Screening And Verification Of Xylanase Interating Protein Of Talaromyces Thermophilus F1208

Alfalfa is a perennial forage,because of its good quality of grass,high yield and nutritional value,all kinds of livestock and poultry like to eat,so it can be used as an important feed crop.GH11 family xylanase is a class of glycoside hydrolases that can hydrolyze the ?-1,4-d-xyloside bond of xylan.It has a wide temperature adaptation range and good heat resistance,which has research value,especially alfalfa feed industry.At present,the research of xylanase at home and abroad mainly focuses on the screening and domestication of xylanase-producing strains,the purification and physical and chemical properties of xylanase,applications,and molecular structure and functional transformation,while the research on screening xylanase interaction interaction proteins by yeast two-hybrid technology has not been reported.In this study,yeast two-hybrid technology was used to screen xylanase interaction proteins,which laid a theoretical foundation for further research on the degradation mechanism of xylan by xylanase,the mechanism of xylanase synthesis and the production of thermostable xylanase.The results are as follows:1.Construction of two hybrid cDNA libraryUsing the SMART method to construct cDNA library,the capacity of cDNA library was 1.50×106,the titer was 4.20×107 cfu/ml,and the fragment size was between200-2000 bp.It is indicated that the cDNA library is qualified and can be used in subsequent experiments.2.Screening of xylanase interacting protein by yeast two-hybridDesign primers based on the target gene sequence to clone the T-Xyn gene.The bait vector pGBKT7-T-Xyn was constructed,and the bait vector was tested for no self-activation and toxicity.The yeast two-hybrid screening experiment,the co-cultured solution was spread on DDO/X/A(SD/-Leu/-Trp/X-?-Gal/AbA)solid plates.For the first time,440 positive clones were screened.After two streaks,preliminary confirmation 191 blue clones were positive.3.Validation of interaction between xylanase and candidate proteinsThe Y2H(yeast two-hybrid)experiment verified again that a total of 40 proteins were screened for interaction with the Talaromyces thermophilus F1208 xylanase.Pull-down experiments were used to verify the interactions between xylanase T-Xyn an glyceraldehyde-3-phosphate dehydrogenase(GAPDH),dienelactone hydrolase(DLH)and glutamine synthetase(GS).In this study,screening and identification of xylanase interaction proteins which provided a scientific basis for exploring the molecular mechanism of xylanase synthesis and the production of thermostable xylanase,the degradation mechanism of xylan by xylanase.