The Study On Cloning Of HVA1 Gene From Barley And Its Genetic Transformation Into Tobacco

Plant growth process requires a lot of necessary environmental conditions, such as the appropriate water, temperature, etc. The plants which survived in the adverse environment can activate a series of mechanisms to resist the adversity in order to better growth. It is very important to study the mechanism of anti-stress, clone and identify the resistance gene, which can improve the resistance of plant. HVA1 gene was cloned from barley, which belongs to LEA gene family. Many studies have indicated that HVA1 gene plays an important role in improving drought resistance and salt tolerance of plants. Tobacco is one of the important economic crops, and it has become one of the model plants in the field of biological engineering because of its easy to operate and the ability to regenerate. In this study, in order to further verify the effect of this gene on plant resistance, and to provide a theoretical basis for improving tobacco genetic traits, the HVA1 gene in barley was cloned into tobacco.The results in this study are as follows:1. Cloning of HVA1 gene in barleyUsing HVA1 gene sequence, a series of experiments were carried out including the HVA1 gene fragment cloning2. Transgenic plants were obtainedIn this study, 650 pieces of tobacco explants were infected, and by a total of 86,and 16 of the resistant buds were obtained by co-culture and screening.3. Detecting of transgenic plants(1) the HVA1 gene was Preliminary detected in the tobacco genome by PCR assay.(2) To further confirm that the HVA1 gene was integrated into the tobacco genome in different copies of the tobacco genome,Southern blotting was carried out.(3) The expression of HVA1 gene in tobacco resistant plants was confirmed by RT-PCR assay, and it was expressed at RNA level.4. Salt tolerance test of transgenic plantsThe salt tolerance test showed that the resistance of tobacco plants exhibited stronger salt tolerance than wild type plants, especially these plants displayed shorter germination stage(40% earlier than wild type), more vigorous of seeding growth under low salt condition and higher survival span under high salt level.5. Drought resistance test of transgenic plantsOur results indicate the transgenic plants displayed more prosperous root system under drought condition. The average length of root and the dry weight of root increase 8.9% and 64.7% compared to wild type plants respectively. The leaf water holding capacity test also indicate that the transgenic plants have more drought resistance ability than wild type as the transgenic plants have more significant leaf water holding capacity(97.4% higher than wild type).