青花菜E3泛素连接酶基因的克隆及表达分析

Cloning and Expression Analysis of E3 Ubiquitin Ligase Gene in Broccoli

为探讨泛素蛋白酶体在高温胁迫下参与蛋白质降解途径相关的生理过程和功能,以青花菜‘KUQ19⁃27’为试验材料进行高温胁迫(38℃)处理,设计特异性引物从青花菜中克隆得到青花菜E3泛素连接酶基因并进行生物学信息分析,并运用实时荧光定量PCR技术分析其表达特征.结果显示:E3泛素连接酶基因的ORF长度为1332 bp,编码443个氨基酸,相对分子量为48.3 kD.亲疏水性分析结果表明:在多肽链中第207位氨基酸时,亲水性最高,为3.38;在肽链中第280位氨基酸时,疏水性最高,为-4.23,是疏水性蛋白.在青花菜E3泛素连接酶基因氨基酸序列中,其C、Y、S值均小于0.5,由此预测其不具有信号肽,不属于分泌蛋白.系统进化分析表明青花菜E3泛素连接酶基因与其他物种,比如拟南芥、草莓、月季、葡萄等苷核酸序列相似性都在60%以上,同甘蓝、油菜、白菜和拟南芥的进化关系较近,同草莓、月季、菜豆、葡萄、刺菜蓟、芝麻和水桔梗的进化关系较远,与其他物种,如拟南芥、草莓、月季、葡萄等核苷酸序列相似性都在60%以上.荧光定量PCR技术结果表明高温处理3 h时,青花菜E3泛素连接酶基因表达量下调30%,胁迫12 h时表达量持续下降仅为对照的20%.研究结果可为进一步深入探讨青花菜E3泛素连接酶的高温胁迫响应机制提供一定的理论依据.

To explore the physiological processes and functions of ubiquitin proteasome involved in protein degradation pathway under high temperature stress.The broccoli‘KUQ19⁃27’was treated with high temperature stress(38 C).And the specific primers were designed to clone the E3 ubiquitin ligase gene from broccoli and biological information was analyzed.The expression characteristics were analyzed by real⁃time fluorescence quantitative PCR.The results showed that the ORF length of E3 ubiquitin ligase gene was 1332 bp,encoding 443 amino acids,and the relative molecular weight was 48.3 kD.The results of hydrophilicity and hydrophobicity analysis showed that the hydrophilicity was the highest at the 207th amino acid in the polypeptide chain,which was 3.38.When the 280th amino acid in the peptide chain,the hydrophobicity is the highest,-4.23,which is a hydrophobic protein.In the amino acid sequence of broccoli E3 ubiquitin ligase gene,the C,Y and S values were all less than 0.5,which predicted that it did not have a signal peptide and did not belong to a secretory protein.hylogenetic analysis showed that the E3 ubiquitin ligase gene of broccoli had more than 60%nucleotide sequence similarity with other species such as Arabidopsis thaliana,strawberry,rose,grape and so on.It had a close evolutionary relationship with cabbage,rapeseed,cabbage and Arabidopsis thaliana,and had a distant evolutionary relationship with strawberry,rose,kidney bean,grape,artichoke,sesame and water orange.The results of fluorescence quantitative PCR showed that the expression of E3 ubiquitin ligase gene in broccoli was down⁃regulated by 30%at 3h of high temperature treatment,and the expression level was only 20%of the control at 12 h of stress.In this study,E3 ubiquitin ligase gene was cloned from broccoli,and its expression level gradually decreased under high temperature stress.The results provide a theoretical basis for further exploring the high temperature stress response mechanism of E3 ubiquitin ligase in broccoli.