Expression Analysis Of Defensin Gene From Cristaria Plicata And Protein Activity Analysis

Defensin cDNA was cloned from freshwater mussle，Cristaria plicata.The full-length cDNA sequence of defensin was514bp, consisting of a5’-terminal untranslated region (UTR) of57bp, a3’-terminal UTR of242bp with apoly (A) tail, and the open reading frame of192bp. The defensin cDNA encoded apolypeptide of63amino acids. The N-terminus had the features consistent with asignal peptide as defined by SignalP3.0software with a putative cleavage site locatedafter position23(MRVAVVLALVLLLAVIDIPQAAA), the deduced mature peptidewas of40amino acid residues. The predicted molecular mass was7.13kDa, and thetheoretical isoelectric point was8.72. The predicted structure revealed that defensingene contained a conserved cysteine-stabilized α-helix and two β-sheet (CSαβ)structural motif.The expression level of defensin gene in haemocytes, mantle, gill andhepatopancreas from C. plicata was estimated by semi-quantiative RT-PCRtechnology. The results showed that defensin expressed in each of tissues and theexpression level of haemocytes and gill were similar, while mantle has highestexpression about1.25times of haemocytes and gill, followed the hepatopancreas.After injected by Aeromonas hydrophila (109cell/mL), the expression level ofdefensin in haemocytes first increased and then decreased, and reached its peak at12;In mantle the expression level of defensin lightly declined and then graduallystabilized; the expression level of defensin in gill didn’t showed evident change; Inhepatopancreas defensin keeped decrease.In haemocytes, after injected by Staphylococcus aureus，the expression level ofdefensin reached its peak at24h, and reached its minimum at48h. In mantle,defensin expression reached a lowest level at12h while appeared a peak at24h afterinjected by S.aureus. In gill, after injected by S.aureus the expression level ofdefensin reached its maximum at12h. After injected by S.aureus in hepatopancreas,defensin expression similarly decreased at12h, and then reached a peak at24h. Inmussle after injected by S.aureus, the expression of defensin descended at12h, while reached a peak at24h, then got a lowest expression at48.In order to further inquire into the functions of defensin, pGex-4T-1prokaryoticexpression system was constructed by double digestion, and prokaryotic expressionplasmid was transformed into E.coli (DE3). The recombinant protein wassuccessfully expressed after IPTG induction and was soluble existed in supernatant.The purified recombinant protein with a certain activity was obtained by affinitychromatography.Bacteriostatic activity of different densities of recombinant defensin protein wasestimated. The results showed that they had different bacteriostatic activities withEscherichia coli, Vibrio anguillarum, A.hydrophila and S.aureus. And0.2mg/Lrecombinant defensin protein performed better bacteriostatic activity, especially withS.aureus.Finally, we studied the bacteriostasic mechanism of defensin using laserscanning microscope and electron microscope. It turned out a “barrel-stave” pore,defensins identified each other and formed into polymers, then inserted intothecytoplasm membrane forming holes, and made the cell contents leaked affectingthe metabolism of bacteria system and then finally killed the bacteria.