| Longan (Dimocarpus longan Lour.) is distributed most abundantly in tropical and subtropical regions. Flowering reversion of longan have been suggested as a main reason that led to a loss of quality and yield since 1980s. Although some physico-chemical methods have played a role in an extent, elaborating the molecular mechanism, cloning the related genes and inhibiting longan flowering reversion by genetic engineering technology have become a way. Using cDNA-AFLP technique, DLchi (Dimocarpus longan chitinase) was found to be up-regulated expression in reversing flower buds. As a pathogenesis-related protein, plant chitinases mainly take part in pathogen resistant, but some researches indicated chitinases might also participate development regulation, vegetative growth, organ abscission and so on, so it is essential to further study chitinase in flowering reversion of longan. The main results were as follow:1. Transmission electron microscope (TEM) was applied to study the differences between normal and revering flower buds in ultrastructure, the results indictated reversing flower buds were characterized by cell wall dissolution, cell membrane invagination, endoplasmic reticulum multiplication, etc. These features were similar to those described in natural abscission.2. Rapid amplification of cDNA ends (RACE) technology was performed to obtain DLchi full-length cDNA (accession no. GU177464). It consisted of 961 nucleotides and encoded an open reading frame (ORF) of 227-amino-acid residues. The estimated isoelectric point and molecular weight of the putative protein were 5.17 and 24.77 kD, respectively. Amino acid sequences showed high identities with other plant chitinases, including Mangifera indica (84%), Citrus sinensis (74%), Pyrus pyrifolia (71%), Galega orientalis (69%), Medicago sativa (67%), Vitis vinifera (68%), Arabidopsis thaliana (67%), Zea mays (65%), Phaseolus vulgaris (63%). DLchi possessed glycoside-hydrolase catalytic domain, but not chitin-binding domain and variable hinge domain, so it might encode a class II chitinase according to conventional classification. This premature protein consisted of a signal peptide of 25-amino-acid residues and the most likely cleavage site was between S25 and Q26. This protein showed no transmembrane signal in TMHMM analysis suggesting to be secreted to cytoplasm. The protein was hydrophilic with a Grand Average Hydropathicity (GRAVY) value of -0.127. Alpha helix and beta turn were predicted to be major construction elements in secondary structure. Besides, there were also some biology activity sits, such as N-linked glycosylation site, Casein kinase II phosphorylation site, Protein kinase C phosphorylation site, ATP/GTP-binding site motif A (P-loop). Although the significance and exact role mechanism of these motifs were unclear, they might involve in signal transduction.3. Using the complete expression cassettes (including CaMV 35S promoter and NOS Terminator) of plant expression vector pBI121, the sense and antisense DLchi plant expression vectors were constructed and transferred into Agrobacterium LBA4404 by freeze thawing method. Model plant, Arabidopsis (Columbia ecotype) was then transformed by floral dip method mediated by Agrobacterium to provide a basis for further study. |
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