The main purpose of this study is to investigate the function of bromelain gene from pineapple and cathelicidin peptide originated from human on enhancing resistance through raising transgenic cabbages, testing gene expression and evaluating resistanc...
The main purpose of this study is to investigate the function of bromelain gene from pineapple and cathelicidin peptide originated from human on enhancing resistance through raising transgenic cabbages, testing gene expression and evaluating resistance to diseases. The experimental results are summarized as follows;
Chapter 1: Expression of a BAA1 gene encoding fruit bromelain in Chinese cabbage enhances resistance to soft rot disease
1. BAA1 gene was isolated from pineapple, build Ti-plasmid vector under control of CaMV 35S promoter in pIG121Hm, and transformed into Agrobacterium tumefacienciens strain LBA 4404. Eight whole plants were regenerated after transformation of cotyledon of Chinese cabbage. The eight transgenic plants were used to bring progenies.
2. Presence of BAA1 gene in the eight transgenic plants was confirmed by PCR analysis and expression of the gene in the plant was also confirmed by RT-PCR analysis. For the exact dose of mRNA, real-time PCR was held and the amount differed between the transgenic plants. The most highly expressed transgenic line was B66-3-1.
3. BAA1 protein was found at 120kDa of the transgenic plant by Western blot analysis, and B66-3-1 line had 0.9% more total protein compared to wild type plants in ELISA analysis.
4. B66-3-1 line harboring the new BAA1 gene showed difference on expression of 2,013 genes in microarray analysis. Among those, over-expressed genes were defense related protein (At1g75830), protein kinase (At2g40560), and protease inhibitor (At5g38195) which were over eight times more expressed compared to non-transgenic plants.
5. To find out the resistance to the soft rot of transgenic cabbage which BAA1 gene was expressed, leaves of the transgenic lines were inoculated with Pectobacterium carotovorum ssp. carotovorum at the density of 2 × 10^(4) and 2 × 10^(6) CFU/ml. Although there was difference in symptom development between the transgenic lines, scarcely no soft rot symptoms developed at 2 × 10^(4) CFU/ml and a little disease developed at 2 × 10^(6) CFU/ml in the transgenic plants. But soft rot symptom was found in 24 hours in the wild type plant even at 2 × 10^(4) CFU/ml.
Chapter 2: Expression of a antimicrobial peptide gene(Cathelicidin LL-37) in Chinese cabbage enhances resistance to both fungal and bacterial pathogens
1. Cathelicidine LL-37 peptide gene was amplified by PCR, and constructed at E. coli expression vector pET 28. As the result of examining the antifungal ability to gram negative E. coli DH5α and gram positive Bacillus subtilis, the clear inhibition zone was found.
2. Plant infection vector VB2 with LL-37 gene was constructed, transformed into Agrobacterium tumefacienciens LBA 4404 line, infected the cotyledon of cabbage and raised 20 transgenic plants. Presence of HPT, 35S promoter, and LL-37 genes in four transgenic plants were confirmed by PCR analysis.
3. LL-37 mRNA was detected in four transgenic plants by RT-PCR analysis and the expected 4kDa sized protein band was found in the four transgenic plants by Western blot analysis.
4. To find out the resistance to the soft rot of transgenic cabbage which LL-37 gene is expressed, leaves of transgenic lines were inoculated with Pectobacterium carotovorum ssp. carotovorum at the density of 2 × 10^(4) and 2 × 10^(6) CFU/ml. After 12 hours, tissues of the leaves of the wild type plant began to soften and soft rot symptoms were found all over the leaves after 48 hours. However, in transgenic lines except line B23, there was no soft rot symptom developed even until 72 hours after inoculation.
5. Transgenic plant harboring LL-37 gene showed 66%, 75%, and 80% more resistance to Colletotrichum higginsianum, Rhizoctonia solani and Fusarium oxysporium, respectively than wild type plant.