Study On Cloning Of CDNA Encoding Na~+/H~+ Antiporter From Tonoplast Membrane Of Helianthus Tuberosus L. And Salt Tolerance Of Its Transgenic Tobacco

Salt stress is mainly caused by Na~+.The Na~+/H~+ antiporters catalyze the exchange of Na~+ for H~+ across the membrane,and they play an important role in the mechanisms of plant salt tolerance.The cloning,expression and studying the function of the Na~+/H~+ antiporter will bring momentous sense to the genetic engineering of plant salt tolerance.In order to isolate the full length cDNA of Na~+/H~+ antiporter gene from the tonoplast membrane of Helianthus tuberosus,RT-PCR and RLM-RACE were carried out,and a pair of degenerate primers were designed according the totonoplast Na~+/H~+ antiporter homologous gene region of other plants.The gene was named HtNHX1.HtNHX1 cDNA (2148bp) included a 269bp 5′UTR,a 235bp 3′UTR,a putative polyadenylylation signal and a 1647bp open reading frame encoding a 549-amino-acid polypeptide which was 80%, 74%,72%,74%,74%,70%to sequences of KcNHX2,OsNHX2,NcNHX1,PhNHX1,InNHX2,ZmNHX5 in amino acid homology respectively.The deduced amino acid sequence included the conserved amiloride binding sites of LFFIYLLPPI.A was considered as the possible transcription initiation site based on the principle of RLM-RACE and the analysis of HtNHX1 5′UTR sequences,and it would establish foundation for the studying of its relating and expression.To amplify the ORF sequence of HtNHX1,Primers were designed according to the full length cDNA sequence of HtNHX1.And the HtNHX1 ORF was insert into the plasnid of pUC19.The right clone which was ensured by sequencing was used for the constructing of the plant expression vector pBI121-HtNHX1.The resultant plasmid pBI121-HtNHX1 was transferred into Agrobacterium tumefaciens(LBA4404) by the liquid nitrogen freezing thaw method.The HtNHX1 gene was transferred into tobacco(Nictiana tabacum L.cv.89) via Agrobacterium mediation.PCR and RT-PCR analysis showed that the HtNHX1 gene was integrated into tobacco genome.The transgenic tobacco could survive on MS medium containing 200 mmol/ L NaCl for 2 weeks but the control could not survive under the treatment of 100 mmol/L NaCl.