Subcellular Localization And Function Analysis Of Wheat Low-affinity Cation Transporter LCT1

Abiotic stresses,such as salinity,drought and extreme high or low temperatures have great impact to wheat growth;largely influence its production and quality.LCT1 gene of wheat,a gene for low-affinity cation transporter,was first cloned from wheat cDNA library which can be complement to yeast K⁺ mutant G19(Schachtman et al., 1997).Wang et al also cloned LCT1 gene and another splicing variant LCT1-2 from full length eDNA pool,LCT1-2 has deletion of 115 base pairs fragment(perhaps intron),that cause early stop code in its mRNA,and LCT1-2 had no effect on Na⁺ transport.We constructed two transient expression vectors 326-sGFP-LCT1 and 326-sGFP-LCT1-2,fused its coding region with GFP gene.After transformed to Arabidopsis thaliana protoplast,the fluorescent signal indicated that LCT1 gene product might be located in the membrane of plasma and chloroplast,but LCT1-2 protein obviously lost its affinity to membrane,most product accumulated in the cytoplasm.In order to study the function of the LCT1 gene performs in the development of wheat or/and under abiotic stresses circumstance,an RNAi vector was constructed and transformed into wheat.We had obtained many LCT1 gene silencing lines which need further analysis.One of the group 3 late embryogenesis abundant(LEA) protein encoded by HVA1 gene of barley which was enriched in the late embryogenesis of seed development and could be induced by draught,salt,cold stress and ABA treatment.Several researches have demonstrated that transgenic plants with HV1 gene could endow high tolerance to dehydration.We also transformed wheat yan2801 through Agrobacterium-mediated shoot apical meristem transformation with barley HVA1 gene driven by RD29A promoter.After carefully check of T₀ generation and T₁ generation,the plants of T₂ generation was stressed long time of drought.But to our surprise we found that all the transgenic plants in T₂ generation appear more sensitive to dehydration than control. Due to there was no HVA1 gene transcription inspected by RT-PCR,the expression level of several members of LEA gene family were also tested but without valuable result.This may be the result of gene silence induced by HVA1 gene transformation, but we need more work to prove it.It must be very useful to understand the mechanism of this phenomenon,which will give a great help to the research of increase plant drought tolerance driven by HVA1 in wheat.