Identification of Salt Tolerance and Function Analysis of NHX1 Gene in Glycine Max, Glycine Soja and Their Hybrid Seedlings
|School||Nanjing Agricultural College|
|Keywords||Glycine soja Glycine max Hybrid NHX1 gene Expressing Vector Salt tolerance Subcellular localization Prokaryotic expression|
The Glycine soja BB52 population, Glycine max N23674 cultivar and their hybrid 4076 strain (F5) were used as the experimental materials in this study. Changes in Na+, K+ and Cl- contents were analysed in leaves and roots of soybean seedlings stressed with NaCl (150 mmol·L-1) for different time. Their salt tolerance was compared by PCR of salt tolerance marker gene for DNA samples extracted from leaves. The relative transcript abundance of NHX1 gene in leaves and roots of the three soybean seedlings under salt stress were analysed using RT-PCR analysis. The two different vectors were constructed in onion epidermal cells and E.coli BL21 for studying the subcellular localization of Na+/H+ antiporter and prokaryotic expression of NHX1 gene, respectively. The results showed that:The contents of Na+ and Cl- in leaves and roots of three soybean seedlings continuously increased when they were stressed with 150 mmol·L-1 NaC1 for 0 h,1 h,4 h,8 h,12 h,2 d and 4 d, while the K+ contents decreased with the prolongation of salt stress time. When compared to N23674 cultivar, the increase of Na+ and decrease of K+ were lower, and more Cl- was accumulated in roots and less Cl- was transported into leaves in BB52 and 4076, especially the former. PCR products of salt tolerant marker gene showed that 2 strips (one 740 bp, the salt tolerant marker; and 620 bp, the salt sensitive marker) were amplified in both BB52 and 4076, while only 1 strip (about 620 bp, the salt sensitive marker) was amplified in N23674.The full-long gene of NHXl was amplified in N23674,4076 and BB52 according to the sequence of GmNHXl (Gen Bank accession number AY972078), and the sequence difference of NHX1 were also compared among the three soybeans. NHX1 gene in N23674 and 4076 exactly showed the same sequence with GmNHX1. However, in BB52, there was a different base:T at the 150th site while C in N23674 and 4076, but it didn’t cause the change in their amino acid sequence. The transcriptional level of NHX1 in the three soybeans under NaCl stress was studied using RT-PCR analysis. Under no salt stress, NHXl mRNA showed higher abundance in N23674 than those in 4076 and BB52. Under 150 mmol·L-1 NaCl treatment for 12 h, an obvious rise of NHX1 mRNA levels were observed in both leaves and roots, and those in N23674 showed faster increase than 4076 and BB52, but the increase in N23674 was less than the others, the changes in 4076 were between their parent. NHX1 was inserted into pBI121-GFP vector to study its subcellular localization, the recombinant constructs were delivered into onion epidermal cells by particle bombardment, and strong signals were observed in tonoplast. Another recombinant vector pET30-NHX1 was constructed to express NHX1 gene in E. coli BL21, the target gene was efficiently expressed in prokaryotic cells by SDS-PAGE after IPTG induction.The above results indicate that, the changes in Na+, K+ and Cl- contents in leaves and roots of soybean seedlings under 150 mmol·L-1 NaCl stress are consistent with the difference of salt tolerance in the three expemental soybean materials. NHX1 is located at vacuole membrane, and the gene expression of NHX1 in different soybeant materials is positively correlated with their salt tolerance. The efficient expression of NHX1 gene in BL21 provides the basis for its antibody making and western blot analysis.