Clone And Functional Analysis Of Glycine Max Na~+/Li~+Tolerant Gene (GmSLT)

Soybean (Glycine max) is one of the most important economic crops in China.Sodium ion is the most important factor affecting the growth and productivity ofsoybean in saline soil.Salt tolerance in higher plant involved complex genesexpression, regulation and plant cellar and physiological regulation for plant copingabiotic stress. Though there are many salt tolerance related genes have been cloned,but detail molecular mechanism still need to be explored.In this study, according to soybean salt tolerance main QTL mappinginformation provided by Institute of Crop Science, Chinese Academy of AgriculturalSciences, one0.5Mb genomic sequence of soybean was targeted. About200ORFs inthis region were predicted and functional assigned by BLAST. Semi-quantificationand real time PCR analysis revealed that spatial temporal expression pattern ofGmSLT was co-ordinated with saline stimulation. Then GmSLT was heterologouslyover-expressed in the Saccharomyces cerevisiae W303-1A, the salt tolerent ability ofW303-1A was improved from100mM NaCl to500mM NaCl presence in the yeastgrowth medium by drop test and growth curve analysis.The functional domains of GmSLT were analyzed. Three domains wereidentified in GmSLT, they are amiloridebinding site (ABS)，transmembrane structuredomain (TM) and zinc-binding site (ZBS). All three domains were deleted by overlapping PCR respectively. Three yeast expression plasmid were constructed withtruncated GmSLT, GmSLT△ABS，GmSLT△TM，GmSLT△ZBS. Additional truncatedplasmid, GmSLT△M303, was constructed as well to delete the N-terminal303aminoacids.The experimental results show that the salt tolerent ability of W303-1Atransformed with truncated construct was reduced to the level of control when TMdomain, ABS domain and N-terminal303amino acids of GmSLT were deletedrespectively. But the salt tolerent ability of W303-1A was not affected when ZBSdomain was deleted. These results revealed that ABS and TM are the key domains forGmSLT function. The function of N-terminal303amino acid is not coordinated withthe AtSLT1in Arabidopis and NtSLT1tobaca respectively, which acting as theautoinhibitory domain reported in literatures. GmSLT fusion with eGFP wereconstrcted to investigate the subcellular localization of the gene. The eGFP signalsindicated that GmSLT protein was located in nucleus. GmSLT protein goes nucleus aswell when ZBS domian was deleted and the function of the gene retained. The fusionprotein of GmSLT goes to cytoplasm and the function of the gene was dispeared whenABS domian was deleted. No eGFP singal can dedected when TM and N-terminal303amino acid were deleted and the gene‘s function was gone as well. These resultssuggested that transported to nucleus was essential for GmSLT to be functional. ABSdomain may be related with the nucleus targeting of GmSLT which has not been reported before. Meanwhile, the TM and N-terminal303amino acid may be relatedwith the expression regulation of GmSLT.Further study need to be carried out to investigate the molecular function ofGmSLT in vivo by transforming the GmSLT into rice, arabidopsis and soybean.Meanwhile further study on molecular mechanism of GmSLT, like how ABS, TMdomain function in the protein, will provide valuable information to elucidate thefunction of GmSLT.