‘红象牙’果实MiHPL基因的表达分析及酶活性鉴定

Expression Analysis and Enzyme Activity Identification of MiHPL Gene in‘Hong Xiangya’Mango Fruit

挥发性醛类是水果挥发物质之一,脂氢过氧化物裂解酶(hydroperoxide lyase,HPL)是植物体内的脂肪酸代谢途径中醛类挥发物质合成下游的关键酶。为探索杧果中挥发性醛的种类及其合成途径进而挖掘相关基因,本研究以‘红象牙’杧果果肉作为实验材料,通过气相色谱质谱联用技术(GC-MS)分析了果实中醛类挥发物,利用同源克隆技术从果实中克隆HPL基因,在原核表达系统中诱导并纯化该酶蛋白,添加前体物质进行酶促反应。结果表明,‘红象牙’果肉中醛的种类和含量较少;从果肉中克隆出一个HPL基因,命名为MiHPL,长1485 bp,编码494个氨基酸,理化性质分析发现Mi HPL属于亲水性蛋白、不稳定蛋白,无跨膜结构,蛋白质分子质量约为54.85 kD;对转录组数据的PFKM值分析显示果实后熟过程表达量下调。生物信息学、系统进化树及原核表达结果表明杧果MiHPL基因可能是属于CYP74B亚族;酶活性鉴定实验表明,该酶具有催化底物13(S)-过氧羟基-(9Z,11E,15Z)-十八碳三烯酸(13-HPOD)、13(S)-氢过氧基-(9Z,11E,15Z)-十八三烯酸(13-HPOT)中第13位的过氧基断裂产生己醛、反-2-己烯醛的活性,属于13-HPL。实验结果为今后进行杧果挥发醛的研究提供参考。

Volatile aldehydes are one of the volatile substances in fruits.Lipid hydroperoxide lyase(HPL),is the key enzyme located in downstream biosynthetic pathway of plant fatty acids metabolism responsible for aldehyde volatile substances producing.In order to explore the species and synthetic pathway of volatile aldehydes in mangoes and mining related genes,the aldehyde volatiles in the fruit of‘Hong Xiangya’mango were analyzed by gas chromatography-mass spectrometry(GC-MS),the HPL gene was cloned from the fruit by homologous cloning technology,and its protein was induced and purified in the prokaryotic expression system.Further,by feeding precursors the enzymatic activity was identified.The results showed that there were less kinds and contents of aldehydes in the pulp of‘Hong Xiangya’.An HPL gene named MiHPL,1485 bp in length,encoding 494 amino acids,was cloned from the pulp.The analysis of physical and chemical properties revealed that MiHPL belonged to hydrophilic and unstable protein,without transmembrane structure,with molecular weight about 54.85 kD.Transcriptomic data demonstrated that the expression was down regulated during fruit ripening.The results of bioinformatics,phylogenetic tree and prokaryotic expression indicate that the Mi HPL gene of mango may belong to the CYP74B subfamily.And it can catalytically break of 13thperoxy group in the substrate 13(S)-peroxyhydroxy-(9Z,11E,15Z)-octadecatrienic acid(13-HPOD),or 13(S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienic acid(13-HPOT)to produce hexanal and trans-2-hexenal,belong to 13-HPL.The results will provide reference for the study of volatile aldehyde in mango fruits in the future.