Fusion, Function Identification Of CNHX1 Gene And Study On Influence Of Transgenic Strawberry Plants To NaCl Stress Tolerance

Stress from environment especial from the salinization and drought is the most importantfactor for plant growth to be limited. Recently,several transgenic plants which exhibitenhanced remarkably salt resistance by constitutive expression Na+/H+ antiporter genes,raised the glory perspective to obtain salt resistance crop plants by using this tactic. Thefunction of NHX gene isolated from fruit tree plants and the NHX gene transgenic fruit treeplants had not been reported up to data. In present research, the complete cNHX1 gene wasfused by using SOEing method, and the ion compartmentation function,ion specific and therestrain effect to programmed cell death induced by high concentration NaCl were identifiedby expression it in multi-gene mutated yeast strain. The results proofed that cNHX1 gene wasa functional vacuole membrane Na+/H+ antiporter. The cNHX1 gene expression character,copy number and several physiological events variation of transgenic strawberry lines undersalt stress were analyzed also. All results mentioned above could be used for reference toutilization of cNHX1 gene to transformation other fruit tree plants. The main result were asfollows: 1. According to the sequencing results of the 5ˊ and 3ˊsegments of cNHX1 gene,4primers ,F01,F02,R01 and R02,were designed adopted to the principle of SOEing method.One 500bp fragment was amplified from vector 1 containing 5ˊsegment by using F01 andR01,another 1200bp was amplified from vector 2 containing 3ˊsegment by using F02 andR02, and the full length cNHX1 gene was amplified by using F01 and R01 from mixture of500 and 1200bp fragments. The sequencing result showed two mutation (T to C) werepresented at 77 and 804bp in plasmid T1,and the first mutation resulted in amino acidsequence variation. The restrict enzyme digestion method was adopted to correction themutation by replacing a 200bp fragments in T1 plasmid in which the 77bp mutation locatedfrom a responding fragment of T10 plasmid, this is the first report on getting complete NHXgene from fruit tree plants. 4山东农业大学博士学位论文 2. Several biological software were used to analyzing sequence characters. Thealignment result showed cNHX1 had high homology to other plant NHXs. Phylogenetic treeconstruction result showed the existence of more close relationship between cNHX1 tofunctional AtNHX2 and AtNHX1, and more distant relationship to non-functional AtNHX4,AtNHX6 and LeNHX2 gene (K+/H+ antiporter). Functional domain search result showed acomplete Na+-H+ exchanger was located in cNHX1 protein from 26 to 449 amino acidresidues. Topology model prediction result showed cNHX1 protein contained 9 TMs.Subcellular localization prediction result showed cNHX1 protein was an IIIa type membraneprotein,no any known localization signal were found in it. 3. The subcellular localization of cNHX1 protein was confirmed further by differentiateblue fluorescence dots that DAPI stained mitochondria emitted from green fluorescence dotswhich cNHX1-GFP fusion protein emitted. The image analyzing results showed cNHX1protein was not located in mitochondria but in vacuole membrane. 4. The ion compartmentation activity and ion specific character of cNHX1 protein wasanalyzed by expression it in AXT3K yeast cells. cNHX1 gene was inserted into pYX212vector to obtain pYXNHX vector,and this vector was transformed into AXT3K yeast cells.Growth curve of W3031B,AXT3K and transformed AXT3K cells under 50mM NaCl stressconditions demonstrated that 50mM NaCl resulted in growth refrain effect,but expressivecNHX1 gene reduced this refrain effect partial to AXT3K cells. Drop assay were performed toelucidate the ion compartmentation activity and ion specific of cNHX1 protein,result showedAXT3K cells contained expressive cNHX1 gene had better growth state then wild AXT3Kcells on YPD solid plate which 50mM NaCl was added. The growth state was obviouslydifferent when the three yeast culture were...