| Summary: | 碩士 === 輔仁大學 === 生物學系 === 84 === Classical defensins are a family of antimicrobial and cytotoxic
peptides existing in the mammalian neutrophil cells. This
research attempts to transfer cDNA fragment encoding mature
human neutrophil peptide-1 (HNP-1) into tobacco Wisconsin 38
(W38) via Agrobacterium-mediated transformation protocol to
develop the transgenic tobacco plants with antimicrobial
properties. Plasmid pBI-mDf was constructed by substituting the
GUS fragment of pBI121 with the segment encoding mature HNP-1,
previously recovered from pUC-DH after double digestion of Hind
III and Sac I. pBI-mDf were subsequently introduced into A.
tumefacieus strain C58C1/pGV2260 by direct transformation.
Tobacco leaf discs were cocultured with C58C1/pGV2260/pBI-mDf
and C58C1/pGV2260/pBI121 cells. Infected leaf discs were then
cultured on MS104 medium contain 250 μg/ml kanamycin and 250 μ
g/ml cefotaxime. A month later, regenerated shoots were
transferred to new selection media. The survived shoots after
another month were transferred to root-inducing media MS0
supplemented with 100 μg/ml kanamycin and 250 μg/ml cefotaxime
for root induction. Plants with well-grown root systems were
finally transferred to green house. Four of the thirty putative
transgenic plants died. HNP-1 and NPT-II cDNA fragments were
proved to exist in the genomes of eight plants examed by the PCR
and their mRNA transcripts were detected using the RT-PCR. The
crude protein extracts from the transgenic plants engineered
with mature HNP-1 cDNA and full length HNP-1 cDNA showed no
antimicrobial activities in the inhibition zone assays against
Escherichia coli and Pseudomonas syringae pv. tabaci. No
apparent band near 3-4 KD region was found on the 15% SDS-PAGE
gel. To further confirm if mature HNP-1 cDNA can yield protein
with antimicrobial activity, a Nco I site was artificially
created using site-directed mutagenesis and an expression
plasmid pET-mDf was constructed by cloning the Nco I(Sac I
fragment of pBI-mDf into pET28b. The growth of BL21/pET-mDf and
BL21/pET-28b were delayed after 0.4mM IPTG induction. Successful
transcription was evidente with the RT-PCR analysis, but no
apparent 3-4 KD protein band was seen on the 15% SDS-PAGE gel.
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