Cloning And Expression Analysis Of Squalene Synthase OeSQS From Olive

Olive（Olea europaea L.）,which belongs to evergreen tree of Oleaceae,is a kind of famous woody oil crop in the world.The oil obtained by using cold-pressed method from olive fruit,which is not only rich in monounsaturated fatty acids,but also has plenty of triterpenoids,such as squalene（SQ）,oleanolic acid（OA）and Maslinic acid（MA）,is famous as the healthiest edible oil.Pharmacological studies show that the SQ,OA and MA have anti-cancer,anti-oxidant,anti-inflammatory and other medicinal activities.And,the content of SQ is one of the important indexes to judge the quality of olive oil.The squalene synthase（SQS）is a key enzyme and plays an important pivotal role in plant triterpene synthetic pathway,and its expression level has an important influence on triterpene metabolic flow in plant..Consequently,the research of cloning and expression of SQS has become a hotspot in plant triterpene biosynthesis in the molecular biology level.Nowadays,the cloning and expression of SQS in Panax ginseng,Panax notoginseng,Withania Somnifera,etc have been studied,but the study of olive SQS has not been reported in literature.In order to identify the structure,function and expression characteristic of olive SQS,two olive SQS genes were cloned by using RT-PCR from olive fruit and analyzed by using bioinformatics.Besides,the prokaryotic expression of olive SQS and the function identication of recombinant protein were carried out to achieve the verification of its biological function.What’s more,in order to reveal the effect of the gene on squalene in the fruit development stages of olive,the correlation between the squalene content and the expression of the gene in olive fruit development stages was analyzed.The main findings are as follows:（1）The cDNA of olive fruit generated by RNA reverse transcriptional was used as the template.The two SQS genes were named as OeSQS1 and OeSQS2,respectively.The bioinformatics analysis showed that the open reading frame（ORF）of the two genes were1245 bp and 1236 bp,which encoding 414 and 411 amino acids,respectively.The calculated molecular weights of OeSQS1/2 were 47.70 k Da and 46.99 k Da,besides,the isoelectric point were predicted as 8.2 and 7.93,respectively.Homologous sequence alignment showed that there are aspartic acid active sites binding to Mg²⁺in the conserved II and IV regions of OeSQS1/2 protein and SQS in other plants.It is speculated that OeSQS1/2 are membrane-bound proteins and have no signal peptide,and the OeSQS1/2 transmembrane domain is concentrated in the VI region.The catalytically active sites of the two proteins are located in the central hydrophobic space shaped by a single domain that formed byα-helical folding.Phylogenetic analysis showed that OeSQS1/2 were clustered in the angiosperm dicotyledon class,which is in accordance with the traditional botany classification.（2）pET30b（+）–OeSQS1,pET30b（+）–OeSQS2 and mutant pET30b（+）-△OeSQS1,p ET30b（+）-△OeSQS2 were constructed and successfully transformed in BL21（DE3）.The results of Western blot proved that the recombinant proteins were successfully expressed.SDS-PAGE analyses demonstrated that soluble proteins could not be obtained in the recombinant strains containing p ET30b（+）-OeSQS1 and p ET30b（+）-OeSQS2 after optimized conditions of low temperature and low concentration of IPTG,while the recombinant strains containing p ET30b（+）-ΔOeSQS1 and p ET30b（+）-ΔOeSQS2 were successfully purified at 16°C with the 1.0 mmol/L IPTG.The results of gas chromatography detection of the enzymatic reaction products showed that the two recombinases lacking the transmembrane domain still have the function of catalyzing the synthesis of squalene,and NADPH is essential in the catalytic process.（3）The correlations between the expression of OeSQS1/2 genes and the squalene content in olive fruit of two cultivars（Koroneiki and Coratina）in five stages（40～160days after flowering）were analyzed by using bivariate correlation statistics.The results showed that there were strong positive correlations between the expression of the two genes and squalene content（P<0.05）.