Cloning And Functional Research Of Citrate Synthase And ATP-Citrate Lyase Of Brassica Napus L.

Brassica napus is one of major oil crops in China and whole world. The mechanism of high oil content in Brassica napus is a very important subject for theoritic and applied studies. As the key enzymes of sugar metabolism, citrate synthase and ATP-citrate lyase are related to fatty acid synthesis by the fact that their production can be used to synthesize fatty acid. It has significant value theoretically to discover the function of citrate synthase and ATP-citrate lyase in oil content accumulation in seed.In this study, we cloned cDNA of citrate synthase, ATP-citrate lyase A and ATP-citrate lyase B from cDNA library or cDNA in Brassica napus. Those products had full open reading frames which were confirmed by sequencing. Alignment analysis indicated that those genes were highly conserved in plants.We designed 16 pairs of EST primers by cDNA sequences of citrate synthase, ATP-citrate lyase A and ATP-citrate lyase B, including 5 pairs of ALCA, 5 pairs of ALCB and 6 pairs of CS, and used them in an oil content segregation F2 population and a different oil content strain population. There were 5 pair of polymorphic primers found in oil content segregation F2 population and 4 pair of polymorphic primers found in different oil content strain population. Correlation analysis between markers and oil content indicated that there was no marker linked to oil content.We detected the expression of citrate synthase and ATP-citrate lyase by semi-quantity PCR and Real-time PCR, and found that the expression of citrate synthase was different in different oil content rapeseed leaves. The expression of citrate synthase was higher in the leaf of high oil content strain than in the leaf of low oil content strain. It suggested that the expression of citrate synthase maybe be related to oil content. And the expression of citrate synthase and ATP-citrate lyase was diverse in organs, so that those enzymes might be indispensable for normal physiological function. The expression levels of those two enzymes were high in early days and digressive latterly in ovule. This fact indicated that they were important in early development of ovule. The expression of citrate synthase gene was increased at different time in treatments of water logging, drought, low or high temperature, IAA, 6-BA, and ABA., and the expression of ATP-citrate lyase gene was increased at different time in treatments of water logging, low temperature, high illumination, and ABA. The expressing analysis of enzymes under stresses with various pattern proved they took a part in these metabolic control and responding to some environmental changes.Then we constructed over expressing vectors and repressive expressing vectors for citrate synthase gene and ATP-citrate lyase A gene, named pMD-tCS, pMN-tCS, Gate-tACLA, pMD-rCS, pMN-rCS, pMD-rACLA and pMN-rACLA. After transfoming above plasmids to Arabidopsis thaliana, we otained a respective transgenic plants of diffenent plasmids except pMN-rCS. The successful transformation was confirmed by fastness filtration and PCR. The properties of transgenic plants of plasmid Gate-tACLA and pMN-rACLA were not been analyzed in this study.Both over or repressive expression of citrate synthase gene made phenotypes changed in transgenic plants. It had thicker side shoots, higher oil content in leaf and seed, and different fatty acid composition in over expression plants compared with wild type. Repressive expression plants were dwarf with miniature leaf, short silique and low oil content. It indicated that citrate synthase played an important role in plant growth, as its expression variety could affect development. And it was one of main regulative enzyme in fatty acid synthesis in plant, because of up or down regulation of its expression resulted in oil content increased or decreased respectively. Furthermore, repressive expression of ATP-citrate lyase A gene led to phenotypes such as dwarfing, leaf miniaturization, shorten silique and oil content decreasing, sugesst that ATP-citrate lyase was also a regulative enzyme in oil synthesis.