番茄醇酰基转移酶基因SlAAT1克隆、序列分析和原核表达

Cloning,Sequence Analysis and Prokaryotic Expression of an Alcohol Acyltransferase(AAT) Gene in Tomato(Solanum lycopersicum)

酯类物质是许多果实香气的主要成分。醇酰基转移酶(AATs)是酯类化合物合成的关键酶。本研究通过反转录PCR,从番茄的成熟果实中克隆了SlAAT1基因(GenBank登录号为JQ070977),其编码一个含有442个氨基酸残基的蛋白,含有醇酰转移酶BAHD家族的H-x-x-x-D和DFGWG保守基序。系统进化分析表明,SlAAT1与苹果MpAAT1,山字草的BEBT及烟草Hsr201等聚在同一分支,进化关系较近。SDS-PAGE电泳分析表明,转化SlAAT1基因的大肠杆菌BL21(DE3)在22℃、0.8 mmol.L-1IPTG条件下可获得大量的可溶性目标蛋白。同时,纯化的SlAAT1大肠杆菌重组蛋白的体外酶活性分析表明了SlAAT1重组蛋白具有醇酰基转移酶活性,可能参与了酯类挥发性成分的合成。

Volatile esters are important aromatic compounds accumulated in many ripe fruits. Alcohol acyhrans- ferase （AAT） plays a key role in the formation of volatile esters. In the present study, a full-length cDNA se- quence homologous with other plant AAT genes was isolated by RT-PCR from tomato ripe fruit, and named as SlAAT1 （ GenBank Accession No. JN398667 ）. Results of sequence analyses showed SlAAT1 encodes a 442-amino acid protein, which exhibits conserved features of the BAHD family of acetyl transferase, such as I：[xxxD and DFGWG motifs. Phylogenetic analysis revealed that S1AAT1 is very closely related to the apple MpAAT1, Clarki- a breweri BEBT, and tobacco Hsr201. The recombinant plasmid was transformed into E. coil BL21 （DE3） to：express the S1AAT1-His6 protein in the optimized condition （22℃, 0.8 mmol · L-1 IPTG）. The purified recombinant S1AAT1 protein was shown to have acetyhransferase activity, suggesting its role in volatile ester formation.