The Expression Of Salt Tolerance Gene SeNHX1 In Tobacco

Se NHX1 gene is a vacuolar Na+/H+ reverse transport protein gene cloned from Salicornia(Saliconia europaea L.), which expresses the Na+/H+ reverse transport protein localed in the vacuole membrane. Vacuolar Na+/H+ reverse transport protein can transport Na+ to vacuoles from the cytoplasm, which plays an important role in maintaining the cytoplasm ion balance, and adjusting the cytoplasmic ph value and maintaining cell osmotic pressure. In the previous research work, transformed tobaccos with salt tolerance gene SeNHX1 have been obtained by using of Agrobacterium-mediated transformation. In this thesis research, transformed tobaccos with SeNHX1 gene as materials, in order to investigate the expression of salt tolerance gene SeNHX1 and its impact on the salt tolerance of transgenic tobacco plants, SeNHX1 gene in tobaccos was identified by PCR,RT-PCR, and real-time quantitative PCR was performed on RNA to detect SeNHX1 gene expressionin in transgenic tobaccos. Transgenic tobaccos with SeNHX1 in physiological and biochemical characteristics under salt stress are tested and analysised.The main research results are as follows:(1)Transformed tobaccos with SeNHX1 gene T0 generation was identified by PCR. The results showed that SeNHX1 gene had been successfully transformed in tobaccos and six transgnic tobaccos with SeNHX1 gene S1, S2, S3, S4, S5, S6 were identified respectively. The transgenic tobacco plants T1 generation detected by PCR, preliminary resuLts showed that SeNHX1 genes were inherited by transgenic tobacco T1 generation.(2)The RNA of transgenic gene tobaccos with SeNHX1 and wild type tobaccos was extracted and reversed transcription.The results of RT-PCR showed that SeNHX1 genes have been expressed in transgnic tobaccos and further identified these tobaccos wer transgenic plants.(3)Transgenic tobaccos with SeNHX1 were determined by real-time fluorescent quantitative PCR. The expression of SeNHX1 gene in non-transgenic and transgenic tobaccos S1, S2, S3, S4, S5, S6, was 0%,20%, 26.7%, 14.29%, 25%, 12.5%, 23.1%.(4) With NaCl salt stress factor, stress concentrations were 0 mmol / L, 100 mmol / L, 200 mmol / L; The change of antioxidant enzymes activity, MDA and chlorophyll content of non-transgenic and transgenic tobaccos under different salt stress concentration were detected. The results were as follows: 1) The SOD, APX, CAT, POD activity of non-transgenic and transgenic tobaccos with Se NHX1 increased with increasing salt concentration, and the antioxidant enzyme activities transgenic tobaccos with SeNHX1 were higher than non-transgenic tobacco. 2) In the absence of salt stress treatment condition, The MDA content of transgenic tobaccos with SeNHX1 and non-transgenic tobaccos are not differences. But under salt stress, the MDA content of all tobacco with elevated NaCl concentration increased, all genetically modified tobacco plants SeNHX1. The MDA levels of transgenic tobaccos with SeNHX1 were lower than non-transgenic tobacco. 3)Under salt stress, the chlorophyll content of all tobaccos declined, and is negative correlation with NaCl concentration. Under the same concentration of NaCl treatment, the chlorophyll content of wild type tobaccos is lower than transgenic tobaccos and downs significantly with increasing concentration of NaCl.(5) T0 generation of transgenic tobaccos with SeNHX1 seeds and wild type tobacco seeds after disinfection were sowed in 1/2 MS cuLture medium with tendency for 0mmol/L, 50mmol/L, 100mmol/L0mmol/L, 150mmol/L, 200mmol/L NaCl. 2 weeks later, the seeds, germination rate was statisticed. 1)The result showed that wild type tobaccos and transgenic tobaccos with SeNHX1 gene seed germination rate was close to 100% in the absence of NaCl. 2)When the concentration of NaCl tendency for 50 mmol/L、100mmol/L, all tobaccos seed germination rate was on the decline and the fall was not very big, but transgenic tobaccos seed germination rate fell more slightly than wild type tobacco. 3)When the concentration of NaCl was tendency for 150mmol/L, all tobaccos seed germination rate dropped substantially, wild type tobacco seeds decreased significantly. 4)When the concentration of NaCl was tendency for 200mmol/L, all tobacco seed germination rate decreased to a minimum; Wild type germination rate was only 8%; transgenic tobacco germination rate was about 25%; Transgenic tobacco plant S3 seed germination rate was the highest.