Identification Of Heat Shock Protein Gene And Functional Study On Cold Hardiness Of Galeruca Dauria(Coleoptera:Chrysomelidae)

Galeruca daurica（Coleoptera:Chrysomelidae,Galerucinae）,mainly feeding on Allium plants of Liliaceae,such as:Allium mongolicum,A.ramosum and A.polyrhizum,has broken out in the grasslands of Inner Mongolia in recent years,seriously endangering the local ecological balance.Insects can reduce their supercooling points by regulating metabolism to improve their cold hardiness.The cold hardiness is related to many factors,such as:the stage of development,the climate change of living environment,the exposure time of low temperature and so on.As a stress-resistant protein produced by organisms under adverse external stress,Heat Shock Protein（HSP）is often used as a molecular indicator for insects to respond to high and low temperature changes.The aimof this study was to explore the adaptive mechanism of cold hardiness of G.daurica larvae mediated by HSP.The biological function of HSP related to cold hardiness of G.daurica larvae was deeply studied.Prokaryotic expression,RNAi technique and thermocouple method were used,which provided a potential new target for scientific prevention and control of G.daurica.The main results are as follows:1.Prokaryotic expression,protein purification and molecular characteristics of Gdhsp10 and Gdhsp60 in G.dauricaProkaryotic expression vectors p ET28a-Gd Hsp10 and p ET30a-Gd Hsp60 of Gdhsp10and Gdhsp60 gene coding regions,were constructed using p ET28a（+）and p ET-30a（+）as expression vector.Recombinant proteins Gdhsp10 and Gdhsp60 were obtained by prokaryotic expression and purified by using the Ni–NTA Agarose affinity column,recombinant proteins.MALDI-TOF-MS was employed to detect the recombinant protein,the three-dimensional structure model predicting,and molecular docking were conducted.The activities of purified protein were tested in vitro,Gd Hsp60 revealed ATPase activity from-20°C to 60°C,and maximum activity was found at 40°C.Gd Hsp60 ATPase activity increased at different temperatures when Gd Hsp10 was added to the reaction system,the maximum increase in activity was found in the temperature range of 0～40℃.Heat-induced citrate synthase aggregation assay was performed to determine the chaperone activity of Gd Hsp60,the recombinant Gd Hsp60 protein exhibited significant molecular chaperone activity in vitro,and the effect was more significant when Gdhsp10 and Gdhsp60 interacted together.2.RNAi effects on Gdhsp10,Gdhsp60 of G.dauricaTo verify the functions of Gd Hsp10 and Gd Hsp60 genes on the cold hardiness in G.daurica,RNAi was conduted on the 2^nd instar larvae in G.daurica using microinjection,and q PCR was used to detect the silencing efficiency.The thermocouple method was used to determine the super-cooling points,freezing points of the 2^nd instar larvae after RNAi.The Ltemp₅₀ and Ltime₅₀ values were used to verify the effect on cold hardiness of the 2^ndinstar larvae after RNAi.The results showed the relative expression of target gene in treatment groups was lower than that in the control group after injection of ds RNA,and the tolerance to low temperature stress also decreased in different degrees,especially when ds Gdhsp10 and ds Gdhsp60 interacted together.3.RNAi effects on Gdhsp70-1 of G.dauricaTo verify the functions of Gd Hsp70-1 genes on the cold hardiness in G.daurica,and the effect of introducing ds RNA in different ways on the effect of gene silence.The ds RNA was introduced into 2^nd instar larvae in G.daurica by dietary and microinjection methods,and the silencing efficiency of target genes were tested.The thermocouple method was used to determine the super-cooling points,freezing points of the 2^nd instar larvae after RNAi.The Ltemp₅₀ and Ltime₅₀ values were used to verify the effect on cold hardiness of the 2^nd instar larvae after RNAi.The results showed that the genes could be knocked down using both dietary and microinjection methods,but the interference efficiency was more higher using microinjection.Moreover,after 2^nd instar larvae was injected with ds Gdhsp70-1,the developmental duration prolonged and the tolerance to low temperature stress decreased.4.Molecular cloning,sequence analysis of Gdhsp70-2,Gdhsp70-3 and the promoter cloningTaking Gd Hsp70-1 as the template of RT-PCR and RACE-PCR,a 130 bp 5’UTR and a 683 bp unknown sequence were obtained by amplification.The gene was identified as the splice isomer of Gd Hsp70-1,named as Gd Hsp70-2（Gen Bank accession number:MZ853083）.The full-length Gd Hsp70-2 c DNA was 2,410 bp,with a 1,974 bp ORF,encoding a protein of 657 amino acids with a calculated molecular weight of 72.90 k D and an isoelectric point of 5.07.The sequence analysis revealed Gd Hsp70-2 has three Hsp70family signature sequences and the predicted amino acid sequence of Gd Hsp70-3 exhibited high similarity to the s HSP from Leptinotarsa decemlineata hsp70（Gen Bank accession number XP＿023015278.1）.The genomic DNA cloning results displayed that Gd Hsp70-2has two introns.Expression profile analysis showed that the expression levels of Gd Hsp70-2 in 2^{n d} instar larvae and pupae were significantly higher than that in other developmental stages,and the relative expression level in wings was the highest in adults.The Hsp70 gene was cloned from G.daurica by RT-PCR and RACE-PCR,named as Gd Hsp70-3（Gen Bank accession number:OK585088）.The full-length Gd Hsp70-3 c DNA was 2,242 bp,with a 131 bp 5’UTR,a 170 bp 3’UTR and a 1,941 bp ORF,encoding a protein of 646 amino acids with a calculated molecular weight of 71.11 k D and an isoelectric point of 5.59.The sequence analysis revealed Gd Hsp70-3 has three Hsp70family signature sequences and the predicted amino acid sequence of Gd Hsp70-3 exhibited high similarity to the HSP from Diabrotica virgifera virgifera hsp70（Gen Bank accession number:XP＿028129506.1）.The genomic DNA cloning results indicated that there was no intron in this gene,and 1 HSF,Nub and 2 Sna binding sites were present in the promoter region.Expression profile analysis showed that the expression levels of Gd Hsp70-3 in pupa and female adults were significantly higher than that in other developmental stages,and the expression level in foot in adults was significantly higher than that in other parts.