The Resistance Of Pathogenesis-Related Protein StPRp27 To Erwinia Carotovora Subsp. Carotovora

Conjac is a perennial herbaceous species belonging to Amorphophallus Blume and has been widely distributed throughout the mountainous areas in Southwest China. Because the corm is rich of polysaccharides, conjac has high value in both agriculture and industry. The soft-rot disease of conjac caused by Erwinia carotovora subsp. carotovora has become one of the most serious diseases in conjac, which even causes destructive damage of the crop sometimes. However, the host resistance has not been identified in cultivated cultivars. Pathogenesis-related proteins (PR) are the proteins induced during the pathogenesis process of plants, which are involved in a broad spectrum to pathogens. PR gene family consists of different embers, individuals may have specific resistance to different pathogen. StPRp27 gene employed in present research is a member of PR-17 family and cloned previously from potato. An assay of antimicrobile was conducted to confirm StPRp27 protein has function against Erwinia carotovora subsp. carotovora. The transformation of StPRp27 gene into conjac was then carried out to lay a foundation for further gene function elucidation. Main results obtained as follows:1. StPRp27 protein has remarkable antimicrobile activity to E. carotovora. Potato pathogenesis-related protein gene StPRp27 was sub-cloned into pGEX-6p-l vector, and the constructed plasmid pGEX-StPRp27 was transformed into E. coli strain BL21 (DE3) for procaroytic expression of the protein. The induction under different conditions showed that soluble fusion protein of GST-StPRp27 was produced when treated with 0.1mM IPTG at 22℃. GST-StPRp27 protein with molecular weight of about 51kD was so obtained through purification and applied for the antimicrobile test. The results indicated that, comparison with GST protein used alone, the fusion protein GST-StPRp27 exhibited a significantly stronger inhibition to the growth of E. carotovora. This impact was enhanced as the concentration of GST-StPRp27 protein increased. Almost no pathogen growth was detected when 0.08mM GST-StPRp27 used, demonstrating that StPRp27 protein has remarkable antimicrobile activity to E. carotovora.2. Selection of transgenic receptor of Amorphophallus rivieri. The induction rate of callus was compared among different parts of petioles excited from A. rivieri plants grown in vito. A 100% of callus induction was observed with morphological base part of the petiole and calli induced were rather large, the callus induction rate of the tip part was just 54.4% with rather small calli in size, whereas the middle part of the petiole showed a moderate ability in callus induction. The results also indicated that a pre-culture for three weeks yielded some calli, but no callus formed in a pre-culture for two weeks. Therefore, the petiole cuttings pre-cultured for two weeks were appropriate explants for the gene transformation.3. After co-cultivation with Agrobacterium tumefaciens and elimination of the bacteria, some petiole cuttings that were lighter than water in term of the density became heavier than the later and lost their capability of callus induction. Adventitious shoots, firstly yielded from the resistant calli during the differentiation culture, were abnormal from which plantlets could not be regenerated. With continuous culture on the differentiation medium, healthy plantlets were regenerated and 28 of total 66 plantlets obtained were positive by the PCR test.