Functionalanalysis Of LeHDAC4 In The Wing Dimorphism Of Lipaphis Erysimi (Kaltenbach) And Its Potential Application

In recent years,the molecular mechanism of insect wing dimorphism has always been one of the hot research areas in entomology.Previous studies in our laboratory found that the histone acetylation level of the wingless aphid was higher than the winged aphid at the end of third age,and silencing histone acetyltransferase gene Le KAT2 B significantly increased the proportion of winged aphid.Therefore,histone acetylation plays an important role in the wing dimorphism of L.erysimi.In order to further explore the role of histone acetylation in the wing dimorphism of L.erysimi,we focused on the function of LeHDAC4 in the wing dimorphism of L.erysimi.The main experimental results are as follows:1.The total length of LeHDAC4 sequence was 4333 bp,and the Open Reading Frame(ORF)was 3171 bp.The gene encodes 1056 amino acids,and a typical Hist-deacetyl functional domain exists in amino acids at position 646-963.The phylogenetic tree showed that this amino acid sequence had the highest homology with the amino acid sequence of HDAC4 gene of Diuraphis Noxia,followed by Myzus persicae and Acyrthosiphon pisum.The total length of Le Ubx sequence was 1922 bp,and the ORF sequence was 1026 bp.The gene encodes 331 amino acids,and a typical HOX functional domain exists in amino acids at position 242-304.The phylogenetic tree showed that the amino acid sequence was closely homologous to Ubx of A.pisum,M.persicae and Rhopalosiphum maidis.2.The expression difference of LeHDAC4 between winged and wingless aphids was analyzed by q RT-PCR.The results showed that LeHDAC4 gene was expressed in all instars of winged and wingless aphid under two induced conditions.There was no significant difference in LeHDAC4 gene expression between the 2nd,3rd and 4th aged aphid induced by winged aphid and the 2nd,3rd and 4th aged aphid induced by wingless aphid.3.The expression of LeHDAC4 gene in L.erysimi was interfered by feeding method.The results showed that feeding ds LeHDAC4-1 and ds LeHDAC4-2 reduced the expression level of LeHDAC4 by 39% and 22%,respectively.After feeding ds GFP,the proportion of winged aphid in the control group was 53%,and that in the treatment group was reduced to 35.8% and 39.6% of the control group,respectively.The proportion of wing is significantly different.After interfering with the expression of LeHDAC4,detected the expression level of wing development genes in L.erysimi — Ubx(Ultrabithorax),Ap(Apterous),Sal(Spalt major)—and the expression level of insulin pathway genes — In R1(Insulin-like receptor 1),In R2(Insulin-like peptide receptor 2),ILP3(Insulin-like peptide 3),ILP5(Insulin-like peptide 5)— by q RT-PCR.The results showed that the expression level of Ubx was significantly up-regulated,the expression level of Ap and Sal was significantly down-regulated,the expression of ILP3 and ILP5 was down-regulated,and the expression of In R2 was up-regulated by more than double.These results showed that LeHDAC4 may regulate aphid wing dimorphism of through two pathways: direct regulation of Le Ubx expression,thereby regulating wing dimorphism;regulation of insulin pathway activity,thereby regulating the expression of Le Ubx,and finally affect the wing dimorphism of L.erysimi.4.Set up the control group ds GFP group,and the treatment group ds LeHDAC4+ds Le GFP group,the ds Le Ubx+ds GFP group and the ds LeHDAC4+ds Le Ubx group,respectively.Interfered with the gene expression by RNAi technology.The results show that there may be a reverse regulation relationship between LeHDAC4 and Le Ubx,that is,the increase or decrease of the expression of one of them will cause the opposite change of the expression of the other one.Observing the results of wing type differentiation,the proportion of winged aphids in the control group was 53%.The results of wing dimorphism showed that the proportion of winged aphid in the control group was 53%.After silencing LeHDAC4,the proportion of winged aphid in the treatment group was reduced to 43.4% of the control group.After silencing the Le Ubx,the proportion of winged aphids was 55%,which was not significantly different from that of the control group,but there is an upward trend.After feeding ds RNA with two genes simultaneously,the proportion of winged aphid recovered to 47%,suggesting that LeHDAC4 may pitching-in wing developmental genes network Le Ubx,thus regulating the wing dimorphism of L.erysimi.5.Interference of LeHDAC4 and Le Ubx gene expression caused lethal effect on the L.erysimi.After feeding ds RNA,the mortality rate of the interference group was the highest in the first four days of feeding ds RNA,and gradually decreased after the fourth day,the results of survival curve analysis were consistent.In addition,the mortality rate of the test insects for feeding ds Le Ubx separately and feeding two kinds of ds RNA simultaneously was reduced to 41.8% and 32.9% of the control group.The lethal effect was better and could be used as a potential target for L.erysimi control.