Construction Of Normalized CDNA Library And Screening Of BnPCS1 Interaction Protein In Ramie

Phytochelatin synthase(PCS)is a key enzyme involved in detoxification and accumulation of heavy metals in plants.PCS synthesizes plant chelating peptide PCs with glutathione(GSH)as substrate in plant and forms chelating compound after chelating heavy metals,which can alleviate the toxicity of heavy metals to plants and plays an important role in the detoxification of heavy metals in plants.In the preliminary analysis of cadmium tolerance in ramie,the main metabolic pathway of cadmium tolerance in Ramie was selected as glutathione pathway PCS1,PCS1 induced heavy metal Cd expression in leaves.In order to further understand the role of PCS1 in the molecular mechanism of Cd tolerance in ramie,a homogeneous cDNA library and pGBKT7-BnPCS1 bait vector were constructed by analyzing the bioinformatics of PCS1 from ramie leaves,the interaction proteins of BnPCS1were screened and validated by yeast two-hybrid screening.The results of this study are as follows:1、According to the GenBank of NCBI,the sequence of PCS1 was predicted to be KF717368.1.The full length of cDNA was 1956 bp.It contained a total of 42phosphorylation sites,a molecular weight of 56.02 kDa,and an isoelectric point(pI)of 6.76bp,the prediction of subcellular localization showed that BnPCS1 was located in cytoplasm.2、The cDNA library of ramie was constructed by DSN homogenization technique.The titer of the library was 3.56×10~6 cfu/ml,which met the requirement of the library.A total of20 positive clones were detected by colony PCR with 24 single colonies randomly selected by AD vector universal primers.The inserted cDNA fragments were between 1000bp and2000bp,the inserted fragments were concentrated at about 1000bp,the recombination rate was 67%.3、A bait vector pGBKT7-BnPCS1 was constructed.The plasmid pGBKT7 and yeast expression vector pGBKT7 were digested by EcoR I and BamH I,which were linked by T4ligase.pGADT7 empty vector and pGBKT7-BnPCS1 bait vector were co-transformed into Y2H Gold yeast strain by lithium acetate method and coated on SD/-leu/-Trp agar plate,the results showed that the recombinant plasmid of bait had no obvious effect on the expression of yeast.The monoclonal cells which grew well on the plate were selected and seeded on SD/-Leu/-Trp,SD/-ade/-his/-leu/-trp agar plate at different dilution concentration,can be used to further filter the library.4、The BnPCS1 interacting protein was screened from ramie cDNA library by yeast Two-hybrid screening.The culture plate of SD/-leu/-Trp was coated and diluted 1000 times.The hybridization efficiency was 8.00%,much more than 2.00%,and the library capacity was 6.98×10~6cfu/ml,much more than 1.00×10~6cfu/ml,the yeast two-hybrid system was proved to be in good condition.Using pGBKT7-BnPCS1 as bait vector,the full-growth cDNA library of Ramie was screened by yeast two-hybrid system,and the positive clones were sequenced and compared,after BLAST comparison,4 proteins that might interact with BnPCS1 were found,which showed that they belonged to 4 different coding genes.The direct interaction between BnPCS1 and Clpb3 and pre-mRNA-processing factor 39 was further demonstrated by the gyration test,which showed that BnPCS1 and these two proteins interacted with each other to perform the function of cadmium tolerance.