Gene Mapping, Expression And Function Analysis Of Wheat Low-affinity Cation Transporter LCT1

Salinlzation and secondary salinizationof soil have been a seriously problem which caused great loss of production of a variety of crops worldwide,and it is still been a bottleneck for the development of agriculture.The salinified soil contained highly concentration of sodium chloride which inhibitory the growth of plant.Studying the Na⁺ related transporter is important to investigated salt tolerance of the plant.The wheat is in one of the important cereal in the world,and is also the main grain crop in China.Increase salt tolerance of wheat and breeding new varieties with high salt tolerance are very important to ensure sustainable development of grain production.Two ion transporter cDNA(high-affinity K⁺ transporter HKT1 and low-affinity cation transporter LCT1)from wheat were indentified by complemented to yeast potassium deficient mutant(Schachtman et al 1992,1997).At present many researchers concentrate their studies on HKT1.The secondary structure of LCT1 suggests that the protein contains 9 transmembrane helices and two hydrophilic amino terminus containing sequences enriched in Pro,Ser,Thr,and Glu(PEST).LCT1 is mainly expressed in roots and leaves of wheat.LCT1 can transport many kinds of cation in heterologous expression system.Apart from K⁺,LCT1 can also transport Na⁺, Rb⁺,Cd²⁺and Ca²⁺.As a low-affinity cation transpoter for Na⁺ and Rb⁺,LCT1 maybe one of the important ways to uptake Na⁺ in the wheat.So the study of the expression pattern and the function of LCT1 gene is very important to salt tolerance of wheat and other crops.In this work,the genomic DNA of the nulli-tetrasomes and ditelosomic lines of Chinese Spring wheat were used as a template,and the specific primers of LCT1 gene were used for gene mapping.The result indicated that LCT1 gene is located on the long arm of chromosome 1 B.In order to study the function and expression of LCT1 gene,the core promoter of LCT1 were cloned by TAIL-PCR.We acquired 263bp genomic DNA sequence that contains many important cis-elements of the core promoter,such as TATA box,MBS,HSE,G-box et al.In addition to the full-length LCT1 cDNA,we-obtained a splicing variant of it(named LCT1-2)from cDNA library, which is lack of a 115 bp fragment(maybe a intron).In order to study the function of this variant of LCT1 gene trancript,we cloned it into an yeast expression vector and a plant expression vector,transform yeast G19(Na⁺-extruding ATPase mutant, disrupted in the genes encoding Na⁺ export pumps and displays salt sensitivity)and Arabidopsis thaliana.After transformation,G19 with LCT1 became hypersensitive to NaC1 and the G19 with LCT1-2 have no change.It suggested that LCT1 enhanced Na⁺ uptake while LCT1-2 didn't.These two gene transcrapts had been transformed into Arabidopsis thalina by the floral dip method.Larger some of seed of T₁ generation had been obtained which were used to select homologous lines and for physiological experiment next.