Cloning And Function Analysis Of ShSWEET1and ShSWEET2in Sugarcane

Sugarcane(Saccharum officinarum L.) is a high photosynthesis efficiency C4plants in tropical and subtropical regions and it is one important sugar and energy crop in world and China. Sucrose, the main product of photosynthesis, is the major form of carbohydrate to transport, distribute and store from source organs to sink organs in sugarcane plants. SWEETs protein is one kind of transport protein which could transmembrane transport sugar from phloem parenchyma cells into the apoplastic space. Besides that, SWEETs play important roles in plant stress adaptation and the interactions of plants and pathogen. Therefore, the isolation and identification of sugarcane SWEET genes will shed light on the distribution mechanisms in sugarcane. In this article, the full-length cDNA of ShSWEET1and ShSWEET2are isolated basing on homologous cloning. Homologous, transmembrane structure, tissue-specific expression as well as the expression pattern analysis under stress conditions and also transgenic analysis are conducted to further understand the important functions of ShSWEET1and ShSWEET2. The main research results are as follows:1. The full-length cDNA of ShSWEET1and ShSWEET2are isolated basing on homologous cloning and bioinformatics analysis reveals that ShSWEET1contains an Open Reading Frame of732bp which encodes243amino acids; ShSWEET2contains an Open Reading Frame of774bp which encodes257amino acids. Both ShSWEET1and ShSWEET2have7transmembrane a-helix and share high identities with maize and sorghum SWEETs.2. Tissue-specific expression profiles of ShSWEET1and ShSWEET2in tissues at various elongation stages in sugarcane are analyzed by RT-qPCR and the results show that: ShSWEET1and ShSWEET2are expressed in roots, immature stems, mature stems, immature leaves and mature leaves, indicating that ShSWEET1and ShSWEET2are constitutive expression in sugarcane.3. The expression profiles of ShSWEET1and ShSWEET2in three kinds of sugarcane diseased leaves are studied which find that the expression levels of ShSWEET1and ShSWEET2in sugarcane leaves infected by brown streak disease, smut disease and red stripe disease are higher compared with those in healthy sugarcane leaves. ShSWEET1has the highest expression level in leaves infected by brown streak which is fairly2.6times of that in healthy sugarcane leaves. ShSWEET2has the highest expression level in leaves infected by red streak disease which is8.2times to its expression level in healthy sugarcane leaves. These results indicate that the expression of ShSWEET1and ShSWEET2 genes may be activated and induced at mesophyll cell membrane upon bacterial infection, causing more sugar efflux into mesophyll intercellular and provide favorable environment for pathogen invasion and propagation.4. The express analysis of ShSWEETl and ShSWEET2in sugarcane plantlets subjected to cold stress shows that:the expression levels of ShSWEETl and ShSWEET2gradually increased as the stress continued within96hours, indicating the important roles of ShSWEET1and ShSWEET2in cold resistance. Thus the expression vectors (pShSWEET1and pShSWEET2) are constructed to transform wild-type tobacco through leaf disc infection method. The transgenic tobacco plants grow faster than the wild-type under cold stress with small and sharp leaves, luxuriant root and vigorous vegetative growth. ShSWEETl and ShSWEET2genes may participate in soluble sugar transport by reducing osmotic potential in sugarcane to maintain the normal growth of sugarcane in under cold stress.5. Genetic transformation of sugarcane through agrobacterium-mediated method obtained4positive ShSWEET2transgenic plants and ShSWEETl transgenic plants.