Study On The Proteins Interacting With BYDV-CP And RdRp In BYDV Resistant Wheat Line

Wheat yellow dwarf caused by barley yellow dwarf virus (BYDV) is one of the most importantdiseases of wheat worldwide. Genes resistant to BYDV originated from Thinopyrum intermediumhave been introduced into the genome of hexaploid wheat, and some BYDV resistant materials such asHW642 have been got in our research group.The BYDV resistant genes from HW642 can inhibit thereplication and movement of BYDV. In order to know the wheat protein interacting with the proteins ofBYDV, yeast two hybrid system was used to isolate the protein interacting with BYDV coat protein(CP) and RNA-dependent RNA polymerase (RdRp) from the cDNA library of HW642. Thestructure of the candidate gene was to be predicted, and its expression model would be analyzed.Theresult of the present research was shown as the following.1. Yeast two hybrid bait vectors of coat protein (CP) and RNA-dependent RNA polymerase(RdRp) of barley yellow dwarf virus (BYDV) were constructed and expressed in yeast. Yeastcontaining bait vector pGBKT7-CP and pGBKT7-RdRp could grow on SD/-Trp culture medium,but couldn't grow on SD/-Ade/-Trp/X-Gal and SD/-His/-Trp/X-Gal medium. The result indicatedthe pGBKT7-CP and pGBKT7-RdRp had no toxicity and autonomous activation in yeast, andcould be used as bait vector in yeast two hybrid system to analyze the proteins interacted with CPand RdRp of BYDV.2. A yeast two hybrid cDNA library of HW642 was constructed (MatchmakerTM LibraryConstruction & Screening Kits,Clotech). The library comprised of a total of 800,000 colonies. Thelibrary titer is 3×10~7 CFU/ml, recombinant rate is about 88％, and the inserted fragment rangesfrom 0.2 to 1.4kb.3. To identify the host proteins that interact with BYDV CP, yeast two-hybrid system was usedto screen a wheat cDNA library. Using full-length BYDV CP as bait, a candidate cDNA clone wasisolated,designated TaSTK1. TaSTK1 encodes a protein shares high homogeneity withserine/threonine-protein kinase (STK). The furtherβ-galactosidase quantitative assay and vectorexchanging test indicated that the interaction between yeast expressing fusion protein of TaSTK1and BYDV CP was specific.4. By using nestle PCR and 5' RACE(rapid amplification of cDNA ends) technique, thecomplete cDNA sequence of TaSTK1 was isolated. TaSTK1 encoded a protein of 460aa, and aconserved domain of STK was found in the N-terminus region of 1-300aa. The result of homologyalignment analysis indicated no other protein similar to TaSTK1 in wheat had been found. Thesecondary structure of TaSTK1 comprised of 21 a helices, 19βsheets and 29 turns. The result ofPrositer analysis indicated 2 ATP binding cite and 1 proton acceptor act_cite were found in theprotein of TaSTK1.By using the system of Swiss-model, three dementional structure of TaSTK1was predicted.5. The result of quantitative real-time PCR indicated that the treatment of BYDV could enhancethe expression of TaSTK1 in BYDV resistant wheat, while it could inhibit the expression of TaSTK1 in BYDV susceptible wheat, indicating TaSTK1 takes an important role in pathogenicmechanisms of BYDV resistance.6.To identify the host proteins that interact with BYDV RdRp, yeast two-hybrid system wasused to screen a wheat cDNA library. Using full-length BYDV RdRp as bait, a candidate cDNAclone containing 2 plasmids named P500 and P350 was isolated.The inserted sequence of P500encodes an auxin response factors (ARF) like protein, and the inserted sequence of P350 encodesunknown protein. Yeast cotransformed with P500, P350 and pGBKT7-RdRp can express reportorgenes. P500, P350 and pGBKT7-RdRp might interact with each other through a three hybrid wayor'guard hypothesis' model.