Screening And Study Of Proteins Interacting With Clathrin Heavy Chain Of Nilaparvata Lugens(St(?)l) And Enolase Of Yeast-like Symbiont

The brown planthopper(BPH),Nilaparvata lugens(St(?)l),is one of the most destrutive pests of rice in Asia.BPH causes serious harm to rice production through feeding exclusively on rice,spawning and transmitting rice virus.The yeast-like symbiont(YLS),living in the abdomen fat body of BPH,provides nutritions for BPH and plays important roles in the development,resistance and virulence change of BPH.Therefore,the purpose of managing BPH can be achieved by controlling the YLS in BPH.YLS can be transovarially transmitted between the generations of BPH,and the entering of YLS into BPH ovary is related to the clathrin mediated endocytosis.Previous studies uncover that clathrin heavy chain(CHC)of BPH and enolase of YLS probably play key roles in the transmission of YLS.These may provide a new direction for the control of BPH by studying the relevant mechanism and function of CHC of BPH and enolase of YLS and their interaction proteins in the process.In this study,the proteins interacting with CHC of BPH and enolase of YLS were screened and studied respectively.The main results are as follows:(1)Screening and study of proteins interacting with clathrin heavy chain of Nilaparvata lugens(St(?)l).Using BPH c DNA as the template,the CHC of BPH was amplified by PCR.Then the p MD19T-CHC vector,p GBKT7-CHC bait vector and p GBKT7-CHC/Y2 H Gold recombinant strain were obtained by double restriction enzyme digestion,ligation and transformation.Then p GBKT7-CHC/Y2 H Gold was hybridized with BPH yeast hybridization library to screen the interaction proteins of CHC,and eleven interaction proteins were screened and identified,including protein PCF11,nucleoporin,mitochondrial ATP synthase lipid-binding protein,cuticle protein 21-like(CP21),myosin regulatory light chain 2(MYL2),mitochondrial thioredoxin(TXN),defensin,proteasome subunit beta type-7(PSMB7)and three uncharacterized proteins.And the functional prediction showed that six proteins interacting with CHC of BPH may be related to the transmission of YLS through ovary,including CP21,MYL2,mitochondrial TXN,PSMB7 and an uncharacterized protein containing SVWC domain.After that,p GBKT7-PSMB7/Y187 recombinant strain was constructed,and then p GBKT7-PSMB7/Y187 was hybridized with p GBKT7-CHC/Y2 H Gold to verify the interaction between PSMB7 and CHC.In addition,PSMB7 of BPH was cloned and analyzed.The ORF of BPH PSMB7 had 834 nucleotides and encoded 277 amino acids.The predicted BPH PSMB7 was deduced with a molecular mass of about 30.02 k Da and a calculated isoelectric point of 7.57.BPH PSMB7 had no signal peptide and transmembrane helix,but contained a proteasome beta type-7 subunit domain and a proteasome beta subunits C terminal domain.Phylogenetic analysis revealed that PSMB7 was conserved in Hemiptera.Immunofluorescence observation showed that the expression of PSMB7 was high in the epithelial plug of BPH ovary,indicating that PSMB7 should be related to the transmission of YLS.The result of ds RNA injection experiment suggested that PSMB7 was related to the normal development of BPH ovary.(2)Screening and study of proteins interacting with enolase of yeast-like symbiont.Using the whole BPH c DNA as the template,the enolase of YLS was amplified by PCR.Then the p DHB1-enolase/NMY51 bait vector strain was constructed.After p DHB1-enolase/NMY51 was hybridized with BPH yeast hybridization library,total 28 interaction proteins of enolase were screened and identified.After that,the functional analysis of these interaction proteins was briefly carried out.There were two proteins that may have strong interaction with enolase,a stress-associated endoplasmic reticulum protein 2that protects unfolded target proteins from degradation during endoplasmic reticulum stress,and a death-associated protein 1.In addition,two proteins that screened in the screening of proteins interacting with CHC of BPH,were also screened,ATP synthase lipid-binding protein(mitochondrial)and myosin regulatory light chain 2.