Isolation And Function Identification Of A Putative Tonoplast Na～+/H～+ Antiporter Gene In Cotton And Its Important Role In Salt Stress

In plants, by the energy provided by transporting proton along its concentration gradient,vacuolar-type Na+/H+ antiporters deliver Na+ into the vacuoles against the electrochemicalgradient generated by vacuolar H+-ATPase and H+-PPiase. This compartmentation of Na+provides an effective mechanism to avert the deleterious effects of Na+ in cytosol andmaintain an osmotic potential by using Na+ in the vacuoles as an osmoticum. The enhancedsalt tolerance of transgenic Arabidopsis, tomato and rice overexpressing tonoplast Na+/H+antiporter genes indicated their important roles in plant salt tolerance. Cotton is one of the oldest and most important fiber and oil crops. It is wildly cultivated insaline-alkali soil of inland or in the sea valley. A great achievement has been obtained.Therefore, cotton as an economic crop cultivated in saline-alkali soil is very important for theusage of saline-alkali soil. But the salt tolerance of cotton is limited, usually not more than0.3% for most of cultivars. So it is valuable to improve the salt tolerance of cotton. However,little is done about studies in the molecular level. It is still interesting to verify whether or notthere exists the compartmentation of Na+ into the vacuoles in cotton. In this thesis, a series ofstudies have been conducted on the isolation, sequence and expression analysis, functionidentification of cotton vacuolar-type Na+/H+ antiporter basis on the materials of asalt-tolerant cotton cultivar ZM3 and two salt-sensitive cultivars ZMS12 and ZMS17. Themain results are as follows: ～4～山东农业大学博士学位论文 1. Two mixed primers were designed to amplify specific DNA fragment using cDNAprepared from the leaves of cotton on the homologous sequences from other plants. Themiddle fragment of interested cDNA was obtained by RT-PCR. The full length cDNA wasfurther isolated by 5'–RACE and 3'-RACE. The clone contains 2485 nucleotides with a5'-untranslated region(5'UTR) of 509 bp, an open reading frame (ORF) of 1629 bpcomprising 543 amino acid residues with the predicted molecular mass of 60.0 kDa, and a3'-untranslated region(3'UTR) of 344 bp. 2. The deduced amino acid sequence showed high identities with other plant vacuolar-typeNa+/H+ antiporters from Arabidopsis, rice and Atriplex, which were 77.6%, 79.7%, 77.1% and80.6%, respectively. But had low identities with plasma-membrane Na+/H+ antiporters frommammalian, Arabidopsis and E.coli, which were 13%, 7.9% and 5.9%, respectively. Thisindicated that the full length cDNA encoding a putative vacuolar-type Na+/H+ antiporter,which was designed as GhNHX1. The phylogenetic analysis of various Na+/H+ antiportersalso indicated that GhNHX1, AtNHX1, OsNHX1, AgNHX1, ScNHX1 share a cluster. 3. By the TMpred software, the deduced protein sequence contained the highly conserved9-12 transmembrane (TM) segments at the N terminal and a C-terminal hydrophilic region. Aputative amiloride binding domain ( 85LFFIYLLPPI94 ) is in TM3 in GhNHX1. TM5 andTM6 seemed not span the tonoplast, which are considered critical for antiporter transportactivity. 4. Northern blot analysis indicated that the mRNA accumulation of GhNHX1 was stronglyinduced by salt stress. The mRNA accumulation was more abundant in the seedlings treatedwith higher concentrations of NaCl. The relative GhNHX1 gene expression did not changemuch from 3 h to 24 h, suggesting that the expression of GhNHX1 in seedlings is inducedrapidly by salt stress. The transcription level of GhNHX1 was also up-regulated by KCl, ABA(abscisic acid), LiCl and SA (salicylic acid) with a higher induced level by ABA, suggesting anon-specific salt stress response and the osmotic responsiveness of the gene isABA-dependent. 5. The expression of GhNHX1 in yeast Na+/H+ antiporter mutant showed functionalcomplementation, confirming the vacuolar localization of GhNHX1. The transgenic tobaccoplants overexpressing GhNHX1 could grow well under 200 mM...