甜瓜CmDWF1基因的克隆及表达分析

Cloning and Expression Analysis of CmDWF1 Gene in Melon

油菜素内酯是调节植物生理活动的重要植物激素,DWF1是油菜素内酯合成途径中重要的合成酶之一。为了研究油菜素内酯合成酶基因DWF1在甜瓜果实发育过程中的功能,从甜瓜中克隆得到CmDWF1基因,并对该基因及其编码蛋白进行了生物信息学分析。利用实时荧光定量PCR方法分析了该基因在甜瓜不同组织器官中的表达。结果表明:CmDWF1基因的开放阅读框片段长为1 701 bp,编码566个氨基酸,预测其编码蛋白的分子质量为66.115 11 ku,理论等电点为6.96,不稳定指数为48.18,总平均亲水性为-0.402,属于亲水性蛋白。CmDWF1蛋白含有FAD-binding-4结构域,推测其具有FAD氧化还原酶特性。该蛋白信号肽序列,在细胞质中的定位系数为9,推测其主要在细胞质中分布。二级结构预测显示,CmDWF1蛋白中α-螺旋的含量相对较高,为37.81%。系统进化分析表明,CmDWF1蛋白与黄瓜的亲缘关系较近;实时定量PCR结果表明,该基因在甜瓜的根、茎及叶中均有表达,但在根中表达量最高,并且在甜瓜果实授粉后25 d内表达量有明显的上升,35~45 d内表达量呈下降趋势。由以上结果可知,CmDWF1基因可能参与甜瓜油菜素内酯的合成,并进一步调节甜瓜的生长发育。

Brassinosteriod is a necessary for plant to regulate the plant physical activities,and DWF1 is one of most critical enzymes involved in brassinosteriod biosynthesis.In order to study the function of DWF1 in melon fruit development,the gene CmDWF1 was cloned from melon for analyzing the nucleotide and amino acid sequences.RT-PCR method was used to analyze the gene expression characteristics.The results showed that the open read frame length of CmDWF1 was 1 701 bp,encoding 566 amino acids.The predicted protein molecular weight of CmDWF1 was 66.115 11 ku,the theoretical isoelectric point was 6.96,the instability index was 48.18 and the GRAVY was-0.402.Signal sequence was not found and the location coefficient in the cytoplasm was 9,which suggested that the protein was localized in cytoplasm.The secondary structure of CmDWF1 protein contained 37.81%of alpha helix,which was relatively high.The CmDWF1 protein contained a FAD-binding-4 domain,which suggested that the protein acted as a FAD oxidoreductase.Phylogenetic analysis showed that Cucumis melo had closer relationship with Cucumis sativus.Real-time fluorescent quantitative analysis indicated that CmDWF1 could express in roots,stems and leaves,but the highest expression level was in roots.And the expression level increased significantly in the 25 d fruit after pollination,but decreased in 35-45 d fruit.These results demonstrated that CmDWF1 might be associated with the biosynthesis of brassinosteriods and then regulated the growth and development of melon.