Cloning And Expression Of Farnesene Synthase Gene From Saraca Dives Pierre

Farnesene is a sesquiterpene secondary metabolite in the main volatile components of fruits and flowers of plants.It is widely used in cosmetics,spices and food additives.Farnesene's hydride farnesane is a new type of clean fuel that has great potential in the development of second-generation biodiesel.In the mevalonate pathway(MVA)synthesis,farnesene synthase(FPS)has the function of converting the substrate farnesyl pyrophosphate(FPP)into farnesene,which is the pathway Rate-limiting enzyme.FPS is currently found in a variety of plants,but the FPS gene of Saraca dives Pierre remains to be studied.This paper attempts to clone the full sequence of farnesene synthase gene from S.dives and perform heterologous expression and enzyme activity detection.It is expected that the farnesene synthase gene from S.dives will be obtained.The cDNA of FPS was obtained from adult leaves of S.dives by RT-PCR technique and cloned into TA vector.The conserved primers were designed and synthesized according to the conservative regions of FPS genes from other sources.The S.dives FPS gene was obtained by PCR.The sequencing analysis showed that the gene size is 1593 bp,the GC content was 38%,and the protein size was about 58.4 KDa.However,the FPS sequences of other species,such as Malus domestica,Pyrus bretschneideri and Citrus reticulata,are incomplete.The 3' and 5' ends lack partial sequences and start and stop codons.The homology with the balsam terpene synthase gene sequence was 87%.The FPS similarity with other species is relatively small,presumably due to species differences.In order to ensure that the cloned ORF structure can express the protein,after adding the start codon ATG and stop codon TAG to the sequence,the gene sequence was cloned into the prokaryotic expression vector pCold TF and the eukaryotic expression vector 605ADH1,respectively,in the Escherichia coli BL21(DE3)and Saccharomyces cerevisiae BY4742 were expressed in SDS-PAGE.The size of the target protein was consistent with the expected size.The protein reacts with the substrate FPP and is detected by GC-MS.The product is farnesane,which is a hydride of farnesene.It is speculated that the reason may be thatthe protein has not been purified,and the structure and function of the enzyme have changed due to the incomplete gene sequence.Subsequent research needs further exploration.