Study On The Function Of Reproductive Regulation Related Genes By RNAi In Insects

Organisms rely on reproduction for propagating their kind.In the long-term evolution process,different species have unique ways of reproduction.Insects,by far are the largest group of animals in the world,mostly depending on sexual reproduction to ensure the continuation of species.Insect reproduction is regulated by a variety of factors,such as the external temperature,humidity,light cycle,nutrient intake,neurotransmitter,juvenile hormone,ecdysone,neuropeptide and pheromone.These factors influence the regulation of related genes,thus affect insect reproduction,including gametogenesis,mating behavior,fertilization behavior of oviposition and development of eggs.Using Helicoverpa armigera and Bactrocera dorsalis as models,we focus on the research of reproductive related genes at a molecular biological level.The main results are as follows:1.Cloning and expression pattern of JH biosynthesis related genes and vg in H.armigeraOne of the final product in mevalonate pathway in insect is JH.By cloning JH biosynthesis related genes and vg,full length of hmgr and vg of c DNA were obtained.The c DNA of hmgr contained 3,329 basic group with one open reading frame(ORF),encoding 837 amino acid(Gen Bank No.GU584103).While the c DNA of vg included 5,636 basic group,encoding 1,756 amino acid(Gen Bank No.JQ723600).Quantitative real-time PCR(q PCR)showed that the maximal value for the relative expression of hmgr m RNA occurred in the 4-days-old pupa,whereas the peak of the expression of vg emerged in the 5-days-old pupa.Futhermore,we cloned other genes in mevalonate pathway in H.armigera,and established expression pattern of these genes.The expression peaks existed in the acct,hmgs,mevk and fpps genes in the pupal stage of female,the expression quantity of mevppd and jhamt were very low in the pupal stage of female.2.The influence of hmgr,vg m RNA expression and oviposition under UV light irradiation on H.armigeraThe objective is to investigate the relative expression of hmgr and vg m RNA of H.armigera adults under UV light irradiation,together with the fecundity variation of UV light treated group.q PCR technique was applied to detect the expression of hmgr and vg of H.armigera for UV light treated group.We recorded the number of eggs produced by female adults after UV light irradiation.The expression of hmgr of H.armigera significantly increased after exposure to UV light from 0.5h to 5h,which was higher than that of control after UV light irradiation,while the vg expression increased 24 h later after UV light irradiation.The results also suggest that the fecundity of female adults significantly increased when UV-treated for 1h to 4h.3.Effects of HMGR inhibitors on the reproductive ability of the H.armigeraHMGR is a key enzyme in the mevalonate pathway,catalyzing the transformation of HMG-Co A to mevalonate.The end-products of mevalonate pathway in insects are JH,dolichol,heme A,ubiquinone and prenylated proteins.To explain the function of HMGR in H.armigera,we used simvastatin,inhibitor of HMGR,to treat the insects.Simvastatin could efficiently reduce fecundity of H.armigera.8μg simvastatin to each female moth inhibited the fecundity about 90%.However,Methoprene,a kind of juvenile hormone analogue,was confirmed to rescue the fecundity after simvastatin treatment.4.RNAi silencing of the hmgr inhibits oviposition in the H.armigeraThe hmgr knockdown using systemic RNAi in vivo inhibited the fecundity of the females,and therefore effectively inhibited ovipostion,and significantly reduced vg m RNA levels.Moreover,the oviposition rate of the female moths was reduced by 98% by silencing hmgr when compared with the control groups.One-pair of experiments showed that both the proportions of valid mating and fecundity were zero.Furthermore,the HMGR-silenced females failed to lay eggs(approximate 99% decrease in oviposition)in the semi-field cage performance.The study demonstrated the potential implications for developing novel pest management strategies using hmgr RNAi in the control of H.armigera and other insect pests.5.Transcriptome analysis of B.dorsalisB.dorsalis is a notorious pest in fruits and vegetables,which caused serious economic loss around the world.Based on the molecular biology technology,we obtained the transcriptome database,sequenced by Illumina Hi Seq 2000.The database included 116,011,686 Raw Reads,111,236,624 Clean Reads and 11.12 G Clean bases.111,113 spliced transcripts was achieved from the database,by which the mean length was 1,150 bp.Unigenes were annotated after aligning with sequences in Nr,NT,SWISSPOT,COG,GO,KEGG databases.Furthermore,64 genes were annotated to JAK-STAT signaling pathway,11 genes were annotated to BMP signaling pathway.6.RNAi silencing of several male reproductive related genes in the B.dorsalisBased on the data of transcriptome of B.dorsalis,the partial of c DNA sequences of lola,ods H,aly,topi,cg5017,dic,nap1,per,orb2,rac,rho,upd,magu were cloned.After RNAi of these genes,the hatching rate of the eggs laid by the females which mated with ds RNA-treated males were significantly reduced.For instance,the rac and rho knockdown oral delivery RNAi in vivo inhibited the fertility of males,that is,the hatching rate was decreased by 61 % and 57 %,respectively and the fertility was decreased by 80 % and 62 %,respectively.Some of these ds RNA would reduce the number and the length of the sperms in the spermatheca.Abnormal enlargement of the head sperms were also observed in the dsrho-treated group.In the semi-field trials,the percentage of infested oranges and the the number of larva emerging were significantly decreased.The study demonstrated the potential implications for developing novel pest management strategies using RNAi of male reproductive related genes against B.dorsalis and other insect pests.In conclusion,hmgr,rho and other key genes regulate the reproductive process of insects at a molecular level.RNAi inhibit the expression of hmgr in vivo which leads to the reduction of insect fertility.In the semi-field trials,ds RNA of rho treatments could significantly decrease the population of pests in next generation.The present studies shed light on the development of novel pest control in terms of RNAi application with regards to reproductive related genes.