Screening Of The Functional Genes Interacting With StNAC262 Gene Of Potato By Yeast Two-hybrid Technique

The NAC designation is derived from the NO APICAL MERISTEM(NAM)gene from petunia(Petunia hybrida)and the Arabidopsis genes ATAF1/ATAF2 and CUP-SHAPED COTYLEDON2(CUC2).NAC protein was one of the plant specificity transcription factors(TFs),most of them was affected by various biological and abiotic stresses,had important effect on plant development and stress response.Reported showed that when transgenic plant was at the period of overexpression,many abiotic stress NAC proteins could promote roots to grow or change the structure of roots.These researches contributed to understand the complex mechanisms of signal transduction controlled by NAC protein and transcriptional reprogramming.But the knowledge about NAC transcription factor target gene was still limited.Thus,this research used host factor which was affected by NAC functional protein,filtrated host factor by yeast two-hybrid technique,to study the drought stress influence responses of NAC protein,realized NAC biological characteristics and drought resistance mechanism,provided theoretical basis to research the drought stress influence responses function of NAC protein in potato.The results showed that:1.Bioinformatics analysis predicts that the predicted molecular weight of StNAC262 is 38.34 kDa,belonged to stability protein,not belonged to secretory protein,subcells were located in the nucleus;structured pEGFP-NAC262 subcellular localization vector successfully,the transient expression vector p EGFP which had StNAC262 genes was introduced into tobacco by agrobacterium-mediated method,obtained 5 transgenic lines,observed green fluorescence localization under laser confocal scanning microscope,fluorescence was found in the nucleus.2.The StNAC262 gene was cloned by PCR,Structured bait vector pGBKT7-NAC successfully,the pGBKT7-NAC plasmid was introduced into the yeast strain Y2 H,the testing result of virulence and self-activation showed that StNAC bait protein did not have virulence and self-activation on yeast strain,which could be screened by yeast two-hybrid system.3.The yeast two-hybrid system was used to screen the plasmid pGBKT7-NAC and the plasmid of potato cDNA library to yeast strain Y2 H.Screened 128 candidate positive clone,confirmed 62 valid positive clones by PCR identification,after sequencing and wiped off repeat clones,there were 23 positive clones which interacted with pGBKT7-NAC didn't have raw frame shift mutation,filtrated 23 candidate host factor which interacted with StNAC.4.Analyzed 23 candidate genes by bioinformatics,the encoded protein included zinc ion binding protein,ubiquitin-protein ligase,auxin flows into the transporter,WRKY transcription factor,cold induced protein and some function unknown proteins could interact with potato NAC protein.