The Function Analysis Of Salt Tolerant Gene HAL1 In Yeast

Salt tolerant gene engineering provide a rapid and effective approaching to select salt tolerant variety in forestry breeding. Compared with traditional breeding, gene engineering breeding not only significantly shorten the breeding period, but also directionally improve the genetic characteristics of forestry. One of the most important elements of the salt tolerant gene engineering is to isolate the salt tolerance gene and to analysis it's function so as to supply excellent gene resources for gene engineering. In this study, the HAL1 gene cloned from yeast AS2.375 was inserted into a binary vector pAHF, and the pAHF was further transformed into Agrobacterium tumefactions, LB4404 by three-factor-across. The HAL1 gene was transformed into tobacco by Agrobacterium through leaf disc transformation and the salt tolerance character of transgenic tobacco was analysised. Moreover, the /L4Ll gene was further transformed into Populus simonii X P. nigra with the expectation to obtain transgenic plants. The main results of these study are as follows: 1. Established the optimal tissue culture system by using tobacco as experimentmaterial; The culture medium inducing bud from the leaf of tobacco was MS +0.5mg/ L 6-BA +0.05 mg / L NAA; The culture medium inducing root was 1/2 MS +0.5 mg/ L NAA; 50 mg/ L Kanamycin was used to be selector. 2. Obtained the transgenic tobacco, the results of PCR amplification of them showedthat 6 of 8 transformants obtained the special bands at 900bp. 3. Salt tolerant tests implied that 481 of 900 transgenic tobacco can rhizogenesis inthe concentration of 0.80%~1.50% NaCl, with rate of 53.45%; In contrast, therooting rate of control can only rhizogenesis in the culture medium with NaCl lowerthan 0.80%, and the rate is 0% in 0.80% NaCl. 4. Took progeny test of open- pollination transgenic tobacco. Observe bourgeon ofbuds in the culture medium contains NaCl, the progeny of transgenic tobacco isoutstanding than the control's. PCR amplification showed that the target gene wasstill in the genome of transgenic tobacco's progeny. 5. Determined the best concentration of kanamycin for HALl gene transform intoPopulus simonii X P. nigra. It is 20mg/L Km to induce bud ; 50 mg/L Km to get the new plants, and 30 mg/L Km to induce root . 6. Obtained the transformants of Populus simonii X P. nigra. PCR amplificationshowed that 8 of 10 transformants have special bands of HAL1 gene. Further test should be made to conform them.