The Expression Analysis Of Apple(Malus Domestic) MdFKs Genes And Prokaryotic Expression

Sugar is the main components of the fruit. As a soluble sugar, fructose is the sweetest one and the main composition in apple. Fructokinase plays an important role in the accumulation and metabolism of fructose. In this study, we analyzed the information of apple fructokinase(MdFK) gene family based on Malus genome database, their expression characteristics and their enzymatic activity by prokaryotic expression. The main findings are as follows:1. Four FK genes were screened out from the apple genome. These MdFKs are highly homologous with Lycopersicon esculentum and Arabidopsis thaliana. MdFK1 and MdFK4 are 1077 bp and the amino acid homology is as high as 92.5%, but MdFK1 is homologous with LeFK1(AAB57733), MdFK4 homologous with LeFK4 on the base sequence, respectively. MdFK2 and LeFK2 were highly homologous and the amino acid homology was 80.9%.2. qRT-PCR showed that four MdFKs were all expressed in apple. MdFK2 has the highest expression abundance, and mainly expressed in shoot tips and young fruit. MdFK1 mainly expressed in mature leaves and fruits. By analyzing the genes associated with suger metabolism in the shoot tips of transgenic apple trees with decreased sorbitol synthesis and studing the exogenous sorbitol and sucrose responding mechanism, we found that MdFK1 and MdFK4’s transcription is regulated by the sorbitol and fructose content: the reduced sorbitol synthesis caused the decreased expression level of MdFK1 and MdFK4, but sorbitol feeding increased the expression level of both. Reduced sorbitol concentration lead to the increasing expression level of MdFK2. This may be related to different affinity of the protein and different functions of fructose in the sink organs.3. Subcellular localization showed that MdFK1 were on the plasma membrane and cytoplasm, MdFK2 located in all the cell, MdFK3 located in the plasma membrane and the nucleus, and also there is a chloroplast shuttle conservative zone, we suspect that it may code a plastid protein.4. MdFKs prokaryotic expression and soluble induction of target protein revealed that: the purpose gene was subcloned into the prokaryotic expression vector pGEX-4T-1(+) and we constructed the restructuring plasmid. The expression product whose molecular mass about 50 kD was checked by SDS-PAGE, which showed that the target protein was successfully expressed in E.coli BL21(DE3). Because the protein always exist in the form of inclusion body, we rebuilt the recombinant bacteria pSUMO-FK2. Due to the help lysis of SUMO tag, the target soluble protein was successfully expressed in E. coli BL21(DE3), and high quality protein of MdFK2 was obtained via one step of the Ni column purification.