| Lymantria dispar is a worldwide pest that has a serious hazard to forests.At present,the prevention and control of Lymantria dispar still depended on chemical control,and the resulting "3R" problem could not be ignored.Therefore,it is important to find new molecular targets to develop new insecticides to descrease environmental pollution.Some researchers have reported that Mth receptors are significantly more resistant to a range of external stress factors including hunger,high temperature and oxidative stresses.Insect Mth receptor structure and function,signal transduction pathway research provide a new way for us to reveal the mechanism of biological aging.The study of endogenous ligands will provide new ideas for us to study the functions of Mth receptors and the development of new pesticide targets.In this study,Asian Lymantria dispar was used to study endogenous ligand peptide gene,Sex Peptide namly LdSP,of Methuselah-like receptor and its bioinformatics analysis for LdSP gene.Effects of LdSP gene on growth,metabolism and stress resistance of insects were investigated by RNAi and transgenic Drosophilatechniques.The results are as follows.1.Endogenous ligand gene sequence of a Methuselah-like receptor was from the transcriptome library of L.dispar,and the full length of LdSP gene was cloned.The open reading frame(ORF)of the LdSP gene is 1263 bp encoding 420 amino acids,the molecular weight of the protein is 48.79 kDa,the pI is 8.79 indicating the alkaline protein without the signal peptide.The LdSP protein is a member of the GPCR B family with 7-transmembrane receptor structure(7-TM).2.The expression of LdSP gene in egg was higher than those of other the developmental stages(except 4,6 and pupae),and the expression of LdSP gene was different between larval stages.The expression level of LdSP gene reached the peak at sixth instar larvae,and the expression level of the LdSP gene in the pupal stage was only 8.80%of that in sixth instar larvae.The expression of LdSP gene in female adult was 5.41 times higher than that of the male adult.These results showed that LdSP gene maybe involved in the aging regulation,pupation and female oviposition function of L.dispar.3.The silencing efficiency of 48.10%for dsRNALdSP at 120 h was the most significant and 240.5-fold of the control group(dsRNAGFP).At all time points,the cumulative growth rate of fresh weight of LdSP gene was lower than that of control group(DEPC and dsRNAGFP),and the relative growth rate,food utilization rate and food conversion rate were slightly lower than those of control group.However,relative consumption rate and approximate digestibility were higher than those of control group suggesting that LdSP gene affects the growth and metabolic regulation of L.dispar larvae.Under high temperature stress,the mortality rate of LdSP gene silencing group was higher than that of control group,the average death time was 104.80 h and is 71.62%of DEPC and 80.62%of dsRNAGFP,respectively(P<0.05).Under the starvation stress,the mortality rate of LdSP gene silencing group was higher than that of the control group at each time point.The average death time of silencing group was 120.67 h and is 89.65%of DEPC treatment and 93.11%of dsRNAGFP treatment,respectively.The LC10,LC30 and LC50 of the LdSP gene silencing group were significantly lower than those of the control group(P<0.05).The mortality of the LdSP gene silencing group was significantly lower than that of the control group(P<0.05).After LdSP gene silencing,the high temperature,starvation and oxidative stress resistance of the L.dispar larvae were reduced indicating that the LdSP gene could enhance the stress resistance.4.The recombinant transgenic plasmid pUAST-LdSP was successfully constructed in this study.The recombinant plasmid pUAST-LdSP was injected into Drosophila embryos to obtain transgenic Drosophila with LdSP gene by microinjection technique.The average life expectancy,half death days and the maximum life-span of transgenic Drosophila expressing LdSP gene were significantly higher than those of the control groups(W1118 and DB),which were 45.55%and 58.72%,22.89%and 40.85%,13.79%and 32.00%respectively(P<0.05).Under 36℃ of the high temperature stress,the survival rate of the transgenic LdSP gene was higher than that of W1118 and DB strains.The average death days was close to that of W1118 and was 1.19 times higher than that of DB.The average death days of LdSP gene was 48.59 h,which was 1.12 and 1.27 times of W1118 and DB strains,respectively.Under the paraquat oxidative stress,the LC10,LC30 and LC50 of transgenic Drosophila expressing LdSP gene were higher than that of W1118 and DB.The mortality rate of transgenic Drosophila expressing LdSP gene was lower than that of W1118 and DB with 54.21%and 65.22%,respectively.The LdSP may be involved in regulating Drosophila longevity,growth and development and resistance.These results indicated that the LdSP gene of Methuselah-like receptor is involved in the regulation of insect growth,metabolism,longevity and stress resistance.The results enriched the functions of insect SP gene and will help us to recognize other insect GPCR B family members and their associated agonists.Meanwhile,it will provide theoretical basis and new ways to explore new pesticide targets to control pests. |
