| Salinity is one of the major factors effects the plants growth.Exposed to high saline condition brings high levels of Na~+ in the plants cell and altered K~+/Na~+ rations,thus disrupts metabolisms processes of plants.Maintaining intracellular K~+/Na~+ ration by ion homeostasis is essential for plants development and salt tolerance.As the regulation role of ion homeostasis are complex and various,isolation and research the K~+ and Na~+ transporter gene from the halophyte provides a model case for understanding the molecular basis of ion transporter responded to salinity in plant cells.In this study,two ion transporter genes encoding vacuolar Na~+/H~+ antiporter(AlNHX1) and high-affinity K~+ transporter(AlHAK1) were isolated from a graminaceous halophyte Aeluropus littoralis(Gouan) Parl,and the hydrophobic and homology of putative proteins were analyzed.Furthermore,the two genes were overexpressed in tobacco and the physiology of the transgenic plants response to salinity was studied.In addition,the promoter of AlNHX1 was isolated and analyzed in detail.Our studies provided theoretical foundation and test basis for application of the ion osmotic regulation genes in improving the salt tolerance of monocot crops via genetic engineering.The research contents and results in detail were stated as follows:1.Vacuolar Na~+/H~+ antiporter gene of Aeluropus littoralis(AlNHX1) was isolated by RT-PCR and RLM-RACE based on the homology.The 2706bp AlNHX1 eDNA sequence contained a 387bp 5'-UTR and a 696bp 3'-UTR,as well as an open read frame of 1623bp encoding a 540 amino acids.The deduced transcripts AlNHX1 shared a high homology with those putative vacuolar Na~+/H~+ antiporters of higher plants.It was predicted that AlNHX1 contained ten putative hydrophobic regions.In addtion,there were two or three copies of AlNHX1 in A:littoralis genome.2.The ORF of AlNHX1 was constructed into plasmid pBI121 and then transferred into tobacco via Agrobacterium-madiated method.The transformed plants were verified by PCR, Southern blot and Northern blot analysis.Overexpression of AlNHX1 in tobacco conferred high salt tolerance to the transgenie plants.The transgenic plants could root in the medium containing up to 300mM NaCl,while the wild-type plants failed to root higher than 150mM NaCl.With different NaCl concentration treatment,the dry weight of transgenic plants were higher than wild-type tobacco,while the relative electronic conductivity and osmotic potential of transgenic plants were significantly lower than that of wild-type tobacco.The analysis of ion contents indicated that under high salt stress,the transgenic plants accumulated more Na~+ in roots and kept a relative high K~+/Na~+ ratio in roots and leaves compared with wild-type plants.3.Northern blot analysis showed that the expression of AlNHX1 was induced by NaCl, ABA or drought treatments,and the increased transcript level of AlNHX1 was much higher in root than that in shoot under salt stress.A 2036bp 5'-flanking region of the AlNHX1 containing a putative promoter was cloned by anchored PCR method.Cis-regulatory elements analysis showed AlNHX1-promoter contained three ABRE and two DRE motifs.The 2036bp promoter and three deletion derivatives were respectively fused to gus reporter gene and transferred into tobacco.Histochemical staining indicated that the GUS expression directed by AlNHX1-promoter occurred in leaves,stems and roots during the entire plant life cycle,but not in reproductive organs.GUS quantitative fluorometric analysis of progressive upstream deletions of the promoters suggested that the ABRE located at -607,and the DRE located at -1506 might involve in the regulation of AlNHX1 under ABA and drought stress.Besides, GUS expression directed by AlNHX1 promoter appeared to be root-specific under salt stress.4.A high affinity K~+ transporter gene(AlHAK1) was cloned by RT-PCR and RLMRACE method based on the homology from Aeluropus littoralis.The 2844bp AlHAK1 cDNA sequence contained 300bp 5'-UTR,213bp 3'-UTR,as well as an open read flame of 2331bp encoding 776 amino acids.The deduced transcripts AlHAK1 shared a high homology with HAK1 of reed and barley,and predicted containing twelve putative hydrophobic regions. Southern blot analysis suggested that there were two or three copies of AlHAK1 in A.littoralis genome.In addition,the expression of AlHAK1 was root-specific and up-regulated by salt stress and K~+ deprivation.5.The ORF of AlHAK1 was inserted into the yeast expression vector pYES2,and then introduced into the yeast mutant strains W△3,which was deficient in high affinity K~+ uptake. AlHAK1 restored the growth of W△3 under micromolar K~+ concentrations and the W△3-HAK could uptake K~+ efficiently with a Km value of 8±0.8μM,indicating that AlHAK1 was a high-affinity K~+ transporter.Compared with W△3-YES,W△3-HAK showed less sensitivity to Na~+.Under 300mM NaCl,the growth of W△3-HAK was less affected by K~+ concentration range from 10μM to 100mM,while the growth of W△3-YES was seriously inhibited, suggesting that AlHAK1 might also mediated a low-affinity K~+ transport.6.The ORF of AlHAK1 was constructed into plasmid pBI121 and transferred into tobacco via Agrobacterium-madiated method.The transformed plants were verified by PCR, Sourthern blot and RT-PCR analysis.Overexpression of AIHAK1 in tobacco enhanced both salt tolerance and potassium nutrition of the transgenic plants.The transgenie plants were capable of rooting under salt stress up to 250mM NaCl in the MS media,while the wild-type plants could not.Under micromolar K~+ concentrations stress,the transgenic plants uptake more K~+,about 2 or 3-fold in wild-type tobacco.Under NaCl treatment,the dry weight and germination rates of the transgenic tobacco were remarkably higher than the wild-type plants, while the relative electronic conductivity and osmotic potential of transgenic plants were lower than wild-type tobacco.Ion analysis indicated that the transgenic plants accumulated more K~+ and maintained a high level of K~+/Na~+ ratio compared with wild-type plants under salt stress.
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