Functional Analysis Of A Fructose-1,6-Diphosphate Aldolase Gene Ald Yin Rice

Fructose-1,6-diphosphate aldolase isoenzyme is one of the most important isoenzymes that participat in the primary metabolic reactions in plant, and catalyzes reversible aldol condensation reaction of dihydroxy acetone phosphate and3-phosphate glyceraldehyde yielding fructose-1,6-diphosphate. This reaction is an important step in carbon metabolism and glucose metabolic pathways in vivo, and has direct impacts on the accumulation of sucrose and starch in plants. With the further advance of functional genomics research, a large number of genes have been clarified the biological function. However, there is few report on rice fructose-1,6-diphosphate aldolase, and their biological functions are aslo not clear. In this study, a mutant of rice fructose-1,6-bisphosphate aldolase (ALD Y) is used as the material, and though the method of reverse genetics combining with forward genetics, we identify and characterize the fructose-1,6-diphosphate aldolase gene ALD Y and its biological functions.The main findings of this study are as follows:1. Isolating the flanking sequence of a yellow leaf mutant in the laboratory, we find that T-DNA is inserted into the5’UTR region of ALD Y. By cosegregation test, we find the T-DNA flanking sequence cosegregates with the phenotypes of mutants. By Real-time PCR analysis, we find that the expression of ALD Y significantly decreased in the mutant, so names mutant ald y.2. Compared with wild-type ZH11, ald y mutant obviously has the phenotype of yellow leaves and semi-dwarfing, the width of seed becomes smaller, the length between each internode and panicle type are also less than ZH11. By mutant complementation experiment, we find that transforming the plasmid ALD promoter Y::CDS into the mutant callus have partially rescued the phenotype of mutant ald y. Moreover, the photosynthetic pigment content and the expression of chlorophyll synthesis as well as chloroplast development and photosynthesis related genes are positive correlation with the expression of ALD Y.3. ALD Y contains a typical type Ⅰ fructose-1,6-diphosphate aldolase domain and is positioned in peroxisome. BLAST search revealed at least seven genes encoding fructose-1,6-diphosphate aldolase (named OsFBAl-OsFBA6and ALD Y) in the rice genome. Predicting subcellular localization find OsFBA5and OsFBA6are likely to locate in the chloroplast, and other members are likely to locate in the cytoplasm. It also indicates that there is certain relationship between the possible subcellular localizations and distributions in the phylogenetic tree of rice fructose-1,6-diphosphate aldolase family members.4. By looking for the microarray data, we find that the members of rice fructose-1,6-diphosphate aldolase have some differents in expression patterns. By Real-time PCR detection and histochemical staining of GUS activity, we verified and confirmed that ALD Y expresses higher in green tissues such as leaves and leaf sheaths, which result is consistent with the microarray data. In addition, ALD Y can respond to various hormones and NaCl stress,5μM ABA and10μM JA can induce the expression of ALD Y, while other treatments showed no obvious regularity.