Cloning, Expression Characterization And Functional Assay Of A Fatty Acyl-coa Reductase Gene In Scale Insect,Ericerus Pela Chavannes(Hemiptera: Coccoidea)

Ericerus pela Chavannes is one of the oldest economically important insects in China,because they can produce a wax-like substance named insect white wax that can be harvested.This insect is a specific species in China,and it has been cultured for thousands of years especially in China.The insect white wax secreted only by the male E.pela has an increasingly wide utilization in many fields such as candle production,printing,medicine,food,cosmetic industries,chemical and precision machinery and so on.The insect white wax is mainly biosynsised by esterification of saturated monobasic alcohols and saturated monocarboxylic acids,the long chain fatty acids are mainly comprised of 26:0,28:0,24:0,30:0 acid,and the fatty alcohols are mainly comprised of26 Alc,28Alc,24 Alc,30Alc,for these esters,26:0-26:0 is the uppermost constituent,followed by28:0-28:0,24:0-24:0,30:0-30:0.In this study,we describe the isolation and characterization of a cDNA,this gene encodes a fatty acyl-CoA reductase?FAR?,and named Ericerus pela FAR?EpFAR?.In order to study the expression profile of this gene and its coding protein,we prepared the polyclonal antibody for EpFAR.And more,we explored the expression profile of EpFAR gene in the five tissues using the qRT-PCR method,and the distribution of EpFAR protein in the body of E.pela using immunofluorescent staining.Further more,an expression vector named pFastBac HT B-FAR was constructed,and were used to product the recombinant baculovirus.Then,the recombinant baculovirus was used to infect the insect cells and formed the target protein.Finally,we explored the function of EpFAR protein.The following,main results were showed.1.Sequence character of EpFAR and phylogenetic analysisThe full-length sequence of EpFAR was obtained by the RACE method.The result showed that the ORF of EpFAR includes 1569 base pairs,and encodes a polypeptide of 522 amino acids.the predicted theoretical molecular mass of EpFAR protein is 60.3kDa.The deduced amino acid sequence EpFAR contained a Rossmann-fold NAD?P?/NAD?P?H binding domain?NABD?,and this domain is one typical constructure feature of FAR gene superfamily,the domain of EpFAR linked with NAD_binding₄?Male sterility protein?,and the conserved motif?I/V/F?-X-?I/L/V?-TG-X-T-G-F-L-?G/A?encompassing residues 24-34 was observed in this NABD domain of EpFAR.Moreover,in the C-terminal of the EpFAR sequence,a Sterile domain was found,its residues are in387-477 amino acid.Using GenBank searches method,we found that,compared with the other FAR genes in the nonredundant protein database,this encoded EpFAR protein shared 35% sequence identity to D.melanogaster WAT and AmFAR1,respectively;and 30% sequence identity to Y.rorrellus pgFAR II,29% sequence identity to OsXIII and 28% sequence identity to BmFAR.These data supported EpFAR as a member of FAR gene superfamily.Phylogenetic relationship analysis showed that the relation ship among the EpFAR and other FARs in plants,animals and microorganisms.In the result,all tested FARs clustered in the twodistinct clades except for the FARs from plant.EpFAR neighbored to Phenacoccus solenopsis FARI,and is near to the D.melanogaster WAT,and is far from the other insect FARs,we speculated that this topology of FAR tree could reflect the functional relatedness among EpFAR and other encoded FAR enzymes.The clade containing the EpFAR seemed to mainly be related to the very long chain fatty alcohol biosynthesis.Both E.pela and Phenacoccus solenopsis are the scale insects,EpFAR neighbored to Phenacoccus solenopsis FARI may be for that the two FAR genes may have the similar function.Phylogenetic data supported EpFAR as a candidate of FAR gene superfamily for wax esters biosynthesis.2.Polyclonal antibody of EpFARIn order to study the expression profile of EpFAR protein in the male E.pela,and explore the subcellular localization of EpFAR we prepared the polyclonal antibody for EpFAR.Th antigen was from artificial synthesized polypeptide which come from EpFAR sequence.The titer of