| Mitogen-activated protein kinase(MAPK)signaling pathway is one of the important signal transduction systems in organisms,which is involved in many physiological and pathological processes such as cell growth,development,division and differentiation.In the study,two important members of Helicoverpa armigera mitogen-activated protein kinase family,c-Jun amino-terminal kinase gene(HaJNK)and p38 mitogen-activated protein kinase gene(Hap38 MAPK),were cloned.And their sequences and expression patterns were analyzed to explore the role of this gene in the growth and development of H.armigera.RT-qPCR and RNAi were used to investigate the role of these two genes in the response of H.armigera to UV stress.The results are as belows:1.Cloning and expression profile analysis of HaJNK from H.armigeraIn this study,a JNK gene was cloned from H.armigera,and named HaJNK(GenBank accession no.: MH719009),which is 2 431 bp in length and contains an open reading frame(ORF)of 1 191 bp,encoding 396 amino acid residues.The putative protein is 45.01 kD with an isoelectric point(pI)of 6.35,and has no transmembrane region and signal peptide.The phylogenic tree indicated that HaJNK shares a high homology with JNK from other insect species.Developmental stage-specific mRNA expression profiling showed that HaJNK were expressed in eggs,1-6 instar larvae,pupae,female adults and male adults.HaJNK was had the highest expression levels in the egg stage.Tissue-specific mRNA expression profiling showed that HaJNK was expressed in head(without antenna and compound eye),chest,abdomen,antennae,compound eye,foot,wing,midgut and ovary of adult.HaJNK was specifically expressed in compound eye,thorax and ovary.The expression level of HaJNK varies at different developmental stages and adult tissues of H.armigera,and it showed that the theoretical basis of HaJNK on the physiological function of H.armigera in response to environmental stress was laid.2.The role of HaJNK in response to UV-A stressIn order to study the role of HaJNK gene in response to UV stress,the expression of HaJNK gene in H.armigera under different UV-A irradiation time was analyzed by real-time PCR.The enzyme activity of SOD,POD,CAT and GR under the UV-A stress of H.armigera was detected by silencing the HaJNK gene by RNAi.The results showed that: The mRNA expression of HaJNK in female adults was induced by UV-A stress,increasing firstly and then decreasing with the increase of the exposure time,and reached the highest expression level at 60 min post exposure to UV-A.SOD activity of adults injected with dsHaJNK solution was significantly lower than that of the control group at 60 min post exposure to UV-A.POD activity of the adults injected with dsHaJNK solution was significantly lower than that of the control group at 30 min post exposure to UV-A.At 60 min,90 min and 120 min post exposure to UV-A,CAT activity of the insects injected with dsHaJNK solution was significantly lower than that of the control group.At 30 min,60 min and 90 min post exposure to UV-A,GR activity of H.armigera injected with dsHaJNK solution was significantly lower than that of the control group.Short-term UV-A radiation can induce the up-regulation of HaJNK expression in female adults of H.armigera,activate JNK signaling pathway,and increase cell survival under DNA damage in response to UV-A stress.When HaJNK is silenced,the JNK signal transduction pathway of H.armigera is blocked.When the H.armigera is subjected to UV-A stress,the defense function of the H.armigera is destroyed,which causes irreversible damage to the insect body.This study preliminarily elucidated the role of HaJNK in response to UV-A stress in H.armigera.This study is important for exploring the signal transduction pathway of insects responding to UV stress.3.Cloning and expression profile analysis of Hap38 MAPK from H.armigeraIn this study,a p38 MAPK gene was cloned from H.armigera,and named Hap38 MAPK(GenBank accession no.MK185422),which is 1 772 bp in length and contains an open reading frame(ORF)of 1 080 bp,encoding 360 amino acid residues.The putative protein is 41.51 kD with an isoelectric point(pI)of 5.93,and has no transmemhrane region and signal peptide.The phylogenic tree indicated that Hap38 MAPK shared a high homology with Hap38 MAPK from other insect species.Tissue specific mRNA expression profiling showed that Hap38 MAPK was expressed in head(without antenna and compound eye),chest,abdomen,antennae,compound eye,foot,wing,midgut and ovary of adult.Hap38 MAPK was specifically expressed in the adult thorax.Developmental stage specific mRNA expression profiling showed that Hap38 MAPK was expressed in eggs,1-6 instar larvae,pupae,female adults and male adults.Hap38 MAPK was had the highest expression levels in the female adult.The differential expression of Hap38 MAPK in different developmental stages and adult tissues of H.armigera laid a theoretical foundation for further study of the functional mechanism of Hap38 MAPK.4.The role of Hap38 MAPK in response to UV-A stressIn order to study the role of Hap38 MAPK gene in response to UV stress,the expression of Hap38 MAPK gene in H.armigera under different UV-A irradiation time was analyzed by real-time PCR.The enzyme activity of SOD,POD,CAT and GR under the UV-A stress of H.armigera was detected by silencing the Hap38 MAPK gene by RNAi.The results showed that: The UV-A stress could induce the mRNA expression of Hap38 MAPK in adults.The expression of Hap38 MAPK gene increased firstly and then decreased with the increase of the treatment time,and was the highest expression level at 30 min.SOD activity and the CAT activity of the adults injected with dsHap38 MAPK solution was significantly lower than that of the control group at 60 min,90 min and 120 min.POD activity of the insects injected with dsHap38 MAPK solution was significantly lower than that of the control group at 30 min and 60 min.At 60 min,90 min and 120 min,the CAT activity of insects injected with dsHap38 MAPK solution was significantly lower than that of the control group.At 30 min,60 min and 90 min,GR activity of the insects injected with dsHap38 MAPK solution was significantly lower than that of the control group.Short-term UV-A radiation can induce adult H.armigera to activate p38 MAPK signaling pathway.Thereby inducing cell growth and proliferation,and improving stress adaptability of H.armigera to respond to UV-A stress.When Hap38 MAPK was silenced,the interaction between p38 MAPK and other signaling pathways led to cell apoptosis,which eventually broke the defense function of the body and caused irreversible damage to the body.This study preliminarily elucidated the role of Hap38 MAPK in response to UV-A stress in H.armigera.This study is important for exploring the signal transduction pathway of insects responding to UV stress.Short-term UV-A radiation can induce the adults of H.armigera to activate the p38 MAPK signaling pathway.Thereby inducing cell growth and proliferation,and improving stress adaptability of H.armigera in response to UV-A stress.When Hap38 MAPK is silenced,the regulation of p38 MAPK and other signaling pathways of H.armigera leads to cell apoptosis,which ultimately breaks the defense function of the insect body and causes irreversible damage to the insect body.This study preliminarily elucidated the role of Hap38 MAPK in response to UV-A stress in H.armigera,and laid the foundation for further revealing the molecular mechanism of insect response to UV-A stress.In this paper,full-length sequences of HaJNK gene and Hap38 MAPK gene of H.armigera were successfully obtained by RACE technology,and their effects on the growth and development of H.armigera and response to UV-A stress were studied by fluorescence quantitative PCR.The enzyme activity assay was used to study the effect of UV irradiation on the antioxidant enzyme system under the low expression of HaJNK gene and Hap38 MAPK gene in H.armigera,in order to explore the role of JNK and p38 MAPK signal transduction pathway in response to UV stress in H.armigera.The role of JNK and p38 MAPK signal transduction pathways in response to UV stress in H.armigera was preliminarily explored,which laid a foundation for further exploring the molecular mechanism of insect response to UV stress. |
