Drought-resistant Gene Hva1 In Genetic Transformation Of Tobacco And Corn

China is the second largest maize producer in the world. It yields more than 100 million tons of maize annually which accounts for 20% of total output of the world. China is also the strongest maize consumer in the world and more than 90 % of consumption depends on domestic production. However, drought which is a general and universal abiotic factor places a serous constraint on maize yield. In our country, over 50% of maize is cultivated in arid and semi-arid regions every year. Drought has greatly got to the growth of maize. It will have a harmful effect to the plant's growth to some extent at the whole stage of growth and development of maize. Lack of water has become a major factor of restricting stable and high yields of maize. Therefore, raising drought tolerance of maize has become one of the key problems to be solved immediately in agricultural development. It can be solved by making a gene that functions in drought tolerance transformed into maize to cultivate new strains with genetic engineering technique.Expression of HVA1 gene from barley is thought to be positive correlation with osmotic intimidation tolerance of plant and the deduced polypeptide contains 213 amino acid residues. The protein which was encoded by barley HVA1 gene belongs to group3 lea protein. The gene includes an open reading frame of 642 nucleotides.The expression of HVA1 is organ-specific. HVA1 mRNA only accumulates in aleurone layers of maturation anaphase seeds, and its level remains high in dry seeds. HVA1 mRNA and protein are highly induced by ABA treatment in all organs of 3-day-old seedlings, including root tip, upper root, coleoptile and leaf. HVA1 mRNA is induced by drought, cold, heat, and salt stress.Presently the gene has been transformed into many plants by researchers for acquiring transgenic plants with drought tolerance. For example, a large number of independent transgenic rice (Oryza sativa L.) plants were generated by introducing HVA1 into rice suspension cells using the Biolistic-mediated transformation method. And second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity. The barley gene HVA1 was also introduced into spring wheat (Triticum aestivum L.) cv. Hi-Line using the biolistic bombardment method and transgenic wheat plants with improved biomass productivity and water use efficiency under water deficit conditions have been acquired. Someone has transformed the gene into oats to get transgenic oat plants with osmotic tolerance. From theabove we can see that it is an effective way to increase drought tolerance of maize in introducing drought-resistance gene into plants.The total RNA of 3-day-old barley seedlings which was treated by 0.1mmol/L ABA was gained. Then the HVA1 cDNA gene was obtained by RT-PCR as a model of the total RNA and was appended digestion sites on both sides by PCR. Then a botanic expression vector pCAMBIA1300-HVA1 which was driven under the ubiquitin promoter was constructed. And the recombinant plasmid was introduced into Agrobacterium tumefaciens LBA4404 by tri-parental mating.Tobacco is a model plant in biological research. There are some advantages for the cultivation of tobacco, such as rapid growth and easy large-scale cultivation. The recombinant plasmid was transformed into tobacco with Agrobacterium tumefaciens-mediated. The genome DNA was extracted from transformed tobacco leaves. And it was proved that HVA1cDNA gene had been integrated into the transgenic tobacco genome by PCR and PCR-Southern blotting analysis. It was confirmed that HVA1cDNA gene had been transcripted into mRNA by RT-PCR analysis, which showed the gene was expressed at transcription level. The results of function validation indicated that the leaf water retaining capacity of transgenic tobacco plants was higher one fold than the control. It implied that there was drought-resistance potential for the transgenic tobacco plants. The above results showed that the construction of the botanic expressing vector pCAMBIA1300-HVA1 was complete and functional.On the basis of above experimental results, the recombinant plasmid was transformed into maize callus and immature embryo with Agrobacterium tumefaciens-mediated. Transgenic plants were acquired by plant tissue culture technology. It was proved that HVA1cDNA gene had been integrated into the transgenic maize genome and was expressed at transcription level by PCR, PCR-Southern and RT-PCR analysis.