The Study Of The Enhanced Drought Tolerance In Tobacco By Transgene Pyramiding

Drought is the most significant environment stress in agriculture worldwide and improving the drought tolerance of plants throught genetic engineering is one of most effective strategies to solve this problem. Single-gene transformation approaches have been used to confer drought tolerance in transgenic plants; however, the tolerance to drought stress of plants is a complex trait that is modulated by multiple genes and requires the coordination of many genes. It is becoming increasingly certain that introducing more than single gene involved in plant drought tolerance into a single plant by gene pyramiding is considered as an efficient approach for extreme drought tolerance.The betA gene from Escherichia coli, which encodes chroline dehydrogenase (CDH), could increase the betaine content in transgenic plants; the TsVP gene from salt cress (Thellungiella halophila), which encodes vacuolar H~+-translocating inorganic pyrophosphatase, would enhance the transport across the vacuolar membrane. It has been reported that overexpressing each gene alone can improve the drought tolerance of plants.In this study, the betA gene, TsVP gene and transgene pyramiding betA×TsVP transgenic tobaccos were named BL, TL and BT, respectively. It is affirmed that the two genes were integrated into the genome and expressed functionally in transgene pyramiding tobacco plants by detecting RT-PCR, the determination of betaine content and V-H~+-PPase hydrolytic activity. The study of the enhanced drought tolerance in the single gene transgenics and transgene pyramiding plants was performing at the whole plant level and cellular level. The results were as follows:The seed germination percentage of transgene pyramiding line BT is higher than that of single-gene transgenic line BL and TL under the same mannitol concentration. But in seedlings growth stage there was no significant difference among them. It suggested that transgene pyramiding could further enhance plant drought tolerance at the seed germination stage..Under drought condition, the TL line and the BT line showed more developed shoot and root systems compared with WT and BT line and the biomass of BT line was the highest. The TsVP-transgenic tobacco plants (TL and BT) showed significantly higher net photosynthetic rate than WT and BL under normal conditions. Drought stress decreased the photosynthetic activities of both transgenic and wild-type plants, while the transgenic plants exhibited higher photosynthetic rate and Fv/Fm ratio than wild-type, implying the higher photosynthetic efficiency in transgenics. The results of the measurements on other photosynthesis features also indicated the transgenic plants had higher photosynthetic capacity than the wild-type ones under drought stress conditions, but the difference in photosynthesis between transgene pyramiding plants (BT) and single gene transgenics (BL and TL) was not significant.No matter before or after drought stress, the TsVP-transgenics and the transgene pyramiding plants have higher soluble sugars content. After stress, the proline content and RWC of the transgenic plants were higher than those of WT. This enable transgene pyramiding plants have lower solute potential and made it keep more water under drought stress.The BT line showed the highest drought tolerance with the lowest MDA content and ion leakage of leaves under drought stress and higher cell viability and better integrity of mitochondrial membrane than wild-type cells under the same PEG treatment. These results suggested the improved drought tolerance in pyramiding transgenic cells.The cytoplasmic and vacuolar pH values were recorded by means of the pH-sensitive, cell-permeant fluorescent indicator BCECF-AM. The transgenic cells, especially TsVP transgenic cells, showed the less change of pH. The vacuolar pH values in TL and BT cells were lower than those in wild-type ones. The membrane potential produced by pH difference across the vacuolar membrane (ΔΨ(H~+)) was calculated using Nernst equation. TheΔΨ(H~+) values of TsVP-transgenic and transgene pyramiding cells were significantly higher than those of wild-type and betA-transgenic cells before and after stress. These results suggested that the V-H~+-PPase could acidify vacuoles and maintain higher proton electrochemical gradient across the vacuolar membrane in TsVP transgenic cells. These suggested that they could maintain the intracellular environment homeostasis better than wild-type under drought stress.