Molecular Cloning And Function Preliminary Analysis Of OsGSTU66 Gene In Rice

GSTs are composed of multiple genes with super family of enzymes that have a variety of functions.GSTs widely exist in animals,plants and microorganisms.GSTs are composed of two homologous or heterologous subunits of dimers.The main function of GSTs combine the catalytic reduced glutathione with some endogenous or exotic harmful substances electrophilic group.The GSTs' hydrophobicity is increasing.Then it is easy to move across the cell membrane and decompose out to the cell,thus reducing the toxic damage to the plant.GSTs can be induced by a variety of abiotic stress and plant hormone,such as drought,high salt,low temperature,high temperature,SA,MeJA and so on.In this study,we clone OsGSTU6 gene which in preliminary study are related to reactive oxygen species.Through the combination of bioinformatics and molecular biology experiment means,we have a preliminary study on OsGSTU6 gene functions.The main research contents and results are:?1?Gene cloning.The OsGSTU6 gene was cloned by RT-PCR.The results show that the length of CDS?Coding sequence?of OsGSTU6 gene is 720 bp encoding 239 amino acids.Bioinformatics analysis show that the gene is located on chromosome 10 in rice.The protein molecular weight is 26.03 kDa and isoelectric point is 4.8.Secondary structure prediction shows that OsGSTU6 has 39.64% alpha helix,20.7% lineage and 39.69% random curl.?2?The subcellular localization.With the expression vector of PTF486 and p35S-eGFP,the fused expression vector of OsGSTU6-GFP were constructed.By the methods of agrobacterium injecting tobacco and PEG mediating rice protoplast,we can see that the fused protein of OsGSTU6-GFP in tobacco cells and rice protoplast are located in the cytoplasm through laser scanning confocal microscope.The results of two experimental methods are the same.So we conclude that OsGSTU6 gene is located in the cytoplasm.?3?The analyses of tissue expression spectrum and induced expression spectrum.The OsGSTU6 gene expression spectrum has been studied by qRT-PCR in the experiment.From the results of tissue expression spectrum,we can see that the gene has been expressed in different organizations of different stages like seeding stage,tilleringstage,booting stage and heading stage.We also find that the expression levels are different in different organizations.The expression levels of OsGSTU6 gene in the ground part of seeding stage is lower,while the 2^nd leaf blade of booting stage is the highest.The expression levels of the young panicles is the lowest in the booting stage.From the induced expression spectrum,we can find that the responses of OsGSTU6 gene are different to the different abiotic stress and hormone treatment.Induced by MeJA,MV and NaCl,the expression levels of OsGSTU6 gene in the ground part of rice rises,while other treatments made no influence on it.Disposed by the low temperature,PEG and MV,the expression levels of OsGSTU6 gene in the underground part of rice rises,while the influences from other treatments are not obvious on it.?4?Cloning and analysis of the promoter.We cloned the promoter of OsGSTU6 by RT-PCR,and the sequences of promoter have 1938 bp in upstream of initiation codon.The sequence of the promoter was analyzed by the online bioinformatics website PlantCARE.Meanwhile,the vector of dual luciferase report system were built,laying the foundation for further function researches of OsGSTU6 gene promoter.?5?The construction of genetic transformation system and the identification of transgenic plants.The expression vectors of OsGSTU6 gene were constructed,and transformed in rice callus by root carcinoma agrobacterium.After stepwise screening,9strains of T0 generation over-expression transgenic plants and 21 strains of RNAi transgenic plants are obtained.The gene expression in T0 generation transgenic plants is analyzed by qRT-PCR.The results show that the expression levels of OsGSTU6 gene in over-expression expression transgenic plants rises about 60 times,while descends in RNAi transgenic plants about 0.2 times.In conclusion,some functions of the OsGSTU6 gene have been studied in this article.The protein of OsGSTU6 locates in the cytoplasm,and expresses in different growing stages of rice.OsGSTU6 gene responds differently to the stress treatment such as drought,MeJA and MV.The transgenic plants of over-expression and RNA interference are obtained in the research,and a solid foundation is established for the study of the gene's metabolic regulation in different stress conditions and rice resistance.