Differential Expression And Function Of Various Vacuolar Na～+/H～+ Antiporter Genes From Different Species In The Salt-and Drought-stress Response

Salt stress is one of the major detrimental environmental factors that affect producitivity of crops around the world. Salt stress disrupts homeostasis in ion distribution of plant cell. High levels of sodium ion (Na~+) in the cytoplasm are toxic to plants because of their adverse effects on cellular metabolism. One important strategy for plants to improve salt tolerance is to reestablish the ionic homeostasis. Ionic homeostasis involves Na~+ entry, efflux, sequestration and long distance transport. To cope with salt sress, plants have developed a variety of adaptation mechanisms. One of them is the compartme- nttation of Na~+ into the vacuoles by the vacuolar Na~+/H~+ antiporters. The imp- ortance of vacuolar sequestration to plant salinity tolerance has been underli- ned by experiments in which constitutive overexpression of different vacuolar Na~+/H~+ antiporter genes can greatly increase salinity tolerance in a wide range of plant species.In this study, we isolated the vacuolar Na~+/H~+ antiporter genes GhNH- X1(GhN1),ScNHX1(ScN1),AtNHX1(AtN1),TaNHX1(TaN1),SsNHX1 (SsN1) from cotton,yeast,Arabidopsis,wheat and Suaeda salsa, respectively. The function analysis demonstrated that these genes play important role in plant salt tolerance. The main results of this study were summarized below:1,According to the sequences of the vacuolar Na~+/H~+ antiporter genes in GenBank, we designed specific primers to isolate vacuolar Na~+/H~+ antiporter genes from different spesies. Five genes which designated as GhN1,ScN1,AtN1,TaN1 and SsN1 were isolated from cotton,yeast,Arabidopsis,wheat and Suaeda salsa, respectively.2,The deduced amino acid sequence alignment showed high identities between plant vacuolar Na~+/H~+ antiporters from cotton, Arabidopsis, wheat and Suaeda salsa, but had low identities with vacuolar Na~+/H~+ antiporter from yeast. The phylogenetic analysis of various Na~+/H~+ antiporters also indicated that GhN1, AtN1, TaN1, SsN1 share a cluster. GhN1, AtN1,SsN1 from dicotylous plant species and TaN1, HvNHX1, HeNHX1 from monocotylous plant species showed much higher homology compared with the overall sequence alignment, respectively. But dicot originated NHX1s are more divergent from the monocotylous ones.3,The expression of GhN1,ScN1,AtN1,TaN1 and SsN1 in yeast Na~+/H~+ antiporter mutant showed functional complementation. But these genes showed distinct levels in functional complementation. Among them, AtN1 confered the yeast mutant with the highest complement ability; But TaN1 confered the lowest complement ability.4,GhN1,ScN1,AtN1,TaN1,SsN1 were introduced into Arabidopsis plants. Northern blot showed that GhN1,ScN1,AtN1,TaN1,SsN1 were expressed in transgenic Arabidopsis. Under 200 mM NaCl treatment the wide-type Arabidopsis were severly damaged. On the contrary, the transgenic plants were less affected. Furthermore, the transgenic plants showed higher rates of photosynthesis rate than wide-type plants under salt stress. After 200 mM NaCl treatment for 7 days, theψp was sustained at normal level in transgenic plants, whereas the wild-type plantsψp was near zero. The transge- nic plants overexpressing different NHX1 genes have different phenotype. For example, after 7-day treatment with 200 mM NaCl, SsN1 overexpressing Arabidopsis showed more compacted size, darker green resette leaves and significantly increased chlorophyl content compared with other transgenic plants. Under drought treatment, different plants also showed different phenotype. The results above indicated that different Na~+/H~+ antiporters from various species can increase the salt- and drought- tolerance of Arabidopsis but in different levels.5,We have made 3'terminal deletion of GhN1 and SsN1. The 3'truncated genes not only complemented the salt-sensitive phenotype of yeast mutant, but also showed an increased salt tolerance compared with the corresponding full-length genes.6,Interactive proteins were screened from cotton leave cDNA library with the N and C terminal of the vacuolar Na~+/H~+ antiporter cloned from cotton as bait protein by Yeast Two-Hybrid System. More than 20 positive clones were identified by PCR colony screening and 12 representative clones were sequenced (Two clones were screened with the N terminal as the bait protein and the other 10 were screened with the C terminal as the bait protein). By blast in GenBank, the prey cDNA inserts were homologous to mRNA sequences in Arabidopsis. RT-PCR analysis indicated that A11, A14, B10 and B14 were induced by 200mM NaCl, and A1 was repressed while no detectable changes were found in A3,A5,C3,C8,N5 and N14. When treated with 100μM ABA for 6h, A1,A11,A14 and B10 were elevated to higher level, A5,C3,C8 and N14 were reduced. The expression level of A3,B14 and N5 were less affected by ABA. These data indicated that some of them are responsive to salt stress and ABA, and presumablely are vital compartments involved in cotton salt stress tolerance and ABA signaling.