Cloning and sequence analysis on cDNA of cysteine proteinase inhibitor gene from Populus tomentosa Carr

Plant cysteine proteinase inhibitors(CPI,phytocystatins) have been widely used in the plant pest-resisting genetic engineers.The aim of this study is to isolate the CPI gene from an indigenous tree,Populus tomentosa Carr.Based on the analysis of the conserved amino acid sequences of reported CPI and poplar expressed sequence tags(EST),two pairs of degenerate and poplar-specific primers were designed for reverse transcription polymerase chain reaction(RT-PCR).A fragment containing 696 base pairs(bp) of nucleotide was amplified from total cDNA of P.tomentosa cambium.Sequencing was carried out through linking to pGEM-T Easy vector.Sequence analysis showed that the cDNA fragment included an open reading frame(ORF) of 417 bp in length,which encoded 138 amino acid residues.Deep analysis of amino acid sequences shows that it contains four conserved motifs region of phytocystatins,which are G residue near the N-terminus and LVI-AGT-RKE-FY-AS-VI-x-EDQV-HYFQ,active site motif QxVxG and PW residues at the C-terminus,these results prove that the CPI gene exists and is effectively expressed in P.tomentosa cambium.Homology analysis of CPI amino acid sequences resulted in 47%-68% similarity scores between P.tomentosa and other trees.