Constructing Of Plant Expression Vector Of Rd22 From Tamarix Ramosissima And Its Functional Verification

The rd22 gene was obtained from Tamarix.sp, which is a drought resistance and salt tolerance plant. It was cloned into the plant express vector pROK II. The recombinant plasmid was transformed into tobacco by Agrobacterium mediated genetic transformation. The purpose is to identificate the function of rd22 gene. 105 kanamycin-resistant buds was obtained in study. Through differentiation, we selected 33 lines to PCR test, 26 lines were positive in PCR. In PCR-Southem hybridization, we checked 6 transgenic plants selected from 26 plants randomly, the results demonstrated that foreign target gene had already been integrated into tobacco genome. The results of reverse transcription-PCR and real-time quantitative RT-PCR approved that the transformed gene can be expressed, the expression level of rd22 gene were 17.12-154.23 times than that of GAPDH gene in the 6 lines.In NaCl and NaHCO₃ stress test, we determined and analyzed the relative conductivity malondialdehyde(MDA) contents the activity of superoxide dismutase(SOD) and peroxidase (POD) between the 6 transgenic tobacco and control tobacco, compared the relative growth . the rate of rooting and salt and alkaline injury index variation, The results as follows:Under different salt and alkaline concentration, the damnification of cell membrane of the 6 transgenic plants was significantly lower than that of control plants, however, the SOD and POD activity of transgenic plants was significantly higher than that of control plants.Under 220 mmol·L^-1 NaCl stress, the growth of control plants was suppressed and can't root normally, the salt-harm index was 76.7%;but the average rate of rooting of all transgenetic plants was 28.9%, of which the highest rate of rooting is the kind of TR3's 40%. The net growths of all the transgenetic plants were more than the control ones while the average net growth was 17.73% higher than the control ones. When the tobacco leaves were cultured under the 220 mmol·L^-1 NaCl stress, the regeneration of tobacoo leaves indicates that the control tobacco leaves were badly harmed under the NaCl condition and can't differentiate while the differentiation rate of transgenetic plants ranged between 20% and 40%.After putting the transgenetic plants' leaves under the NaHCO₃ stress, the result indicated that the control plants' rooting was suppressed and can't root normally under 30 mmol·L^-1 NaHCO₃ stress condition while all other transgenetic plants could grow better root and the average rooting rate was 28.9%, of which the rooting rate of TR3 was highest, 40%. All varieties of transgenetic plant' growth were more in comparision with the control ones' 54.96%.All the experiments results above indicated that the expression of external gene rd22 in transgenetic tobacco enhanced resistance to bad condition of tobacco and significantly reduced the harm caused by salt, which absolutely proved the rd22 gene from Tamarix.sp has thethe harm caused by salt, which absolutely proved the rd22 gene from Tamarix.sp has the funtion of increasing ability of tolerance to NaCl and NaHC03.