Response Of Hsp23-like In Lymantria Dispar To Carbaryl Stress And Its Functional Analysis Using RNAi Technology

Gypsy moth?Lymantrla dispar?was a kind of wide distribution pest.At present,the main method of preventing and controling of gypsy moth is application of chemical insecticides.However,use of chemical pesticides is easy to cause insecticide resistance and environmental pollutions.Therefore,we need to explore new effective and friendly control strategy.With the rapid development of molecular biology,the exploration of molecular targets will contribute to find novel control ways.The ideal molecular target is the gene product which involved in vital physiological function.By interferring the expression of this typical gene,we can seriously affect the normal physiological mechanism of pests.Heat shock protein gene is this kind of genes participating in important protection mechanism.Under stress situation,small heat shock protein?smHsp?genes play an important role in response to xenobiotics.Based on the gypsy moth as the object,we vestigated the expression of LdHsp23-like under stress of carbaryl.We further determinated the effects on growth of gypsy moth after LdHsp23-like gene silence.It will help reveal the gene function of LdHsp23-like.1.Through the analysis of the gypsy moth transcriptome library,we got the full-length of LdHsp23-like gene.The reading frame?ORF?length of LdHsp23-like gene is 600 bp encoding 199 amino acids.The LdHsp23-like protein contain a-crystallin domain including 83 amino acids and multiple ?-sheet.The multiple sequence alignment and phylogenetic tree analysis showed LdHsp23-like has close relationship with Bombyx mori smHsp?XP₀04923862.1?.2.The leaf dip method was used to measure the insecticidal toxicity to 1st and 2nd instar larvae of Lymantria dispar.The results showed that 24 h-and 48 h-LC50 values of carbaryl were 59.31 mg/L and 33.62 mg/L for the 1st instar larvae and 74.04 mg/L and 31.48 mg/L for the 2nd instar larvae,respectively.3.After the larvae were treated the 2nd instar larvae with three sublethal concentrations of carbaryl?48 h-LC5,48 h-LC10,48 h-LC30?,we analyzed the specific expression of LdHsp23-like gene in 2nd instar larvae of Lymantria dispar using real-time PCR.The results showed that LdHsp23-like increased expression in the three sublethal doses of carbaryl at 6,12,24 h while significantly decreased at 48 and 72 h time points.4.After dsRNA injected into 3rd instar L.dispar larvae,the nutrition utilization was measured after dsRNA injection for eight days.The mortality and larvae weight were recorded every day.Compared with ddH20 and dsRNA GFP treatment,the ECI,RCR,AD of gypsy moth larvae treated by dsRNA LdHsp23-like were significantly descreased while RGR was increased.Compared with ddH2O treatment,the fresh weight of 3rd instar gyspy moth larvae treated by dsRNAGFP and dsRNA LdHsp23-like slowly after injection during 3 days while those of three treatments are in descreasing order of ddH2O>dsRNALdHsp23-like>dsRNAGFP.RNAi does not cause larval death and development period of L.dispar.5.After dsRNA injected into 3rd-instar L dispar larvae,we investigated the expression of LdHsp23-like using real-time PCR.The results showed that LdHsp23-like gene silence effect is remarkable at 6 h.After injected dsRNA LdHsp23-like,the expression of LdHsp23-like higher than normal control group at 1,2,4 d,while lower at 6 d.After injected dsRNAGFP,the expression of LdHsp23-like lower than normal control group at 6 h,1 d,2 d,6 d,only higher at 4 d.The trend of the expression of LdHsp23-like is down-up-down.From the above results can be seen LdHsp23-like gene can response carbaryl stress.Silencing of LdHsp23-like gene does not cause larval death,but reduce Efficiency of conversion of ingested food?ECI?and Approximate digestibility?AD?.These results will provide a theoretical basis to further explore the function mechanism of LdHsp23-like gene.