Cloning And Function Analysis Of Different Sucrose Transporter Genes In Sugarcane

Sugarcane is an important sugar-producing crop in the world. In sugarcane plants, sucrose is the main product of photosynthesis and molecular for carbohydrate translocation, partitioning and storage. During sucrose transporting from source to sink tissues, sucrose transporters (SUT) mediate sucrose transmembrane movement. In plants, the gene family of sucrose transporter includes three subgroups:SUT1type, SUT2type and SUT4type. They are of different functions on sucrose export in source tissues, phloem loading and unloading and carbohydrate accumulation in sink tissues. In this thesis, sucrose transporter genes were cloned by RT-PCR and RACE techniques, and their functions were clarified through expression in yeast mutant SuSy7, subcelluar location analysis, gene expression pattern analysis and transgenic sugarcane, the main results followed as:At the first, one SUT4type gene ShSUT4(GenBank accession numbers GQ485583.1) with full length1611bp, encoding501amino acids including12transmembrane a-helix and highly homological with ZmSUT4, HvSUT2and OsSUT2, was isolated from sugarcane stem cDNA. Yeast mutant SuSy7/ura3was used to identify the sucrose uptake activity of ShSUT4. It showed that ShSUT4had sucrose transporting activity. Both subcellular locations in onion epidermal cells and tobacco mesophyll cells protoplast showed that ShSUT4-GFP located on the tonoplast. The results of expression pattern analysis indicated that ShSUT4expressed in leaves, stems and roots of sugarcane and most highly in leaves. With the detection of ShSUT4expression in different internodes, it showed that ShSUT4most highly expressed in rapid sucrose accumulation internodes. So ShSUT4may play an important role on sucrose accumulation in mature sugarcane stems. In order to clarify ShSUT4specific function in sugarcane, we constructed its over-expression and antisense expression vectors. These vectors were transformed into Agrobacterium strain EHA105. Then sugarcane embryo callus were transformed by these Agrobacterium strains carrying expression vectors. After PPT selection and the survival plants were detection by PCR,6PCR positive transformed sugarcane plants were received.Secondly, one SUT1type gene ShSUT3with full length1791bp, encoding533amino acids including12transmembrane a-helix and highly homological with ZmSUT5and OsSUT5, was also isolated. At the same time SuSy7/ura3was used to identify the sucrose uptake activity of ShSUT3. It demonstrated that ShSUT3also had sucrose transporting activity. Subcellular locations in onion epidermal cells showed that ShSUT3-GFP located on the plasma membrane. The results of expression pattern analysis indicated that ShSUT3expressed in leaves, stems and roots of sugarcane and most highly in stems. With the detection of ShSUT3expression in different internodes, it showed that ShSUT3most highly expressed in rapid sucrose accumulation internodes. So ShSUT3may also have an important function on sucrose accumulation in mature sugarcane stems.In order to further clarify the specific function of ShSUT3in sugarcane, we constructed its over-expression and antisense plants expression vectors which were transformed into Agrobacterium strain EHA105. Then sugarcane embryo callus were transformed by these Agrobacterium strains. After PPT selection and the resistance plants were detected by PCR,17PCR positive transformed sugarcane plants were received.Thirdly, two SUT2type genes, ShSUT2A and ShSUT2B with full length1791bp and2072bp, encoding598and592amino acids both including12transmembrane a-helix and highly homological with ZmSUT2and OsSUT4, were also cloned successfully. Both of ShSUT2A and ShSUT2B could transport sucrose when they were expressed in SuSy7/ura3. Subcellular locations in onion epidermal cells showed that ShSUT2A-GFP and ShSUT2B-GFP located on the plasma membrane. The results of expression pattern analysis indicated that ShSUT2expressed in leaves, stems and roots of sugarcane at the same level and there were no expression variety in different internodes. So ShSUT2may be responsible for sucrose uptake from apoplast space in the whole sugarcane plants.In conclusion, we have isolated three type sucrose transporter genes from sugarcane. All four analysed sucrose transporters had sucrose transporting activity. They involve in sucrose transmembrane movement in sugarcane plants and play an important role in mature stem sucrose accumulation. Further studies on their detailed function will clarify the net regulation mechanism and control points of sucrose accumulation and provide theoretical basis for breeding high sugar-content sugarcane.