Isolation And Expression Of Antifungal Proteins And The Related Genes In The Seeds Of Jatropha Curcas

Jatropha curcas is a member of the family Euphorbiaceae. The seeds represent an abundant source of proteins. Purification to antifungal proteins is important for improving resistance of plants against pathogenic fungi. In our study a β-1, 3-glucanase was isolated and purified from J. curcas seeds, which were collected from Panxi area, Sichuan province. It exhibited a molecular weight (Mr) of 65.5 kD consisting of three subunits with non-covalent bond conjugate. The isoelectric point (pI) of 8.54 was determined. The optimal temperature and pH of the β -1 3-glucanase activity were 40℃-50℃ and 7, respectively. The enzyme was stable at pH 6-8 and temperature between 4℃- 55℃. The specific ultraviolet spectrum, fluorescence spectrum and amino acid composition were determined. Antiserum to β -1 3-glucanase recognized three bands of 20 kD, 22 kD and 23 kD on Western blot of proteins extracted from seeds. No band was showed on Western blot of roots, stems and leaves. We suggest that the β -1 3-glucanase is expressed specifically in seeds.The β -1 3-glucanase exhibited different antifungal activity toward hyphal growth, spore formation and germination of Pyricularia oryzae Cav., Rhizoctonia solani Kuha, Curvularia lunata (Walk) Boed and Gibberelle zeae (Schw.) Petch., ofwhich the inhibitory effect on Pyricularia oryzae Cav. was the strongest and could be maintained for a long time. The calabash-shape hyphae of P. oryzae Cav. and the yellow balls in hyphal cells of G. zeae (Schw.) Petch. were observed under light microscope. Under transmission electron microscope, the common effect of the -1,3-glucanase on hyphal growth inhibition could be detected in the presence of cell wall. The cell walls of hyphae treated by -1,3-glucanase became thin and weak. We suggest that the antifungal activity of -1,3-glucanase might be related to hydrolyzing fungal cell walls and changing fungal metabolic pathway. -1,3-glucanase was slightly toxic to mice , LD50 2.2 g/Kg (95% confidence limits). It could agglutinate duck red cell under polyethylene glycol (PEG) induction condition.Curcin could obviously inhibit hyphal growth and spore formation of Pyricularia oryzae Cav., Pestalotia funereal, Rhizoctonia solani Kuha, Sclerotinia sclerotiorum (Lib) de Bary, and Collectotrichum gloeosporioides (Perz.) sacc. Modified hyphae treated with curcin were observed under light microscope. Some protein bands of treated hyphae disappeared on SDS-PAGE. We suggest that the inhibitory effect of curcin on pathogenic fungi might be related to protein disappear of treated fungi. Antiserum to curcin recognized one band of 28.5 kD on Western blot of proteins extracted from seeds. No band was showed on Western blot of roots, stems and leaves. We suggest that curcin should be expressed specifically in seeds.An open reading frame (ORF) encoding mature protein of curcin, which was almostly corresponded to its cDNA sequence, was first cloned from total DNA of Jatropha curcas. It was indicated that no introns existed. The ORF was transferred into tobacco (Nicotiana tabacum L.) via Agrobacterium mediation. The curcin ORF was identified to be integrated into tobacco genome, and to be expressed at transcription level by PCR and RT-PCR. The transgenic tobacco plants exhibited the antifungal activity enhance toward Rhizoctonia solani.The cDNA sequence encoding JFIP was first cloned from J. curcas leaves, which were stressed by PEG, temperature and fungi infection. Its ORF, which contains a signal peptide of 42 amino acid residues, had 927 bp that encoded a precursor protein of 309 amino acid residues with calculated Mr of 34.9 kD and thepredicted pI of 6.56. The ORF that encoded the mature protein with 267 amino acid residues had 801 bp. The calculated Mr was 30.1 kD and the predicted pI was 5.25. There are specific hydropathy plot and chemical modification point in the deduced amino acid sequence. The predicted secondary structure of JFIP was mainly a -helix. JFIP includes six a -helixes and six -foldings in 3D-structure. There is high similar...