Cloning And Function Analysis Of ATP-citrate Lyase Gene(SoACLA-1) From Sugarcane

ATP-citrate lyase plays an important role in organisms. In recent years, there are four mainly functions of ACL have been reported:(1) generate intracellular acetyl-CoA;(2) regulate enzymes for fatty acid synthesis;(3) to improve plant resistance and;(4) involve in regulation of sexual reproduction. But previous relevant ATP-citrate lyase researches focused mostly on the enzyme activities and less on functional studies. The present study investigated the relevant functions of ACL gene from sugarcane. The main results were as follows.1, Cloning SoACLA-1gene. According to SoACLA-1the sequences of both ends of the ORF gene of sugarcane (accession number JQ292843.1), the specific primers were designed for PCR, and the1272bp full-length cDNA fragment of SoACLA-1gene coding region was cloned.2, Constructing binary vector. The cloned cDNA fragment was inserted into PRI101-AN, a dicotyledonous plant expression vector, and the product was designated as SoACLA-1-PRI101-AN. The cloned cDNA fragment inserted in the opposite direction of PRI101-ON, a monocot plant expression vector, and the product was designated as SoACLA-1-PRI101-ON.3, Getting transgenic tobacco and transgenic sugarcane. SoACLA-1-PRI101-AN and SoACLA-1-PRI101-ON were transformed into tobacco (Nicotiana tabacum L. cv. K346) and sugarcane variety ROC22respectively by Agrobacterium-mediated genetic transformation. Fourty four transgenic tobacco plants and21transgenic sugarcane plants were obtained, and they have been confirmed by PCR and Southern blotting. 4, Real-time RT-PCR analysis showed that the expression of SoACLA-1gene was significantly higher in the transgenic tobacco than in the wild-type plants. With increasing drought stress, the gene expression grew in parabola state. Under moderate drought stress, the activities of SOD, POD and CAT increased in varying degrees in the transgenic tobacco compared to the wild type plants, and the differences in activities of SOD and CAT were statistically significant. With increasing drought stress, the tobacco plants began to wilt, but the transgenic tobacco plants delayed leaf wilting for4-5days compared to the wild-type tobacco plants. It has been shown that over expression of sugarcane SoACLA-1improves the drought tolerance of tobacco.