Studies On Salt-tolerance Of Transgenic Nicotiana Tabaccum And Arabidopsis Thaliana With The Vacuolar H~+-pyrophosphatase Gene From Halophyte Chenopodium Glaucum

Salt stress is the primary effect that limits the growth and development of plant, the higher salt concentration in the soil what can arouse many damages for the plant, for example, the loss of ion balance, oxidation damage, the deficiency of water, the lack of nutrition, which lead to the destroy of biology molecule, the slower of growth and the faster death of plant.Ultimately, those could result in the fall of plant yield or on harvest.That is why plant has to reduce cellular Na~+ and keep K~+/Na~+ balance when exposed to salt stress.Under salt stress, Na~+ influx into cells occurs via Na~+ permeable transporters which in turn elevates the cytoplasm sodium concentration and causes toxicity.Na~+ sequestration into vacuoles was not only avoid to destroy the cytoplasm, keep a higher K~+/Na~+ balance in cytoplasm, but also can be used as an osmolyte in the vacuole to help to achieve osmotic homeostasis.So, it is necessary to adapt the salt stress for plant, Na?~+ compartmentation meansby the energy provided by transporting proton along its concentration gradient,vacuolar-type Na~+/H~+ antiporters deliver Na~+ into the vacuoles against the electrochemical gradient generated by vacuolar H~+-ATPase and H~+-PPase.The salt tolerance of plant can be advanced by accumulate to a higher level Na~+ in the cytoplasm through enhance the over expressing of vacuolar H~+-PPase.The enhanced salt tolerance of transgenic Arabidopsis, tomato, rice by overexpressing vacuolar H~+-PPase genes indicated their important roles in plant salt tolerance. Chenopodium glaucum L. is a Chenopodiacea halophytic plant species, which widely distributes in Xinjiang.It is the typical salt-tolerant plant, and it can grow normally under 100~600mmol/L NaCl treatment.The appearance of the whole plant of C.glaucum has neither chylocaulous stem, nor also no any specialized leaf, relatively closer to that of glycophyte.Therefore, it is more valuable to study the mechanism of salt tolerance and for applying on glycophyte to enhance the salt tolerance.Based on all of these, in this thesis, a binary expression vector pCAMBIA1301.1-CgVP1 which harbored CgVP1 gene was constructed and introduced into Arabidopsis thaliana and Nicotiana tabaccum plant by Agrobacterium-mediated method.The function analyses of CgVP1 transgenic plants have been done by molecular identification and some physiological indexes assay.The key results were as follows:1. The binary expression vector pCAMBIA1301.1-CgVP1 was introduced into A. thaliana by Agrobacterium-mediated method.T3 homogenous plantlets were obtained by screening hygromycin-resistant seedlings.PCR and RT-PCR analysis indicated that CgVP1 have been integrated into the genome of A.thaliana and expressed at transcriptional level.Overexpression of CgVP1 gene in A. thaliana led to enhanced tolerance to salt stress.The relative growth rate of wild-type was restrained markedly under 150mmol/L NaCl treatment.The germination rate of transgenic plants was 91.7%, 89.2%, 84.2%, repectively, while the wild-type was 67.5%, 53.3%, 35.8%, respectively when exposed to 100 mmol/L, 150 mmol/L, 175 mmol/L NaCl.The result of the Na~+, K~+ determination suggested that sodium was compartmentated in the root in order to decrease the sodium concentration of the leaf in transgenic plants under salt stress.Moreover, the relative water and chlorophyll content assay indicated that the relative water content of transgenic plants was higher than that of wild-type,and a higher chlorophyll content was also detected in transgenic plants under salt stress.Our data showed that overexpression of CgVP1 gene could enhance the capacity of salt-tolerance in transgenic A.thaliana.2. The binary expression vector pCAMBIA1301.1-CgVP1 was introduced into tobacco by Agrobacterium-mediated method.T0 plantlets were obtained by screening the hygromycin- resistant seedlings.PCR and RT-PCR analysis indicated that CgVP1 gene have been integrated into the genome of tobacco and expressed at transcriptional level.The relative growth rate of both wild-type and transgenic plants were restrained lightly under salt treatment.The K~+ /Na~+ rate and the relative water content of transgenic plants were higher than that of wild-type, and the electrolyte leakage rate was lower than that of wild-type after being exposed to 400 mmol/L NaCl for 30 days.The drought tolerance assay indicated that transgenic plants performed better than that of wild-type, such as growth status, almost no hurt after being drought stress for 45 days.But the water loss was too serious in wild-type to be restored.Our data showed that overexpression CgVP1 gene could enhance the capacity both in salt and drought tolerance in transgenic tobacco.