Cloning,sequencing and expression of a cysteine proteinase inhibitor gene(PbCPI) from Pyrus betulaefolia Bunge

Plant cysteine proteinase inhibitor(CPI) has played more and more important roles in the fields of plant genetic engineering for resistance to adverse environments.It is one of the hot issues to isolate and validate CPI gene functions in the stress-tolerance gene engineering at present.The objective of this study was to illuminate the role of CPI gene in the defense mechanism of birch-leaf pear(Pyrus betulaefolia Bunge).The full-length cDNA and DNA sequences of a CPI gene were cloned from birch-leaf pear seeds by rapid amplification of cDNA ends(RACE) and PCR.Its expression patterns under different stresses were analyzed by semi-quantitative reverse transcription polymerase chain reaction using cross-introns primers.The results showed that PbCPI cDNA sequence length was 987 bp,which contained a 738-bp-length open reading frame(ORF) and encoded 245 amino acid residues.Sequence analysis indicated that PbCPI protein contained a signal peptide(26 amino acids) and a mature peptide(219 amino acids).Predicted isoelectric point and relative molecular mass of PbCPI protein were 6.68 and 27 190,respectively.PbCPI genomic DNA sequence consisted of 3 exons(1-302 bp,401-772 bp,1 615-1 897 bp,respectively) and 2 introns(303-400 bp,773-1 614 bp,respectively).Subcellular localization result indicated that PbCPI protein was localized in the endoplasmic reticulum through the PSORT software analysis.PbCPI deduced polypeptide had the primary structure which was absolutely necessary for inhibitory activity of plant cysteine proteinase inhibitors.This structure included two glycine residues(Gly46-Gly47),assumed response areas QXVXG(Q90-V91-V92-A93-G94) and A/PW motif(P120-W121).PbCPI protein contained three typical motif domains,LARFAVQEHN,QVVAG and YQAKVWVKPW,which usually exists in plant cysteine proteinase inhibitors superfamily.PbCPI and other CPI proteins from Rosaceae plants belonged to the same branch in the CPI phylogenetic tree.Homology analysis exhibited that it had the highest similarity(95.92%) with Malus domestica MdCPI(AAO19652).PbCPI gene expression was inducible and its transcription abundances climbed up quickly after high-temperature(30 ℃),low-temperature(4 ℃),NaCl,mechanical damage,MeJA or ABA treatments for 4 h.These results indicated that PbCPI transcription response was induced under temperature stress,salinity,mechanical damage or exogenous hormone treatment.Conclusively,PbCPI was involved in the defense mechanisms against biotic or abiotic stress in birch-leaf pear.