Molecular Cloning And Preliminary Functional Analysis Of Gadd45 Gene In The Diamondback Moth, Plutella Xylostella (L.)

In recent years,more and more attentions has been paid to the research on the regulation mechanism of cell cycle and cell apoptosis of agricultural pests,which can provide a new perspective and theoretical foothold for integrated pest control.The gadd45 gene has the functions of inhibiting cell growth and inducing DNA damage which is one of the key factors in response to exogenous stress.However,for Plutella xylostella,a major agricultural pest,the role of its encoded GADD45 protein in a series of physiological processes,including DNA damage repair,cell proliferation and apoptosis,and its regulatory mechanism are still unclear.Based on the successful cloning of the open reading frame(ORF)of gadd45 gene in Plutella xylostella,we analyzed the changes of its expression at different instars and under various stress sources,such as heat shock,irradiation of ultraviolet and pesticide treatments by qPCR.Meanwhile,its prokaryotic and eukaryotic recombinant vectors were constructed for prokaryotic and eukaryotic expression and its characteristics at the protein level were analyzed with relevant prediction software.Besides,dsRNA expression vector of gadd45 gene of Plutella xylostella was constructed and qPCR was used to analyze the expression level of gadd45 gene and related genes after RNA interference feedingtest,so as to preliminarily analyze the function of gadd45 gene of Plutella xylostella.The main results of the present study are shown as follows.According to the data of GenBank and Plutella xylostella genome database,we designed the primers of gadd45 ORF and cloned its sequence of the coding region with the adult cDNA of Plutella xylostella by using PCR.The sequence length of the nucleotide coding region of the gadd45 gene was 495 bp,with a total of 164 amino acids encoded,the predicted protein molecular weight was 17.94 kd,and the isoelectric point was 4.884.The derived amino acid sequence analysis showed that diamondback moth GADD45 had a higher homology with Plutella xylostella GADD45 protein,and a lower homology with other insects and chordate,which was consistent with its taxonomy status.Through fluorescence quantitative PCR,the gadd45 relative expression levels at different instars of plutella xylostella were detected,it was found that the relative expression level of plutella xylostella gadd45 gene in the pupa stage was relatively high,which was significantly different from that in other stages(P < 0.05).Meanwhile,the relative expression levels of gadd45 gene under different exogenous stresses were studied,and it was found that the relative expression levels of gadd45 gene were different under different exogenous stresses.Its relative expression reached the maximum value of 12.73 after 4h of heat treatment,reached themaximum value of 4.23 after 8h of ultraviolet treatment,reached the maximum value of 6.35 after treatment with 1mg/mL tebufenozide for36 h and reached the maximum value of 16.71 after 24 h treatment with10mg/mL tebufenozide,which showed that plutella xylostella gadd45 gene could respond to different exogenous stresses,and could play a role in DNA damage repair and cell apoptosis according to the degree of cell damage.After the construction of gadd45 recombinant plasmid and the prokaryotic expression of it,protein electrophoresis(SDS-PAGE)detection found that the GADD45 fusion protein could be expressed in E.colistrain,and the molecular weight was approximate 18 kD,which was closed to the software prediction.While the eukaryotic expression in vitro results showed that the cell damage was serious,a large number of cells were broken,and the morphology was irregular after transfection with gadd45 recombinant plasmid.It was found that the pupation peak of Plutella xylostella larvae was delayed after dsRNA was fed to larvae of Plutella xylostella for RNA interference.The results of qPCR showed that the relative expression level of Plutella xylostella gadd45 gene decreased and the expression of cyclin dependent kinase inhibitor(CDK inhibitor)gene related to gadd45 also changed,which indicated that the RNA interference mediated by feeding test was feasible,and the gadd45 gene of Plutella xylostella might be involved in the physiological activities such as DNA damage induction,cell cycle arrest and apoptosis.This study laid a foundation for revealing the relationship between gadd45 of Plutella xylostella and cell cycle regulation and apoptosis mechanism,which also provided new clues for the comprehensive control of diamondback moth.