Screening Of Interaction Protein From CDNA Library Of Isaria Fumosorosea And Nilaparvata Lugens Against Ifcdp1 Protein From Isaria Fumosorosea

Nilaparvata lugens is one of the most important piercing-sucking mouthparts insect pests on rice,has showed high resistance to many commonly used chemical insecticides.Isaria fumosorosea is one of the important entomopathogenic fungi to control piercing-sucking mouthparts insect pests.In order to evaluate the role of Ifcdp1 protein during the course of I.fumosorosea infection N.lugens nymph,the yeast two-hybrid c DNA library of N.lugens nymph induced by I.fumosorosea was constructed for screen target protein interaction with Ifcdp1 protein from I.fumosorosea as bait,at the same time target protein interaction with Ifcdp1 protein was screened from c DNA library of I.fumosorosea.The results are as followed:(1)Construction of the yeast two-hybrid c DNA library of N.lugensTotal RNA was extracted from N.lugens nymph induced by I.fumosorosea.Double-stranded c DNA was obtained by PCR amplification with c DNA,reverse transcription from the total RNA,as template.The double-stranded c DNA was cloned into p GADT7-Rec vector via homologous recombination and then transformed into yeast Y187competent cell to generate yeast two-hybrid c DNA library of N.lugens.The results showed that the conversion efficiency of library was 2.9×10~6 cfu/3μg p GADT7-Rec,the titer was3.42×10~7 cfu/m L,the recombination rate was 100%,the library complexity was 2.2×10~6cfu,and the length of inserted fragment in c DNA library was among 500 to 2000 bp.These data indicated that the c DNA library with high quality can be used to screen the interaction protein.(2)Construction of p GBKT7-cdp1 expression vectorThe cdp1 gene from I.fumosorosea was cloned into p GBKT7 vector by homologous recombination to generated p GBKT7-cdp1 expression vector and then was transformed into Y2HGold yeast competent cell to harvest a bait strain.The bait strain with p GBKT7-cdp1 plasmid grew well on selective medium indicating that the cdp1 gene expression in yeast has no auto-activation and no protein toxicity.The results indicated that the bait strain with p GBKT7-cdp1 plasmid can be used to hybrid with the yeast two-hybrid c DNA library.(3)Screening of target proteins interaction with Ifcdp1 protein from c DNA library of N.lugens nymphTo evaluate the function of cdp1 gene in the process of I.fumosorosea infection N.lugens nymph,mating the bait strain contain p GBKT7-cdp1 plasmid and c DNA library of N.lugens was operated for yielding positive colonies or interaction target protein on selective defective media.The results showed that 89 blue colonies were harvested on selective medium and then were identified by PCR and sequenced to determine the target gene in c DNA library expression protein interaction with Ifcdp1 protein.Finally,4 samples are probably the target proteins for the further study of the interaction between I.fumosorosea and N.lugens.The No.34 sample was Ras protein of N.lugens via PCR amplification and BLAST identification in NCBI website,and Ras protein of N.lugens was the target gene in c DNA library expression protein interaction with Ifcdp1 protein.(4)Screening of target proteins interaction with Ifcdp1 protein from c DNA library of I.fumosoroseTo determine how many protein having close relation to the Ifcdp1 protein secretion from fungi during the process of I.fumosorosea infection N.lugens nymph,mating the bait strain contain p GBKT7-cdp1 plasmid and c DNA library of I.fumosorose was operated for yielding positive colonies or interaction target protein on selective defective media.The results showed that 35 blue colonies were harvested on selective medium and then were identified by PCR and sequenced to determine the target gene in c DNA library expression protein interaction with Ifcdp1 protein.Finally,2 samples are probably the target protein for the further study of the interaction with Ifcdp1 protein.The No.6 sample was dynamin-related protein of I.fumosorosea via PCR amplification and BLAST identification in NCBI website,and dynamin-related protein of I.fumosorosea was the target gene in c DNA library expression protein interaction with Ifcdp1 protein.