Agrobacterium Tumerfaciens Transformation Of Wheat By E.coli OtsA Gene And Barley HVA1 Gene

Wheat is one of the most important crops, whose productivity and quality are guarantees on food supplies throughout the world. Abiotic stresses, such as drought, salinity, extreme temperatures and oxidative stress are serious threats to wheat growth and seed production. Thus, it has great significance to culture new wheat varieties with abiotic stresses resistance. Plant improvement in stresses tolerance is mostly based on the manipulation of genes that protect and maintain the function and structure of cellular components. In this thesis, two genes, trehalose-6-phosphate synthase gene (otsA) from E.coli and HVA1 gene from barley, are isolated separately, transformation of wheat by either of them are able to improve its resistance to abiotic stresses. We expect to gain higher stresses tolerance wheat and valuable varieties through transformed wheat with these genes.Trehalose is a kind of indeoxidize disaccharide coupled with two glucose by a, α-1,1 bond, which plays an important physiological role as an abiotic stress protectant in a large number of organisms, including bacteria, yeast, and invertebrates. Trehalose has been shown to stabilize enzymes, proteins, and lipid membranes and protect biological structures efficiently in dry condition. Besides, recently researches have shown that trehalose is necessary for plants growth and development , regulation ofglucose sensing, carbon metabolism and photosynthesis.In Escherichia coli, trehalose-6-phosphate synthase (TPS), which encode by otsA gene, catalyze UDP-glucose and glucose-6-phosphate to form trehalose-6-phosphate. We designed a pair of primers according to otsA sequcence that published on Genbank and gained the gene through PCR. Two binary plasmids, pRAL-otsA and pR29A-otsA , had been constructed , and the former was drived by constitutiveexpression promoter ubiquitin, the latter was drived by drought-inducible promoter RD29A. Both of plasmids have excised the selectable marker genes in order to pass biology safety test easier. Agrobacterium AGL1 was transformed by the two constructed expression vectors respectively by freeze-thawing method. 300 wheat plants of cultivars yan2801 were infected by Agrobacterium bearing pRAL-otsA plasmid through shoot apical meristem transformation. 214 of To generation plants and 1250 seeds were obtained. We used the method of PCR to detect otsA gene in 140 To generation and there were 9 PCR-positive plants. 276 of Ti generation were growed in greenhouse after vernalization. The result of PCR showed that there were 21 positive plants. At the same time they exhibited many visible variations but the reason is unclear now. All of these will be benefit to study the function of trehalose gene in wheat and can been used to enhance the tolerance to abiotic stresses.Another gene HVAl was isolated from barley through RT-PCR, which encode the group 3 late embryogenesis abundant (LEA) protein. HVAl gene can be induced by dehydration, salt stress, cold stress and ABA. Several researches have demonstrated that transgenic plants with HVAl gene could endow high tolerance to dehydration. So we constructed two plant expression vectors without selectable marker genes, pRAL-HVAl and pR29A-HVAl, and then transformed yan2801 with them through Agrobacterium-mediated shoot apical meristem transformation. We have obtained 100 and 36 To generation separately.This study make a steady basis for acquiring drought, salinity stress tolerance transgenic wheat plants and for researching the function of trehalose and HVAl protein in wheat resistance stresses.