菠萝Ac4CL5基因的克隆及表达分析  

Cloning and expression analysis of Ac4CL5 gene in pineapple fruit

作　　者：宋康华[1] 洪克前[1] 侯晓婉 谷会[1] 姚全胜[1] SONG Kanghua;HONG Keqian;HOU Xiaowan;GU Hui;YAO Quansheng(South Subtropical Crops Research Institute,Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Hainan Province for Postharvest Physiology and Technology of Tropical Horticultural Products,Zhanjiang,Guangdong,524091,China)

机构地区：[1]中国热带农业科学院南亚热带作物研究所/海南省热带园艺产品采后生理与保鲜重点实验室/农业农村部热带果树生物学重点实验室,广东湛江524091

出　　处：《中国南方果树》2025年第2期91-98,共8页South China Fruits

基　　金：海南省自然科学基金(321QN0921);中央级公益性科研院所基本科研业务费专项(1630062023005、630062022006)资助。

摘　　要：了解Ac4CL5基因在菠萝果肉酚类物质合成中的作用及调控机制,为利用该基因遗传改良菠萝果实品质和抗逆性奠定基础。以“巴厘”菠萝果肉为材料,克隆菠萝4-香豆酸辅酶A连接酶(4-coumaric acid:coenzyme A ligase,4CL)Ac4CL5基因,并进行生物信息学分析,同时对Ac4CL5基因进行进化分析、原核表达分析和上游调控因子筛选。结果表明,Ac4CL5编码区全长1659 bp,比基因组获得序列少92 bp,推测由于品种差异导致;Ac4CL5具有4CL基因家族典型的保守结构域,亚细胞定位预测Ac4CL5定位于过氧化物酶体;菠萝Ac4CL5属于Class I家族Type I类,推测Ac4CL5参与菠萝果实木质素生物合成;利用原核表达系统成功诱导出可溶性Ac4CL5目的蛋白,对纯化蛋白的酶活性和最适催化底物分析表明,Ac4CL5的最适催化底物为香豆酸,咖啡酸次之。利用Ac4CL5基因启动子进行酵母单杂交筛库发现,溴结合域蛋白GTE9和C2H2型锌指蛋白可能是调控Ac4CL5表达的上游调控因子。Ac4CL5蛋白主要以香豆酸和咖啡酸为底物,其超量表达与菠萝黑心病发生中该类酚类物质的大量积累密切相关。Ac4CL5可能受到溴结合域蛋白GTE9和C2H2型锌指蛋白转录因子调控。To understand the role of the pineapple Ac4CL5 gene in phenolics synthesis in pineapple flesh and lay the foundation for improving the fruit quality and stress resistance by this gene,Ac4CL5 gene(4-coumaric acid:coenzyme A ligase,4CL),was cloned from Comte de Paris pineapple flesh,and bioinformatics was analyzed.The phylogenetic analysis,prokaryotic expression analysis and upstream regulatory factor screening of the Ac4CL5 gene were carried out.The results showed that the full length of coding region of Ac4CL5 was 1,659 bp,and 92 bp less than the sequence obtained from genome database,which was presumably due to differences in variety.Conserved domains of 4CL were found in Ac4CL5.Subcellular localization prediction showed that Ac4CL5 was localized in peroxisomes.Ac4CL5 belonged to type I of Class I 4CL gene family and was inferred to be involved in the lignin biosynthesis.The soluble target protein of Ac4CL5 was successfully induced through the prokaryotic expression system.Analysis of the enzyme activity of purified protein with different substrates showed that the optimal catalytic substrate of Ac4CL5 was coumaric acid,followed by caffeic acid.Using the promoter of Ac4CL5 gene for yeast single hybrid screening,it was found that the bromine binding domain protein GTE9 and C2H2 zinc finger protein may be the upstream regulatory factors regulating the Ac4CL5 expression.The Ac4CL5 protein mainly uses coumaric acid and caffeic acid as substrates.Overexpression of Ac4CL5 is closely related to the accumulation of these phenolic substances in the occurrence of pineapple black heart disease.Ac4CL5 may be regulated by the transcription factor of bromine binding domain protein GTE9 and the C2H2 zinc finger protein.

关 键 词：菠萝 4-香豆酸辅酶A连接酶(4CL) 序列 原核表达 转录调控 

分 类 号：S668.3[农业科学—果树学]