The Gene Cloning,Expression And Biochemical Characterizat Ion Of The FAR3,A Key Enzyme Responsible For The Synthesis Of White Wax

The Chinese white wax is mainly secreted by the second instar male larvae of Ericerus pela.The white wax is a kind of natural ultra-long monoester.It is widely used in food,chemical,pharmaceutical,cosmetics industry,and precision instruments.It has been proved that the biosynthetic pathway of wax esters is conservative in plants and animals.The pathway is mainly catalyzed by two key enzymes,which includes fatty acyl-Co A reductase(FAR)and wax synthase(WS).Since there are many kinds of FAR in organisms,their physiological functions are different.It is necessary to study the protein expression and biochemical characteristics of FAR to reveal the mechanism underlying wax ester synthesis.In this study,the third generation sequencing of full-length transcriptome was used,and one far gene was identified,which has the identical sequence with the overlapped part with the previously screened far3 gene fragment,and was named far3 after compared with our previous studies..The far3 gene c DNA cloning and sequence analysis,prokaryotic expression,antibody preparation and protein expression analysis in male and female,and biochemical characteristics analysis were performed on.The results showed that the obtained FAR3 was highly expressed in the males in the second instar lavae,which was much higher than in the females.The preliminary biochemical analysis showed that the optimum temperature of the enzyme was consistent with the environment temperature during the production of white wax.The substrate is consistent with the main components of the wax.The main results are as follows:(1)The third generations sequencing of full-length transcriptome was successfully performed on the second-instar males at the early stage of wax secretion of E.pela,a total of81 far genes were obtained,and 6 far genes consistent with the wax secretion of the insect were screened.One of the far genes was selected,and was named far3.(2)The full length of far3 c DNA and ORF were obtained successfully.By sequence analysis,FAR3 has the conservative structure of FAR?C superfamily.Phylogenetic analysis showed that FAR3 was clustered together with FAR which were involved in the synthesis of wax esters in Human sapiens,Mus musculus and Drosophila melanogaster,indicating that FAR3 was likely related to the synthesis of wax.(3)The FAR3 prokaryotic expression vector p ET-30a-e GFP/Epel FAR3 was successfully constructed,and a large amount of FAR3 protein was obtained by inducing expression.Enzyme activity analysis in vitro showed that FAR3 protein expressed had no biological activity.(4)The monoclonal antibody of FAR3 was successfully prepared by using FAR3 protein induced by prokaryotes.And the expression of FAR3 in male and female was analyzed by using the monoclonal antibody,it was confirmed that FAR3 was highly expressed in male during the second larval stage and slightly expressed in female.(5)The FAR3 eukaryotic expression vector p IZ/V5-FAR3-Linker-e GFP was successfully constructed and successfully transferred into Bm N silkworm cells and successfully expressed.The cell line stably expressing FAR3 was screened.(6)FAR3 protein was obtained by expression of silkworm cells,and the biochemical characteristics of FAR3 were studied by in vitro enzymatic activity.The FAR3 protein expressed by silkworm cells was determined to have biological activity.It was confirmed that FAR3 can catalyze the reduction of C28 fatty acyl-Co A to C28 fatty alcohol.The optimum p H value of the enzyme reaction is 3.0,and the optimum reaction temperature is 35 ?,which is consistent with the characteristics of wax secretion of E.pela.This study confirmed that FAR3 was involved in the synthesis of some main component of the white wax,which provided a reference for the study of other FARs of E.pela.It is of great significance for the study of the biochemical mechanism of white wax secretion by E.pela.