Transformation Of Malus Domestica With Mhrar1and Mhsgt1Gene From Malus Hupehensis For Resistance Of Physalospora Piricola

RAR1and SGT1are tow important signaling components in plant disease resistance approach mediated by resistance (R) gene which may play their roles between R protein downstream and eactive oxygen species release. The main purpose of this research is to study the expression characteristics in different plants and gene functions of plant disease resistance reaction of Rarl and Sgtl.We studied the the role of ribosomal DNA-internal transcribed spacer (rDNA-ITS) in classifying and identifying the Botryosphaeria berengeriana pathogen, and the correlation between sequence mutations and biological characteristics and pathogenicity. Conditions were screened for conidiospore induction method in in vitro cultures of the pathologic pathogens, which could provide material for the disease detection.Based on previous transformation system, we would transfer MhRarl and MhSgtl, cloned from Malus hupehensis, to Nicotiana tabacum and Nagafuji No.6. The transgenic plants would be infected by Botrytis cinerea Pevs. and Botryosphaeria berengeriana de Not f. sp. Piricola (Nose), in the purpose of investigating the effects of the tow genes on disease resistance for tobacco and apple.The research results were as follows:1. Ten fungal strains that cause apple ring rot disease(Physalospora piricola Nose) were used as experimental material. PCR amplification were performed for the rDNA-ITS regions with universal primers ITS1and ITS4. After sequence alignment there were variant bases found in the sequences in five strains. Through bioinformatic analysis a phylogenetic tree was constructed,and all ten strains were to be located in the I sub-variety of Botryosphaeria berengeriana through homology comparisons. The growth status, sporulation ability and phylogenetic effect of different variant strains were determined. The strains with a higher degree of variation performed better in terms of biological characteristics and were more pathogenic than the strains with less variation and normal strains, which suggested the variations in the ITS1region might have more predictive power. The influence of culture medium types, pH, temperature and light and the effect of mycelia scraping were investigated. The appropriate developmental conditions for ring rot colonies were determined to be25-30℃on a potato-based medium at a pH5.0-7.0. Alternating irradiation of12h/d black light and12h/d fluorescent light and scrape treatment of fungal mycelia were best able to induce conidiosporangia and sporulation.2. Through leaf disc method, MhRarl and MhSgtl were transferred into tobacco by Agrobacterium, with the induction frequencies were61.02%and55.58%. After PCR and RT-PCR amplifications on Hyg positive plants,11MhRarl strains and8MhSgtl strains were finally screened. We determined the changes of antioxidant enzymes (SOD/POD/CAT) activities and MDA content in transgenic plants after Botrytis cinerea inoculation, to investigate the resistance effect of the over expression of MhRarl or MhSgtl in tobacco. The results showed that both kinds of transgenic tobacco plants could display more rapidly increase on all enzymes activities and gentle MDA changing waves, which could be beneficial to the resistance reaction. The excised transgenic leaves could display better resistance to Botrytis cinerea, as well as the Ti generation young seedlings grown from self-fertilized seeds with Hyg resistance, which suggested that these two over expression genes have influenced the tobacco resistance ability, which could be heritable.3. With similar method, MhRarl and MhSgtl were transferred into apple by Agrobacterium, with the induction frequencies were2.47%and1.53%. After PCR and RT-PCR amplifications on Hyg positive plants,6MhRarl strains and3MhSgtl strains were screened. We determined the changes of antioxidant enzymes (SOD/POD/CAT) activities and MDA content in transgenic plants after Botryosphaeria berengeriana inoculation, to investigate the resistance effect of the expression of MhRarl or MhSgtl in Fuji apple. The results showed that both kinds of transgenic tobacco plants could display more rapidly increase on all enzymes activities and gentle MDA changing waves, which could be beneficial to the resistance reaction.