Cloning Of A Vacuolar Na~+/H~+ Antiporter Gene NnNHX1 From Nelumbo Nucifera And Preliminayr Study On Salt Tolerance Of Transgenic Tobacco

Salt stress seriously affected the growth and development of plant, while the vacuolar membrane Na+/H+ antiporter could transport and compartment the excess Na+ into vacuoles, playing an important role in plants for salt tolerance.The cDNA of NHX1 gene which was then designated as NnNHXl was successfully isolated from 'Donghe'by RT-PCR and RACE cloning method. The full-length cDNA of NnNHX1 was 2237 bp, included a 426 bp 5'UTR, a 194 bp 3'UTR, and a 1617 bp ORF which was predicted to encode a 59.12 kDa protein with 11 highly hypothetical transmembrane domains in the N-terminal part while a hydrophilic tail in the C-terminal part. The bioinformation analysis of NnNHX1 indicated that the amino acid sequence NnNHXl showed highly homologous with NHXls from Vitis vinifera, Oryza sativa and Arabidopsis thaliana respectively, and all of them had a high degree conserved amiloride binding-site sequence LFFIYLLPPI. The phylogenetic tree analysis showed that NnNHX1 shared the same group with vacuolar Na+/H+ antiporter protein, but distinguished from the plasma Na+/H+ antiporter protein.A plant expression vector for NnNHXl gene pCAMBIA-1301-220-NnNHX1 was constructed and was transferred into wild Nicotiana tabacum by leaf disc transformation, then 14 tobacco plants with hygromycin-resistant were obtained after co-culture, sub-culture and root screening. Then 7 hygromycin-resistant tobacco plants were determined to be transgenic plants by PCR analysis, which were named as T-N1-T-N5,T-N9 and T-N10 respectively. The fluorescence quantitative primers were designed based on the conservative sequences of NHX1 and the specific sequences of NnNHX1, the results showed that the expression level of NnNHX1 and total NHX1 in transgenic plant T-N1,T-N2 and T-N10 were higher than those in control. The salt stress of tissue culture seedlings showed that when the NaCl concentration was 100 mmol/L in culture medium, control and transgenic plants could both root, but the growth condition of transgenic plant was better than that of control. when the NaCl concentration was 200 mmol/L, control couldn't root, meanwhile the leafs and stem etiolated, verging on dying, but transgenic plant colud still growth slowly. Salt stress on potted seedlings of control and transgenic lines T-N1, T-N2 and T-N10, and then we measured its related physiological indexes, the results showed that under normal condition, each physiological indexes in leaves of control and transgenic lines were approximately same, and under salt stress, the chlorophyll content in all plants were both significantly decreased, but the decrease in transgenic plants were less than control; the content of MDA and relative conductivity had risen, and the increment in control was a little higher than transgenic plants; the content of Pro and SOD and POD activity all increased both in transgenic lines and control plants, and compared with control, change range in transgenic plants were more significant.The germination rate results of transgenic tobacco lines under salt stress showed that transgenic tobacco seed germination was better than control, indicated that the salinity tolerance trait could be inherited.