Isolation And Identification The Interaction Proteins Of StBAM9 In Potato

Potato(Solanum tuberosum L.)is widely used due to its rich and comprehensive nutrition.Its high-value-added products such as French fries and chips in the processing industry are very popular because of their unique flavor.However,the cold-induced sweetening in potatoes has seriously hindered the development of fried products.Starch is the main energy storage form in potato tubers.Previous studies have shown that starch is the source of starch-glucose metabolism,and is one of the main factors that cause cold-induced sweetening.One of ?-amylase(StBAM9)with undetectable amylase activity plays an important role in cold-induced sweetening.The functional mechanism of these ?-amylase-free proteins is still unclear.In this study,we have separated the interaction proteins from StBAM9 and then verified to analyze the interaction mechanism between them.The main results are as follows:1.Through screening of the StBAM9 interaction protein using the cold-induced sweetening yeast library,a total number of 92 potential interaction proteins were obtained by using StBAM9 and StBAM9-P as baits.Among them,12 proteins are the same.Through WEGO analysis,we found that the potential interaction proteins of StBAM9 and StBAM9-P are similar in positioning,molecular function,and biological pathway.They are mainly located in cells and most of them are proteins with catalytic activity and binding function,and many of them are concentrated in cells,metabolic,biological regulation and biological response to stimuli.Through yeast two-hybrid verification,four interacting proteins were initially identified as StDUF842,StTPR01660,StTPR22129 and StTPR45174.2.To clarify the expression patterns of StDUF842,StTPR01660,StTPR22129,and StTPR45174 in different tissues of potato cultivar E3 or tubers under low temperature treatment,we detection of gene expression by qRT-PCR and found that the relative expression of StDUF842 and StTPR22129 were high in leaves,StTPR01660 was high in leaves and mature potato tubers,and the relative expression of StTPR45174 was high in mature potato tubers.Among them,StTPR01660 and StTPR45174 are similarto StBAM9 that they have a high expression levels in mature tubers.The leaves are the main productive organs of the potato,and tuber is the main energy storage organ of the potato.It is speculated that they are all likely to participate in the degradation of starch.3.To clarify the subcellular locatization of interaction protein,StDUF842,StTPR01660,StTPR22129 and StTPR45174 were fused with GFP and StAmy23 were fused with RFP.And then GFP-StDUF842,GFP-StTPR01660,GFP-StTPR22129,GFP-StTPR45174 were co-expressed with RFP-StAmy23 and the free RFP in Nicotiana benthamiana leaf cells,respectively.The results showed that DUF842,StTPR01660,StTPR22129,StTPR45174 are both located in the cytoplasm.4.To further clarify the interaction site between interaction protein and StBAM9 in plant cells,we use the BiFC to verify the interaction between StDUF842,StTPR01660,StTPR22129,StTPR45174 and StBAM9 and the results show that only StTPR01660 interacted with StBMA9 on starch granule.Meanwhile,StTPR01660 interacted with StBMA1(that located in the plastid matrix)in cytoplasm.In addition,the results of GST pull down showed that StTPR01660 interacted with StBAM9 and we also found that StTPR45174 had a strong interaction with StBAM9.The previous studies showed that StBAM9 was localized on starch granule.Therefore,we speculated that StTPR01660 may interact with StBAM1 in the cytoplasm to form a protein complex and then arrived to the starch granules through the recruitment of StBAM9 to degrade starch.In addition,whether or not other proteins such as StTPR45174 are involved in this process remains to be further determined.5.In order to clarify the interaction segments between StTPR01660,StBMA1 and StBMA9,we performed a Yeast two-hybrid experiment with different StTPR01660 short segment and StBMA9.The results showed that the complete TPR domain segment of StTPR01660(from 181 to 277 amino acid segment)can interact with StBMA9.Through the prediction domain of StBAM1 by NCBI,it was found that there was a plastid transit peptide from the 1 to 85 amino acid segment and a glucosyl hydrolase domain from the 111 to 542 amino acid segment.By truncating the hydrolysis domain,we obtained a 330-413 amino acid segment of StBAM1 caninteract with StBAM9-P.Through the prediction domain of StBAM9 by NCBI,it was found that there was one plastid transit peptide from the 1 to 63 amino acid segment and the glucosyl hydrolase domain is from the 88 to 506 amino acid segment.By truncating the hydrolysis domain,we obtained that the 238-386 amino acid segment in StBAM9 interact with StBAM1.There were two identical substrate binding sites(lysine and histidine)in the short segments of StBAM1 or StBAM9,indicating that StBAM9 may degrade starch by StBAM1.6.The results of the amylose content and amylopectin content in StBAM9 interference strains showed that the degradation rate of amylopectin was slower than that of amylose under the condition of 30 days at 4°C,and we can see that StBAM9 show a favor in amylopectin.We can further add the protein in vitro to analyze the possible functional mechanisms of interaction proteins and StBAM9.In addition,the starch phosphate content showed that there was no significant difference between the interference strain and the control E3,indicating that StBAM9 regulated starch degradation may not be through the starch phosphorylation pathway.