Construction Of CDNA Library Of Isaria Fumosorosea And Screening Of Interaction Factors From Library Against Serpin Fromplutella Xylostella

Plutella xylostella is one of the most important insect pests on cruciferous vegetables,has developed high resistance to many commonly used broad-spectrum chemical insecticides.Isaria fumosorosea is one of the important entomopathogenic fungi for effective controlling the increase of P.xylostella population.It is reported that p roteins such as subtilase proteinase,serine tryptase,chitinase and antimicrobial peptides were involved in the infection insect host.The expression of serpins gene was significantly increased when P.xylotella infected by I.fumosorosea.This indicates that there is immunological interaction between I.fumosorosea and P.xylotella during the infection process,including the response of virulence genes to receptor.In the present study,the yeast two-hybrid c DNA library of I.fumosorosea induced by P.xylostella was constructed for screen target gene with Serpin gene from P.xylostella as bait,in order to evaluate the interaction between the I.fumosorosea and P.xylostella.?1?Construction and examination of the yeast two-hybrid cDN A library of I.fumosoroseaTotal RNA was extracted from I.fumosorosea mycelium after incubated in MM medium with P.xylostella larvae as induced resource.Conversion of RNA into double-stranded c DNA was performed according to the operation guideline of C lontech's Make Your Own?Mate&Plate?Library System K it.The yeast two-hybrid cDN A library of I.fumosorosea was constructed via transformation p GADT7-Rec vector,homologous recombination with target double-stranded c DNA,into yeast Y187 competent cell.The results showed that the length of inserted fragment in c DNA library was among 300 to 2000bp,the conversion efficiency of library was 2.2×10⁶ cfu/3?g p GADT7-Rec,the recombination rate was 100%,the titer was 3.12×10⁷ cfu/mL and the library complexity was 2.2×10⁶ cfu.These data indicated thatthe c DNA library with high quality can be usedto screen the interaction protein.?2?Constructionof plasmidp GBK T7-serpin and examination of the autoactivation and protein toxicity of plasmid p GBKT7-serpin expression in recombination yeastThe Serpin gene was amplified by PCR,from P.xylostella infected by I.fumosorosea,using c DNA as template.The cDNA fragment was inserted into p GBK T7 vector by homologous recombination yielding p GBKT7-Serpin expression vector and then the expression vector was transferred the into Y2HGold yeast competent cell harvesting recombinant yeast.The recombinant yeast with p GBKT7-Serpin plasmid grew well on SD/-Trp,SD/-Trp/X-?-Gal selective medium and the colony was white color,however,no colonies of the same yeast were observed on SD/-Trp/X-?-Gal/AbA selective medium,which indicating that the Serpin gene expression in yeast with pGBKT7-Serpin plasmid has no autoactivation.There were no differences of the colony size and number between the two recombinant yeasts with p GBKT7-Serpin and p GBK T7 plasmids,respectively,incubated on the SD/-Trp selective medium,which indicated serpin expression in yeast with no protein toxicity.The results indicated that the recombinant yeast with pGBK T7-Serpin plasmid expression no protein toxicity can be used to hybrid with the yeast two-hybrid c DNA library of I.fumosorosea.?3?Screening of target proteins interaction with Serpin gene expression protein from c DNA library and the relative gene to the target proteinsMating the recombinant yeast with p GBKT7-Serpin plasmid and yeast two-hybrid cDNA library of I.fumosorosea was operated according to C lontech's Matchmake?Gold Yeast Two-Hybrid K it yielding positive colonies on selective defective media.The clover-shaped diploid cells were observed on selective medium,indicating that the yeast two-hybrid was successful.Blue colonies on selective medium were identified by PCR and sequenced to determine the target gene in cDNA library expression protein interaction with Serpin protein for the study of the immune interaction betweenI.fumosorosea and P.xylostella.