Screening For PVY NIb Interacting Proteins By Yeast Two-Hybrid System

In order to explore the interaction between viral protein NIb and host tobacco G28,this paper constructed a bait protein vector and constructed a tobacco G28 yeast two-hybrid cDNA library by artificially synthesizing PVYMSNR-NIb and PVYN-NIb genes.By using the yeast two-hybrid system technology to use the NIb protein as a bait protein,the PVYMSNR-NIb and PVYN-NIb interaction factors were screened from the tobacco cDNA library,and different candidate proteins were screened.In order to further study the basis of NIb’s role in virus infection and pathogenesis,it provides a scientific basis for the application of yeast two-hybrid system in plant virus research.Below are key research findings:(1)The PVYMSNR-NIb and PVYN-NIb genes were synthesized to ensure the sequence was accurate,and the target fragment was constructed on the yeast two-hybrid bait vector pGBKT7.Through the detection of bait protein PVYMSNR-NIb and PVYN-NIb yeast toxicity and self-activation,the results showed that the yeast can grow normally on the selection medium,indicating that the bait protein NIb is not toxic to the host yeast Y187,and can be used for subsequent screening experiments.(2)The extracted total RNA of tobacco G28 was determined by UV spectrophotometer,the concentration was 1.2181 μg/μl,A260/280=2.20,A260/230=2.17.28S/18S=1.9,RIN=8.6,the quality satisfied yeast two-hybrid experiment On request,follow-up experiments can be performed.(3)An Uncut G28 yeast two-hybrid cDNA library was constructed using tobacco G28 mRNA as a starting template.The results showed that the primary cDNA library titer was 2.4×106 cfu/ml the total number of clones was 9.6×106 cfu,and the recombination rate was 91%.The secondary library titer was 1.61×106 cfu/ml and the total library capacity was 1.6×107 cfu.The recombination rate was approximately 100%;the tobacco cDNA library plasmid was transformed into yeast transformants,and the density of the cells exceeded 3 × 107 cells/ml.The PCR clones were identified by randomly selecting 24 clones.The results of PCR identification indicated that the length of the target gene fragments was more than 1 KB on average.(4)The yeast two-hybrid technique was used to screen the protein interacting with PVYMSNR-NIb,and finally 9 positive clones were obtained.After sequencing,after comparison with the GenBank database,there were 7 candidate proteins,respectively including ATP synthase delta chain,chloroplastic[Nicotiana sylvestris],dnaJ homolog subfamily B member 1-like[Nicotiana tomentosiformis],ribulose bisphosphate carboxylase small chain S41,chloroplastic-like[Nicotiana sylvestris],protein MID1-COMPLEMENTING ACTIVITY 1-like[Nicotiana sylvestris],transcription repressor OFP1[Nicotiana tomentosiformis],protein BONZAI 1-like isoform X1[Nicotiana tomentosiformis],carbon catabolite repressor protein 4 homolog 1-like[Nicotiana tabacum].(5)The yeast two-hybrid technique was used to screen the protein interacting with PVYN-NIb,and finally 8 positive clones were obtained.After sequencing,after comparison with the GenBank database,there were 6 candidate proteins,respectively including cell wall integrity protein scwl isoform X1[Nicotiana tomentosiformis],NADP-dependent malic enzyme,chloroplastic[Nicotiana attenuata],cell wall integrity protein scwl[Nicotiana attenuata],pupal cuticle protein Edg-91-like[Nicotiana sylvestris],keratin,type I cytoskeletal 9-like[Nicotiana tabacum],ribulose bisphosphate carboxylase small chain S41,chloroplastic-like[Nicotiana sylvestris].(6)Yeast two-hybrid rotation verification showed that ATP synthase delta chain,chloroplastic[Nicotiana sylvestris],carbon catabolite repressor protein 4 homolog 1-like[Nicotiana tabacum]and PVYMSNR-NIb protein have an interaction.