Characteristic Of A Ca²⁺/H⁺ Antiporter Of Puccinellia Tenuiflora,PutCAX2,Transporting Ca²⁺ And Ba²⁺

This study cloned a Cation/H+antiporter gene(PutCAX2)of Puccinellia tenuiflora from its cDNA.Sequence analysis of PutCAX2 shows,the amino sequence and structure of PutCAX2 is the most similar to that of OsCAX1a and PutCAX2 belongs to I-B of CAX family.The recombinant plasmids of PutCAX2 and its truncated forms were transformed into yeast mutant strain K667.The cation responsing experiment showed,yeast strain of GFP-ANPutCAX2 and GFP-?NCPutCAX2 could grow on medium containing high concentrations of Ca2+ or Ba2+while the others could not grow at all.This demonstrates only N-terminal truncated form of PutCAX2 could enhance the resistance of K667 on Ca2+or Ba2+,it is thus clear that PutCAX2 has N-terminal autoinhibitory.Subcellular localization showed,the fusion proteins of GFP-PutCAX2,GFP-?NPutCAX2 and GFP-?NCPutCAX2 were located on the endoplasmic reticulum of K667,and the fusion protein of GFP-?CPutCAX2 was located on the Golgi of K667.This means the resistance of K667 on Ca2+ or Ba2+ is not affected by the changing subcellular localization of PutCAX2 and its truncated forms.Further verification revealed that Under Ba2+ stress,Ba2+ contents of yeast strains expressing GFP-?NPutCAX2 and GFP-?NCPutCAX2 were lower than those expressing GFP-PutCAX2 and GFP-?CPutCAX2.?NPutCAX2 and ?NCPutCAX2 efflux a portion of intracellular Ba2+to in vitro,and this helps to reduce the toxicity of Ba2+.Then we observed the subcellular localization of PutCAX2 and its truncated forms in Arabidopsis thaliana.The fusion proteins of GFP-PutCAX2,GFP-?CPutCAX2 and GFP-?NCPutCAX2 were located on the Golgi of Arabidopsis protoplast,and the fusion protein of GFP-ANPutCAX2 was located on the endoplasmic reticulum.It means that N-terminal truncation changes the subcellular localization of PutCAX2 in Arabidopsis while C-terminal truncation doesn't.To further understand the mechanism of PutCAX2 and its truncations against Ba2+ stress,we also carry on functional analysis In Arabidopsis.Under Ba2+stress,PutCAX2 and its truncations could improve the germination rate of the transgenic Arabidopsis thaliana.The root length of transgenic Arabidopsis were longer than wild type,and the N-terminal truncation showed higher Ba2+ resistance.