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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。
Chitinase is an important disease-related protein that plays an active role in plant disease resistance. In order to study the resistance of chitinase to the infection of Penicillium in cantaloupe, we cloned the cDNA of chitinase gene from cantaloupe (Hami melon) using RT-PCR and analyzed the relative fluorescence quantitative (SYBR Green) Its expression level. The cloned chitinase gene fragment HmCHI was 1153 bp in length with a maximum open reading frame of 445 nucleotides encoding a protein of 145 amino acids belonging to the 18 family Class III chitinase gene. It was presumed that the molecular weight of the encoded protein was 15.04 ku , The theoretical isoelectric point 8.82, as a hydrophobic protein; predicted that there is a signal peptide, non-transmembrane protein, located in the intercellular space; HmCHI amino acid sequence and the closest genetic relationship with cucumber. Fluorescence quantitative analysis showed that the expression peak of experiment group appeared later and the time of high expression level was longer, and the amplitude of increase was obviously larger than that of control group. The results showed that HmCHI may play an important role in the fungal stress response of cantaloupe fruit.