CDNA Cloning, Expression And Functional Identification Of Sucrose Synthase From Ornithogalum Caudatum

Numbers studies showed that glycosylation modification of drugs has played a crucial role in enhancing drug activity, increasing bioavailability and reducing toxic and side effects. However, the nucleotide sugar donors which used in glycosylation modification of drugs have few problems, such as the scarity of content, tedious extraction and difficulty in synthesizing. These problems limit the further study on the glycosylation of drugs, development of innovative drugs and the needs of social sustainable development. But these problems can be solved by synthetic biology. According to analysis of the biosynthesis pathway, the informations of related enzymes can be gotten. Finally, the mentioned nucleotide sugar donors can be obtained by study and assembly the gene of key enzymes.With the analysis of biosynthesis pathway of nucleotide sugars, it has been found that UDP-glucose and UDP-glucuronic acid are precursors of the mentioned nucleotide sugars. Therefore, the further analysis of biosynthesis pathway of UDP-glucose and UDP-glucuronic acid may be a key to synthesis nucleotide sugars by synthetic biology. Three nucleotide sugar biosynthesis pathway have been found through documentary analysis, and the sucrose synthase pathway is the shortest. Through this pathway, UDP-glucose can be generated by one step reaction. Besides, it has been found that UDP-glucose is at the core position of biosynthesis pathway of nucleotide sugars, it can be converted to a variety of nucleotide sugars. Thus, the study of sucrose synthase is a foundation of synthesis nucleotide sugars using synthetic biological technology.1, Cloning and expression of sucrose synthase gene familyTotal RNA was extracted from sterile bulb tissue of O.caudatum, and been use as a template for reverse transcription to obtain the cDNA. Three sucrose synthase genes were cloned using nested PCR. And then, according to the method of In-Fusion, primers were designed for expression vector construction of target genes. Soluble expression of OcSus1, OcSus2 and OcSus3 was obtained by direct expression or co-expression with molecular chaperone.2, Functional identification of sucrose synthase gene familySince, sucrose synthase is an enzyme catalyzing both the synthesis and cleavage of sucrose in a reversible manner. Sucrose synthase activity was, therefore, determined in both directions. OcSus1-3 was initially determined to have the function of synthesizing sucrose using the anthrone method. In the direction of cleavage of sucrose, reaction product UDP-glucose was determined by ion-pair reverse-phase high-perfomance liquid chromatography (RP-HPLC). And it was further confirmed by mass spectrometry and co-inject of reaction products and UDP-glucose standard. A final conclusion can be got that, both OcSusl, OcSus2 and OcSus3 have sucrose synthase function.3, Substrates preference of sucrose synthase gene familyTo investigate the substrates preference of OcSus isoforms, other nucleotide substrates like ADP, CDP, GDP and TDP were also used to incubate with sucrose in the sucrose breakdown direction. The results showed that, only UDP facilitated sucrose cleavage for the both three sucrose synthases. Besides. TDP is much less effective than UDP and able to only act as the substrate of OcSus1.4, Characterization of OcSusl-3 in the sucrose breakdown directionThe optimum temperature and most suitable pH in the sucrose breakdown direction of OcSusl-3 were not same. OcSusl and OcSus3 displayed maximum activity at 50℃. However, the optimum temperature of OcSus2 was 37℃. For the optimum pH, OcSus1 and OcSus2 were 7.0. while OcSus3 was 5.0. Besides that, the effect of different concentration of divalent metal ion on sucrose breakdown reaction of OcSus isoenzymes were also determined. Except Ca2+, all the divalent metal ion used in experiment had a inhibitory effect on sucrose cleavage activity. We also determined the kinetic parameters of the OcSus 1-3 in the breakdown direction. The results showed that, the three Km values for UDP were all smaller than that for sucrose, suggesting a higher affinity for UDP than sucrose in the three Sus isoforms. In addition, OcSus2 showed the highest affinity for UDP, while OcSusl has the highest affinity of sucrose and highest catalytic efficiency among the three isoforms.5, Expression pattern of sucrose synthase gene familyExpression profiles of OcSus genes were performed based on RT-qPCR to elucidate their possible physiological functions. The resist showed that, OcSusl and OcSus2 were assumed to be responsible for the biosynthesis of the four glucose-containing polysaccharides, while OcSus3 may play a role in plant growth.