The Sources And Molecular Mechanisms Of The Biosynthesis Of Aphid Alarm Pheromone

Aphids are among the most important and widely distributed agricultural pests all over the world.Most aphids can release alarm pheromones to warn their conspecifics of potential threat when they are attacked by natural enemies,which may be used for development of new control measures for aphids.(E)-?-farnesene(E?F),a type of sesquiterpenes,is the major component of the alarm pheromone of many aphid species.But the biosynthesis mechanism of E?F in aphids is not clear.In this study,a series of experiments were conducted to preliminarily analyze the source and candidate genes for biological synthesis of E?F in aphids.The results were as follows:1.The possible biosynthesis sources of aphid E?F were identified:host plants,endosymbiont bacteria and aphids themselves.Firstly,the Myzus persicae and Aphis gossypu were fed with pure artificial diet,and then we analyzd the tissue volatiles of the two species aphids by GC-MS and found that the diet-reared aphids could still release E?F.It excluded the possible effects of host plants on EPF biosynthesis.On the other hand,we treated aphids with antibiotics to eliminate Buchnera and found the aposymbiotic aphids were infertile but could still release E?F.This suggested that Buchnera was not the biosynthesis source of E?F,too.In conclusion,the host plants and obligate endosymbionts bacteria were not the sources for biosynthesis of E?F,indicating that the aphids probably synthesized E?F by their own genes.2.The ispA gene of Buchnera in A.gossypii was identified.Although we excluded the possible effects of endosymbionts bacteria on E?F biosynthesis,but the concentration of the E?F(ng/mg aphid)of aposymbiotic aphids was reduced significantly compared with the normal aphids feeding with plant.We hypothesized that the ispA of Buchnera may provide some precursor FPP for the synthesis of E?F.So we cloned and constructed the prokaryotic expression vector pGEX-6p-1-ispA of the ispA gene and successfully expressing and purificating of ispA.But the enzyme activity analysis experiment showed that the ispA had no activity of synthesis FPP in vitro.This further excluded the possibility that Buchnera was involved in the biosynthesis of aphids E(3F.3.We analyzed transcriptome of the A.gossypii released droplet and the aphids without any stimulation,and assembled de novo into 60,614 unigenes.Gene differential expression analysis showed that 1201 genes were up-regulated and 729 genes were down-regulated.GO enrichment analysis found that metabolic and biological processes and catalytic activity were significantly activated.KEGG enrichment analysis found that the biosynthesis of amino acids,glutathione metabolism and lysosomal pathway were significantly activated.We verified 5 key genes on aphids isoprenoid pathway by qPCR.AgFPPS gene,AgGGPPS gene,ispA and AgDPPS gene appeared different response patterns after the aphids releasing droplet within 2h and the results showed that the response patterns of AgFPPS and AgGGPPS gene were similar.And it is likely to be more closely correlated with the biosynthesis of E?F than other genes.4.In this study,we interfered with four key IPPS genes(AgFPPS,AgGGPPS,AgDPPSl and AgDPPS2)of isoprene biosynthetic pathway to study their function in biosynthesis of E?F in A.gossypii.It can elicit higher silencing effect after respectively ingesting 150 ng/?L dsAgFPPS and 250 ng/?L dsAgGGPPS in succession for 2 d,while 200 ng/?L dsAgDPPSl and dsAgDPPS2 can significantly inhibit the expression of target gene after continuous feeding of 4 d.The biological characters of aphid were basically not affected significantly after the four genes separate interference,but quantitative measurement of E?F showed that the concentration of E?F as per the weight of aphid was drastically decreased to the inhibition of FPPS gene,whereas the inhibition of GGPPS gene significantly raised the concentration of E?F,while there was no significantly difference between aphids feeding with dsDPPS and control.Our experimental results provided the first evidence in vivo that AgFPPS play crucial roles in biosynthesis of E?F.5.The prokaryotic expression and enzyme activity analysis of AgFPPS gene were carried out to further determine the relationship between AgFPPS gene and E?F synthesis.We constructed the prokaryotic expression vector pET-28a(+)-97AgFPPS of the AgFPPS gene and successfully expressing and purificating of FPPS.The enzyme activity analysis showed that the recombinant protein had double function of GPPS and FPPS,but we didn't detect the product of E?F.It proved that AgFPPS didn't have the function of the E?F synthetase under certain conditions in vitro.This study provided the first molecular evidence for the close relationship between FPPS and GGPPS genes with the biosynthesis of aphid E?F.This study had laid a solid foundation for the study of the biosynthesis and regulation mechanism of aphid E?F,and also provided new ideas for the sustainable control of aphids.