Functional Analysis Of Glycoside Hydrolase-Like Gene AtGHL In Arabidopsis Thaliana

As one of the four major organic molecules, sugars are important energy sources and structural components that act not only as metabolic substrates but also as signaling molecules between cell to cell. The structural and functional diversity of carbohydrates implies a vast spectrum of enzymes involved in their synthesis (glycosyltransferases), modification (carbohydrate esterases) and breakdown (glycoside hydrolases ).Glycoside hydrolases (also called glycosidases or glycosyl hydrolases) catalyze the hydrolysis of the glycosidic linkage to release smaller sugars. Glycosyl hydrolases have been classified into 130 families based on amino acid sequence similarity. However, there are also some sequences could not be assigned to any family. 401 genes that encoding glycoside hydrolases were identified in Arabidopsis thaliana, which belong to 35 families. Biological function of glycosidases was widely studied because they form the major catalytic machinery for the synthesis and breakage of glycosidic bonds.In this research, we used the method of the reverse genetics to study the function of glycoside hydrolases-like genes AtGHL1 and AtGHL2 by genetic transformation, RT-PCR and other technologies. We found that:1. Result of bioinformatic analysis, both amino acid and nucleotide sequence of these two genes’similarity are over 90%. And both of them have the same Six-hairpin glycosidase-like and DUF1680 domains. But they could not be assigned to any family, so they maybe belong to the new glycoside hydrolases.2. By PCR and RT-PCR, gkl1 and ghl2, two knock-out mutants were characterized respectively for AtGHL1 and AtGHL2. Then RNAi vector was constructed and transformed for getting the mutants which both of these genes were knock-down. Deficiency of the stamen in the mutants implied that AtGHL1 and AtGHL2 maybe involved in stamen development.3. Several transgenic expression vectors were constructed for tissue and subcellular localization of AtGHL1 and AtGHL2 genes. The transfusion tissue of seeding, leaves and flower expressed a higher level of AtGHL1 and AtGHL2 by GUS staining. The two genes were located in the cell wall by fluorescence microscopy. 4. RT-PCR analysis indicated that AtGHL was induced by sugar starvation. In MS without sugar, survival rates of ghl1 and ghli were reduced to 20%.