Study Of HrmA And Ferritin Gene Transgenic Rice Plants In Disease Resistance

hrmA gene is an avirulance-like gene isolated from Pseudomonas syringae pv. syringae. It was constructed into a prokaryotic cell expression vector pET-29(b). The recombined pET-hrmA plasmid was transformed into Escherichia coli BL21 strain. Recombinant protein was induced by IPTG treatment, and expressed as inclusion bodies. Hypersensitive responses were observed around of the infiltration sites 5 days after treatment of the recombinant HrmA. The result indicated that the HrmA protein was expressed as an active form, and the protein can be used in the study of rice cells. The burst of active oxygen species (AOS) is one of the early defense responses of plant against pathogen attacks. The burst was observed and reached to its maxmum 20 minutes post HrmA treatment of rice suspension-cultured cells. The fast burst of AOS by HrmA treatment was quite similar to the treatment of an elicitor isolated from yeast cell wall. Similarly, the transcription of PBZl gene, a pathogenesis-related (PR) gene was induced in rice cells with both HrmA and YE treatments. However, there was no significant transcriptional induction of glucanase gene with the same treatment in the time period of our study, and the result of Northern blot analysis indicated a low level of constitutive expression of the gene in cultured rice cells. These results suggest that HrmA protein can arise defense responses in rice cells and may play a certain role in rice resistance against pathogens.Based on the results obtained above, HrmA gene was constructed under the control of CaMV35S promoter and a rice PAL promoter, respectively, into a binary vector (pCambialSOl) for plant transformation. Meanwhile, the signal peptide of PRlb was fused into the N-terminus of HrmA protein to lead the expressed HrmA protein to apoplast. Both the constructs were transformed into rice by particle bombardment. In this study, 48 independent transgenic lines were obtained, in which 30 lines were with the control of PAL promoter and 18 lines with CaMV35S. During the processes of plant regeneration, there were not unusual phenomena with both the promoters. However, a few of plants with CaMV35S promoter showed necrosis dots on the leaves, and even died in the greenhouse.The transgenic plants were confirmed PCR analysis, and 20 of 21 lines were determined as PCR positive. The transcripts of hrmA gene was demonstrated by Northern blot analysis. The results showed the mRNA of hrmA gene can determine in transgenic plants but can not determine in non-transgenic plant. The transgenic plants exhibited more resistance to Magnaporthe grisea comparing with the non-transgenic plants.Ferritin is an iron binding and storage protein. In this study, a pea ferritin gene in Tl transgenic plants was confirmed by PCR analysis. Photosynthetic function of the transgenics with 60 umol/L paraquat treatment was determined by measurement of the maximal yield of the photochemical reaction (Fv/Fm value) and the chlorophyll content. The results indicate that transgenic plants exhibited a variety degree of tolerance to oxidative stress. Accumulation of mRNA of Ferritin gene was demonstrated by Northern blot analysis. The results showed the accumulation of mRNA level was elevated in 5 of 9 plants determined. T2 progeny of transgenic plants showed resistance to M. grisea comparing with the non-transgenic plants. The average number of lesion in transgenic plants leaves inoculated with M. grisea was decreased significantly. These results suggest the overexpression of Ferritin gene in rice enhance its resistance to oxidative damage and M. grisea infection.