆及表达分析

Cloning and Expression Analysis of Chitinase Gene in Hami Melon

摘要:

几丁质酶是一种重要的病程相关蛋白，在植物抗病中发挥积极作用。为研究哈密瓜中几丁质酶对青霉菌侵染的抗性作用，本研究采用RT-PCR的方法，克隆一段哈密瓜中几丁质酶基因的cDNA，并采用相对荧光定量法（SYBR Green）分析其表达量。克隆得到的几丁质酶基因片段HmCHI长1153 bp，最大开放阅读框为445个核苷酸，编码145个氨基酸，属于18家族Class Ⅲ几丁质酶基因；推测其所编码蛋白分子量为15.04 ku，理论等电点8.82，为疏水性蛋白；预测其有一个信号肽，为非跨膜蛋白，定位于细胞间隙；HmCHI氨基酸序列与黄瓜的亲缘关系最近。荧光定量分析显示，实验组的表达高峰出现时间较晚，维持高表达量的时间相对较长，上调幅度明显大于对照组。研究结果表明HmCHI可能在哈密瓜果实真菌胁迫响应中发挥重要作用。

Abstract:

Chitinase, an important pathogenesis-related protein, plays an active role in disease resistance in plants. In order to explore the resistance of chitinase in Hami melon to Penicillium, real-time polymerase chain reaction (RT-PCR) was used to clone the cDNA of chitinase, and relative fluorescence quantitative PCR (SYBR Green) was used to analyze its expression. A gene fragment of 1153 bp was cloned, and was denoted as HmCHI. The largest open reading frame in HmCHI was 445 nucleotides long, encoded 145 amino acids, and belonged to Class III of Family 18 chitinase genes. Using software prediction, the encoded protein was determined to be a hydrophobic non-transmembrane protein distributed within the intercellular space, with a molecular weight of 15.04 ku, isoelectric point of 8.82, and one signal peptide. The amino acid sequence of HmCHI had the closest similarity with Cucumis sativus (XP_004145916.1). Fluorescence quantitative analysis showed that the expression peak in the experimental group appeared later and was maintained for a longer time, and that the magnitude of upregulation was significantly higher than the control. These results indicated that HmCHI in the Hami melon plays an important role in fungal stress response.