producted antiserum against EpFAR was determined by ELISA method,and the final titer was 1:512000.The concentration of the polyclonal antibody were 0.7mg/ml which was determined by BCA KIT.This polyclonal antibody was successfully prepared,which laid a foundation for further study of structures and functions of EpFAR protein.3.Tissue specificity of EpFARThe expression profile of EpFAR gene was studied using the qRT-PCR method.The tested five tissues were excised from the living second instar of male E.pela at the peak of white wax secretion,included cuticle,fat body,digestive tube,testis,and Malpighian tubule.The mRNA expression of EpFAR in these tissues was quantified using the standard curve quantification method.The known plasmids containing EpFAR were prepared in order to generate the absolute standard curve.As shown in the result,the mRNA was expressed in all five tested tissues,and it was preferentially expressed in the cuticle,and fat body,Malpighian tubule,testis and digestive tube in turn,minimally in the digestive tube.The expression level in the cuticle was the most highest than it in other tissues.There was significant difference in the these tissues?P<0.05?.The distribution of EpFAR protein in the body of E.pela using immunofluorescent staining technique.One whole insect body of living male E.pela which were secreting wax in peak level was selected and embedded in the tissue freezing medium to slice.EpFAR protein combined with anti-antibody was observed using Laser Scanning Confocal Microscopy,suggested that EpFAR protein localized in the testis and wax gland,but not in digestive tube,fat body and Malpighian tubule.The distribution profile of EpFAR protein was inconsistent with the distribution profile of mRNAof EpFAR gene.4.Expression of EpFAR gene in insect cells and its functionThe recombinant donor plasmid named pFastBac HT-FAR was constructed by PCR method and restriction enzyme digestion,and the recombinant vector was transformed into competent DH10 bac E.coli competent cells and the recombinant bacmid plasmid DNA was obtained.The insect cell Sf9 was transfected and the recombinant nuclear polyhidrosis baculovirus was obtained.The results of SDS-PAGE and immunofluorescent staining suggested that EpFAR gene had been expressed in insect cells.The EpFAR enzyme activity assay was performed in a enzyme reaction system in vitro,and the production in the reaction system was detected using GC-MS.The extracted protein from the insect cells was incubated in the presence of Hexacosanoyl Coenzyme A?26:0-CoA?.GC-MS analysis indicated the retention time and mass spectra of novel peaks of the products matched those of TMS derivative of 26:0-OH standards,showed that hexacosanol was produced,but no corresponding alcohol products were observed in the controls,indicating that EpFAR has reductase activity.To further characterize the enzymatic properties of EpFAR,series assay were performed in a cell-free system using 26:0 fatty acyl-CoA as a substrate,and one reaction condition was changed in per assay and under the anterior optimized condition,and the protein was extracted from the infected cells.The result showed that the TMS derivative of 26:0-OH product was detected when NADPH was included,whether or not was NADH,indicating that EpFAR required specifically NADPH as a cofactor.buffer systems with different pH value were used to evaluated the pH optimum,and the result showed that TMS derivative of 26:0-OH product was not detected in pH6.4 system,indicated slant acidity might not be adapt to the reducibility of EpFAR.The temperature properties of the enzyme were determined from a range of 25?to 40? with 5? interval,almost unanimously,every assay produced detectable amounts of TMS derivative of fatty alcohols,suggested the diversity of temperature is not an obvious factor for the enzyme activity.To examine the substrate perference of EpFAR,three fatty-acyl COA were supplied in the reaction system,including 22:0-CoA,24:0-CoA and 26:0-CoA.The result showed that the peaks of derivation production corresponding to 26:0-OH was identified,24:0-OH and 22:0-OH are either absent or below the detection limit.