Cloning Of The CDNA Encoding The Polygalacturonase Inhibiting Protein (PGIP) In Prunus Persica (L.) Batch And Its Expression In E.coli

Polygalacturonase inhibiting proteins (PGIPs) are extracellular glycoproteins located in plant cell wall. It is believed to play an important role in the defense against plant pathogenic fungi. PGIPs can specially bind and inhibit or reduce the hydrolytic activity of polygalacturonases (PGs) secreted by plant pathogenic fungi, and limit the growth of plant pathogens, as well as eliciting defense responses in plant. Furthermore, PGIPs belong to the super family of Leucine-rich repeat (LRR) proteins which also include the products of several plant resistance genes. They are considered to be important candidate genes for genetic engineering to obtain transgenic plants with increased tolerance to fungal infection, which decrease the use of insecticide. Therefore, the basic research of transgenic engineering for that purpose is to isolate and identify the pgip genes.Through reverse transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers based on the conserved region of the pgip genes of genus Prunus, a fragment about 1,000 bp was obtained from the total RNA extracted from the leaves of mature peach seedling. After cloning it into pMD18-T vector, the linked DNA was transformed into Escherichia coli JM109. The positive clones were picked out and sequenced. The results showed that the cloned cDNA was 1,053 bp and a real pgip gene of peach designed as Pppgip in this thesis with an accession number EF409977 in GenBank. The cloned cDNA contains an open reading frame of 330 amino acid residues with a molecular mass of 36.4 kDa and a pl of 7.42, and a hydrophobic region of 24 amino acid residues in the N-terminal which was considered to be a signal peptide. The deduced amino acid sequences contains 7 potential N-glycosylation sites. Although the cloned cDNA exhibits a homology of the nucleotide and deduced amino acid sequences of 93.2～94.6% and 89.7～92.7% respectively, which was significantly similar to other pgip genes from the genus Prunus, the homology tree shows that it obviously distinguished from the others. And the phylogenetic tree shows that it indeed far from its near species, and belongs to a different branch with the P. mahaleb L.. The three-dimensional structure of protein coding by the gene was predicted using the ESyPred3D software. The three-dimensional model looks like a horseshoe-shaped structure, and contains 11α-helices and 21β-sheets which make up 8β-sheet/β-turn/β-sheet/α-helix regions, and the center LRR structural domain is composed of 10 tandem LRR motifs.The fusion and non-fusion expression plasmids were constructed by inserting the coding region of Pppgip mature peptide into the expression vector pET-32a(＋). The recombinants were transformed into E. coli Rosetta-gami(DE3)pLysS respectively and induced by IPTG. The SDS-PAGE shows that the recombination proteins were mainly appeared as inclusion bodies, a small portion of solvable proteins in the supernatant of the post-sonicated thalli also.The results mentioned above lays out a foundation for investigate the function of peach PGIP and its interaction with PGs, and provides an important reference for molecular plant breeding of fungi-resistant.