Effect Of Heliothis Virescens Ascovirus 3h On Chitinase Activity And Expression In Spodoptera Exigua

Chitinase is an important participator and regulator in insect molting process.The normal metabolism of chitinase is crucial for insect growth and development.Ascoviruses,as a class of insect viruses with great biological control potential,were reported to prolong the host larval stage,inhibit the host larval molting,and fail the hosts' pupating.All these results indicated that the metabolism of host larval chitinase might be severely disrupted by the infected with ascoviruses.In order to detect the changes of chitinase transcription after Heliothis virescens ascovirus 3h(HvAV-3h)infection,the differentially expressed chintinase genes of Spodoptera exigua after infected with HvAV-3h were selected from the transcriptome data.Then,through analyzing the activity,transcriptional and expressional patterns of chitinase in infected larvae and mock-infected larvae,thereby exploring the regulation of HvAV-3h on S.exigua chitinase.The main results of this study are as follows:1.Cloning and phylogenetic analysis of S.exigua chitinase genes: Totally five S.exigua chitinase genes(SeCHIT)and one S.exigua chitin-binding domain gene(SeCBD)of HvAV-3h induced significant differential expressioncan be screened from the transcriptome data and identified by Reverse Transcription-Polymerase Chain Reaction(RT-PCR).Molecular characteristics analysis and phylogenetic analysis of the five SeCHITs(SeCHIT7,SeCHIT11,SeCHIT12,SeCHIT13 and SeCHIT14)showed that SeCHIT7 was belong to Groups III chitinase,while SeCHIT11,SeCHIT12,SeCHIT13 and SeCHIT14 were belong to Groups VIII chitinase.2.Prokaryotic expression of SeCHITs and preparation of its polyclonal antibody: The SeCHIT7N(the N-terminal of SeCHIT7),SeCHIT11,SeCHIT12 and SeCBD were successfully expressed in Escherichia coli.Chitinase activity were detected from three expressed SeCHITs with an order as SeCHIT7 N > SeCHIT11 > SeCHIT12,while SeCBD had no chitinase activity,but it can increase the activity of SeCHITs.Polyclonal antibodies of SeCHIT7 N,SeCHIT11 and SeCBD were prepared.3.Effect of Hv AV-3h on chitinase activity: the chitinase activity in different developmental stages and tissues of healthy S.exigua was detected.The results showed that the chitinase activity was increased 9.4 times from egg to 1st-instar larvae.The chitinase activity of 4th-and 5th-instar larvae was higher than that in 1st,2nd and 3rd-instar larvae.The chitinase activity of pupae was the highest(113.77 Unit).The order of chitinase activity in four tissues was fat body > cuticle > midgut > hemolymph.Then the chitinase activity in different tissues and developmental stages between infected and mock-infected larvae was compared.The results indicated that after HvAV-3h infection,the infected larval chitinase activity was 13.20%,6.37%,5.63%,10.49%,11.79% and 7.94% that of mock-infected at 48,72,96,120,144 and 168 hpi,respectively.The chitinase activity of infected larvae was greatly lower than mock-infected at 168 hpi in the hemolymph,fat body,midgut and cuticle,which was reduced by 87.95%,96.96%,76.84% and 95.86% at 168 hpi,respectively.4.Effect of HvAV-3h on transcriptional levels of SeCHITs: The transcription levels of three SeCHITs in different developmental stages and tissues of healthy S.exigua were detected.The results showed that SeCHIT7 was the most abundant transcribed in 5th-instar larvae.SeCHIT11 and SeCHIT12 showed similar transcriptional patterns in larval stage.The transcriptional levels from 1st-to 3rd-instar larvae gradually increased,but finally decreased.SeCHIT11 had the highest transcriptional level in eggs,while SeCHIT12 had the highest transcriptional level in pupae.Three SeCHITs were transcribed in all four tissues,mainly in the midgut,then in the fat body.The transcriptional levels of three SeCHITs in different developmental stages and tissues between infected and mock-infected larvae were compared.The results showed that HvAV-3h reduced the transcriptional levels of SeCHIT7 decreased dramatically at 6,9,12,48,72 and 96 hpi,while the transcriptional levels of SeCHIT11 and SeCHIT12 decreased markedly at 3-96 hpi.The transcriptional levels of three SeCHITs increased significantly at 120-168 hpi,respectively.SeCHIT7 was decreased by 99.94% at 96 hpi and was more than 1300 times higher at 120 hpi compared with mock-infected;SeCHIT11 was decreased by 99.98% at 6 hpi and increased by 25.15-fold at 168 hpi compared with the mock-infected;SeCHIT12 was completely inhibited by HvAV-3h at 6 hpi and increased by 40.10-fold at 120 hpi compared with the mock-infected.HvAV-3h significantly affected the transcription of three SeCHITs in four different tissues,especially the transcription of SeCHIT7 in fat body and cuticle,and the transcription of SeCHIT12 in fat body were significantly inhibited.In this study,the molecular characteristics of five SeCHITs were analyized,the chitinase activity in infected and mock-infected larvae was compared,and the spatiotemporal specificity of activity and transcription of SeCHIT7,SeCHIT11 and SeCHIT12 were detected.The results obtained in this study revealed the regulation of chitinase in S.exigua larvae during the infected with HvAV-3h,which can lay a foundation for the further study on the pathogenesis of HvAV-3h.