Molecular Cloning,prokaryotic Expression,Purification And Functional Characterization Of Cysteine Proteinase Inhibitor(CPI) From Solanum Tuberosum

Potato?Solanum tuberosum.?,rich in nutrition and widely planted,is the fourth most important crop food in the world.It is widely accepted in the market as a dual-use crop.Salt and drought stress are two major factors that hamper potato growth and development,which seriously restrict the development of potato and having a detrimental effect on its yield and quality.It is important to find stress resistance and related genes in potato.Cysteine protease inhibitor?CPI?is widely distributed in plants,participate in response of plant to environmental stress,programmed cell death?PCD?and regulation of metabolism.The potato CPI gene StCPI was cloned from commercial tetraploid potato cultivar“Netherlands 15#”and its protein sequence were analyzed by bioinformatics software.StCPI protein were expressed in Escherichia.coli BL21?DE3?And function of StCPI gene were validated in E.coli under high salinity and drought stress.The transgenic plants showed stronger ability in resisting high salt stress and drought stress.This research obtained experimental results as follows:1.The potato CPI gene StCPI was cloned from potato cultivar“Netherlands 15#”,the StCPI gene contains 666 bp ORF and was predicted encoding 221 amino acid.Ex PASy ProtParam tool predicted that its coding protein molecular formula is C₁₁₂₃H₁₇₇₀N₂₉₀O₃₂₄S₈,molecular weight is 24.7 KD,isoelectric point is 6.88,adipose index is 101.26,instability index is 25.32,average hydrophobicity is 0.006.The results showed that StCPI was stable hydrophilic protein.SignalP predicted a signal peptide which containing 23 amino acids exist at the N-terminal.TMHMM predicted the amount of transmembrane amino acid is 9.1363,indicates that StCPI did not have transmembrane domains,neither receptors on the membrane nor located on the membrane.2.The prokaryotic expression recombinant plasmid pET28-StCPI was successfully constructed,transformed and expressed in E.coli BL21?DE3?,then verified by western blot.Fusion protein expression in inclusion body form.After dialysis,StCPI significantly inhibited papain activity.The results showed that StCPI which expressed in E.coli had CPI activity in vitro.3.Overexpression of StCPI gene can enhance the ability of E.coli to resist salt stress and drought stress.Through spot assay experiment under 0.25 M,0.5 M,0.75 M,1 M NaCl stress and 15%,20%,25%,30%PEG stress and determination of growth curve under 0.5 M NaCl stress,the results showed that E.coli which expressing pET28-StCPI plasmid can tolerate high salt stress of 0.5 M NaCl while the E.coli expressing empty pET28 vector can not grow under same culturing conditions.The former growth condition was significantly better than the latter under high salt stress and drought stress.4.Overexpression of StCPI gene can enhance the ability of potato to resist salt stress and drought stress.The overexpression StCPI potato lines was obtained then verified by qRT-PCR and PCR methods.The OE3 lines which with the highest expression level was selected for stress test.High salt and drought stress treatment was processed at OE3 and wild potato plants.Through 0.2%NaCl stress treatment,most of the wild plants died after 3 d.The plant's withering is caused by the shrinkage of plant leaves.The OE3 lines can survive and grow normally,However,the top and edge of some leaves appear withered and yellow under the influence of salt stress.After 10 d drought stress,the growth of transgenic plants was significantly better than wild type.The above results indicated that the overexpression of StCPI in diploid potato plants can enhance its resistance ability to high salt stress and drought stress.