Biochemical Characteristics And Functional Analysis Of Chitin Deacetylase HcCDAs From Hyphantria Cunea Larvae

The fall webworm,Hyphantria cunea?Drury??Lepidoptera:Arctiidae,Hyphantria?is not only an important invasive pest,but also major harmful to forest resources and agroforestry production in China.Chitin deacetylase?CDA?,the member of carbohydrate esterase family 4,is a chitin extracellular modification enzyme involved in the metabolism of chitin in insects.In this study,the H.cunea was used as a model insect for biochemical characteristics and physiological functions of chitin deacetylases,which provided a theoretical basis for new insecticide targets.The main results are as follows:1.The hccda1?KF975504?,hccda2a?KT781841?,hccda2b?KT781842?and hccda4?KX822766?genes were identified and cloned by using RACE-PCR method combing with transcriptome database from H.cunea midgut.The open reading frames were 1632 bp,1629 bp,1611 bp and 1494 bp,respectively.The deduced HcCDA1,HcCDA2a,HcCDA2b and HcCDA4 proteins were synthesized as preproteins of 543,542,536 and497 amino acids.After removal of the signal peptide,the secreted HcCDAs were predicted to have molecular weight of 62 kDa,61 kDa,61 kDa and 56 kDa,respectively.The isoelectric point?pI?were 4.75,5.20,5.27 and 5.11,respectively.HcCDAs contained a signal peptide in the N-terminus,multiple N-glycosylation sites and five conserved motifs.HcCDA1?HcCDA2a and HcCDA2b containing a chitin-binding functional domain?ChBD?,a low-density lipoprotein receptor domain?LDLa?and a catalytic domain?CDA?,belonged to the Group I CDA protein.HcCDA4 containing a chitin-binding functional domain?ChBD?and a catalytic domain?CDA?,belonged to the Group III CDA protein.2.The cloned hcactin gene?KT781843?provided a reference gene for the expression analysis of hccdas gene in H.cunea.qRT-PCR results showed that hccda1,hccda2a and hccda4 genes were higher expressed in the first day and second day of 5th instar larvae,and were higher expressed in head?integument and hindgut tissues;hccda2b gene was higher expressed in the first day of 5th instar larvae,and was higher expressed in head and hindgut.Western blot results showed that,the trend of HcCDAs protein expression was consistent with hccdas genes transcription.3.HcCDA1?HcCDA2 and HcCDA4 were successfully expressed as 80 kDa?75 kDa and 70 kDa recombinant protein in insect cells?HighFive/Sf9?.Chitin binding experiments showed that the recombinant protein HcCDAs?HcCDA1?HcCDA2 and HcCDA4 protein?have chitin-binding activity in vitro.Results of catalytic activity experiments showed that HcCDAs protein have deacetylation function in vitro.The optimum reaction temperature was 50?and optimum pH was 8.0,respectively.Under the optimum reaction conditions,Mg²⁺and Ca²⁺showed inhibition on HcCDA1,HcCDA2 and HcCDA4 protein;Fe²⁺showed activation on HcCDA1,HcCDA2 and HcCDA4 protein;an increasing Zn²⁺concentration would lead to the enhancement of inhibitory effect on HcCDA1 protein,but decreased activation on HcCDA4 protein;Mn²⁺showed inhibitory effect on HcCDA2protein,but an increasing Mn²⁺concentration showed decreased activation on HcCDA4protein;Co²⁺showed activation on HcCDA1 and HcCDA2 protein,with increasing Co²⁺concentration,activation effect decreased;while,Co²⁺showed a trend of inhibition on HcCDA4 protein,with increasing Co²⁺concentration,inhibition effect increased.4.Pathogenic microorganisms had effects on the expression level of hccdas genes.Treatment of 3rd larvae with the Cry1Ab35 toxin,hccda1,hccda2a,hccda2b and hccda4genes were down-regulated significantly at the 9 h⁴8 h time point,but hccda2a and hccda2b genes were up-regulated significantly at the 24 h time point,as well as hccda1and hccda4 genes were up-regulated significantly at the 36 h time point.Treatment of 3rd larvae with the HcNPV virus,the expression levels of hccdas genes showed up-regulated first and then down-regulated significantly at 1⁷ d.5.RNAi was used to explore the physiological function of HcCDAs.The hccdas genes were specifically silenced after dsRNA-hccdas injected into the 5th instar larvae of H.cunea by qRT-PCR analyse.The larvae injected with dshccda1,dshccda2,dshccda2a and dshccda4 were all underwent abnormal in molt and the larval mortalities were 93%,80%,85%and 87%,respectively.However,the larvae injected with dshccda2b molt properly,just a larval mortality of 30%.In addition,the survivors displayed pupation abnormal and even death,while the larvae injected with dsgfp could pupate normally.Western blot and immunohistochemistry analyses showed that the HcCDAs protein signal were significantly attenuated after dsRNA-hccdas injection.Chitin content detection suggested that the silent expression of hccda1,hccda2,hccda2a and hccda4 genes would significantly decrease chitin content of H.cunea larvae except hccda2b gene.