Study On Mhtga2Gene Transformat Of’Nagafu No.6’Apple

Apple genetic is high heterozygous, the traditional cross-breeding of apple to breed improved strains is extremely difficult and long breeding period, plant genetic engineering provides a new way for developing resistant cultivars. TGA2is a member of the TGA class subfamily in the bZIP transcription factor gene family, as bZIP gene family members are involved in several biological processes, including plant growth, flower development, seed maturation, aging, the optical signal, injury and a variety of stresses response. The function of MhTGA2gene was analzied through the transgenic tobacco plants. In the hope of getting strong resistance apple cultivars, MhTGA2gene was introduced into ’Nagafu No.6’by Agrobacterium-mediated transformation. The main results are as follows:1、In order to optimize malus plant regeneration system and establish the genetic transformation system of apple’Nagafu No.6’, taking leaves of rooted seedlings and secondary seedlings of apple varieties’Nagafu No.6’and rootstock varieties M hupehensis Rehd., M robusta Rehd. as materials, the leaf regeneration differences between Malus plants rooted seedlings and secondary seedlings and the effect of different dark culture time on Malus plant rooted seedlings leaf regeneration were studied. Taking leaves of’Nagafu No.6’rooted seedlings as receptor materials, the effective factors on genetic transformation for apple ’Nagafu No.6’were discussed in this study, the methods PCR and RT-PCR were used to detect hygromycin-resistance shoots and the induction rate was calculated. The results showed that:the leaf regeneration ability of rooting seedlings was better than secondary seedlings, the dark culture time for21d was the best condition; the highest transformation efficiency was obtained when the OD600nm of Agrobacterium tumefaciens solution was0.5, the explants were infected in the bacterium solution for9min, the co-culture time was3d, and the Hyg concentration was1mg/L, PCR and RT-PCR analysis indicated that the exogenous gene likely was transferred into’Nagafu No.6’and in transcription level of expression.2、Three weeks old M. hupehensis leaves were sprayed with0.01mM abscisic acid (ABA) respectively for4h,12h and48h, with water treatment as control. Two years old M. hupehensis seedlings, grown in the glasshouse under the temperature of25℃, were treated by different stress factors:high salinity (200mM NaCl), high osmotic pressure (10%PEG6000), and low temperature (4℃). Leaves were sampled at0,4,12, and48h after each treatment. The results of qRT-PCR analysis showed that:The MhTGA2gene was clearly induced by low temperature and NaCl, weekly induced by PEG-6000and ABA.3、Take seeds and seedlings of transgenic tobacco as materials, we analyzed the resistance of transgenic tobacco seeds and seedlings. The results show that:Overexpression of MhTGAl gene in the transgenic tobacco plants confers enhanced resistance to NaCl in the stage of seeds generation and seedlings studied, and resistance to mannitol in the stage of seeds generation. These results supported that:MhTGA2gene involved in systemic acquired resistance and has enhanced the functionality of plants to environmental stress endurance.4、For the cultivation of high resistance of apple’s new lines, the Agrobacterium tumefaciens method was used to importe MhTGA2gene imported into apple ’Nagafu No.6’. There were seven PCR positive plants and five RT-PCR positive plants were detected by PCR and RT-PCR. By RT-PCR-positive strains and non-transgenic plants to NaCl stress test, preliminary proved that the import of gene MhTGA2enhanced plant tolerance to salt stress.