Study On The Transformation Of Populus SimoniiXP.nigra With The Salt-tolerance Gene(Bet-A)

Some salt梩olerant microbes and plant cell can maintain osmotic balance and water in the plant through active accumulation of compatible solute under osmotic stress, such as glucide, polyol, osmotin etc. One of the osmoregulatory is glycine betaine which is considered as one of the most important plant osmoprotectant at present, but glycine betaine can not be accumulated in some important corps and trees. If we transform synthetical gene of glycine betaine into plant梒ell, we might obtain high salt梩olerant transgenic plants. In plant, glycine betaine is synthesized by two step oxidation from choline to glycine betaine . The proceeding is choline梑etaine aldehyde梘lycine betaine, which the first step is established by choline monooxygenase(CMO), and the second step is done by betaine aldehyde dehydrogenase(BADH). Choline dehydrogenase gene isolated from Escherichia co]i, is coded by Bet桝 gene which is one of the Bet gene family . The Bet桝 gene is consist of 1 668 nucleotides, that is capable of encoding a protein in 61.9 KD, choline dehydrogenase(CDH). Since CDH has both function of CMO and BADH in catalyzing reaction, it is a necessary enzyme in the way of betaine synthesis. In this study , we have introduced the Bet桝 gene into Popu]us simonhi XP. nigra by agrobacterium ?mediated transformation. lYe hope to see that glycine betaine can be accumulated and enhance salt stress tolerance in transgenic plant in the future. Firstly, the leaves of this hybrid variety is used as explants and the medium is added some plant hormones, such as NAA and BA in different concentration as experiment component. The optimal medium of cell polarization and multiplication is MS + 0. 5mg/L BA + 0. lmg/L NAA. The best medium of rooting is 1/2 MS + 0. lmg/L NAA. Kanamycin is used as selective agent in transformation, because Bet桝 and NPT II genes is connected in the vector. It needs to test the sensitivity of the explants to Kanamycin in order to decide a reasonable selective pressure for transformation. The critical concentration of kanamycin to the cell polarization is 50 mg/L. That is to say, the lowest concentration of kanamycin in cell polarization is 50 mg/L, and 4Omg/L during transplants rooting in Populus simoniiXP. nigra. Secondly, bet桝 gene was introduced into leaves of asepsis plants of poplar by agrobacterium-mediated transformation. The efficient system of transformation via agrobacterium ?mediated is established by regulating the crucial factor, such as the bacterial concentration, the soaking time, the co梒ulture time, respectively. Then, transgenic plants are analysed by PCR and southern blotting. The result shows that Bet桝 gene has been introduced into the genome of Populus simoniiXP. nigra.