Screening Of The Regulatory Proteins Interacting With StSOD1 Gene Of Potato By Yeast Two-hybrid Technique

Superoxide dismutase（SOD）plays a key role in the process of eliminating ROS among those antioxidant enzymes,which is widely existed in aerobic prokaryotes and eukaryotes.It can rapidly disproportionate superoxide anion radical（O₂·^-）into hydrogen peroxide（H₂O₂）and oxygen.In the subsequent reaction,the H₂O₂ is decomposed to water and oxygen by catalase,various peroxidases and ascorbate-glutathione circulatory system.In all,SOD plays an very important role in scavenging oxygen free radicals,preventing free radicals from damaging cellular tissue structure and functional integrity,and protecting cells from oxidative stress damage.Studies have shown that potato tubers produce a large amount of reactive oxygen species（ROS）when breaking dormancy.These excess reactive oxygen species will seriously inhibit the growth of potato after germination.Therefore,it is very important to study the mechanism of regulating SOD expression.Since SOD is a functional protein,it is still unclear how upstream regulators,regulatory mechanisms,and signal transduction pathways that regulate its expression.Therefore,this study used yeast two-hybrid technique to screen upstream regulators that regulate StSOD1gene expression,thereby better studying the mechanism of regulation of SOD expression and signal transduction pathways,thus providing a certain theoretical basis response to the action of reactive oxygen species in potato breaking dormancy.The main research results obtained are as follows:1.Successfully amplified StSOD1 gene by PCR technology,then connected with pGBKT7 vector though reaction of double enzyme digestion and connection,thereby successfully constructed the yeast two hybrid bait expression vector pGBKT7-StSOD1.After then,converted the recombinant plasmid into yeast cell of AH109 to detect the toxicity and auto-activation using different defective mediums.The results showed that the bait expression vector had no toxic effect on the yeast strain and no self-activation effect on the reporter gene.2.The plasmid of pGBKT7-StSOD1 and the cDNA library of potato tuber dormancy were co-transformed into yeast AH109,using the characteristics of yeast strains to screen on different defective medium.85 positive candidate clones were preliminarily screened.After PCR detection,42 effective positive clones were identified and then sent to GENEWIZ Biotechnology Co.,Ltd for sequencing.Finally,By comparing,analyzing and eliminating duplicate clones,six protein factors interacting with StSOD1 were finally determined.3.Through bioinformatics analysis of six candidate regulatory factors,it was found that the encoded proteins were mainly arginine decarboxylase,cysteine protease,low temperature response factor,amino acid transporter,GID1-like gibberellin receptor and function unknown protein.4.Since there is low temperature response factor among the regulators interacting with StSOD1 screened out by yeast two-hybrid technique.Therefore,in this study,relative expression level of StSOD1 gene were measured.qRT-PCR analysis also showed that the relative expression of StSOD1 gene in potato plants increased under low temperature treatment,and the expression levels increased with treatment time.By observing the phenotype of potato plants,it was found that OE strains showed strong low temperature tolerance,while RNAi and NT strains showed decreased SOD activity and poor response to low temperature.5.The physiological indexes of transgenic plants with overexpression of StSOD1were determined under low temperature treatment.The results showed that under4℃low temperature treatment,the activity of superoxide dismutase in potato plants also increased correspondingly with the increase of treatment time.Among them,the over-expressed strain（OE lines）increased the most and increased about 1.38 times compared to non-transgenic（NT lines）;the activity of POD and CAT also increased under low temperature stress.In addition,malondialdehyde（MDA）levels,which are often used as an indicator of membrane lipid peroxidation,were found to increase by2.02 and 1.78 times in RNAi and NT lines compared to untreated control,respectively,while the changes of OE lines was small.This indicated that overexpression of StSOD1 could improve the activity of potato superoxidase and reduce the content of malondialdehyde,thus improving the tolerance of potato to low temperature stress.