Cloning And Functional Analysis Of A 9-Cis-Epoxycarotenoid Dioxygenase Gene (CrNCED3) In Citrus Reshni

In China, Citrus is important in fruit industry. ABA is proved to have an important role under citrus stress conditions. In our experiments, we cloned the 9-cis-epoxycarotenoid dioxygenase (NCED) gene that encodes a key enzyme in ABA biosynthesis from Citrus reshni using homology-based method, investigated the expression of CrNCED3 under different stress like low temperature (4℃), drought, salt and endogenous ABA treatment using SqRT-PCR, and confirmed the copy number using southern blot. By gene manipulation and genetic transformation, CrNCED3 gene was transformed into tobacco (Nicotiana nudicaulis) to confirmd the function. The results were as follows:1. An opening reading frame of 1821 bp of CrNCED3 was isolated encoding proteins of 606 amino acids. No intron was shown in its sequence. At the amino acid level, the sequence of CrNCED3 shows high identity of over 60% with other species. The closer relationship between CrNCED3 and AtNCED3 was shown by the generation of a phylogenetic tree. The iPSORT prediction showed a detection of a N-terminal chloroplast transit peptide in the sequence. Southern blot indicated there was a single copy of NCED in Citrus reshni.2. The SqRT-PCR was used to detect the NCED expression in different organs and stress conditions, taking citrusβ-Actin as inner conrtol. Under non-stress conditions, CrNCED3 was highly expressed in shoots and leaves, but having a relatively low expression in roots. The expression of CrNCED3 showed a organ-specific patterns. Under stress conditions, CrNCED3 was induced in leaves by drought and endogenous ABA and the expression increased as the treatment continued. As the seedings returned to normal conditions, the expression of CrNCED3 in leaves declined immediately. The expression of CrNCED3 induced by drought and endogenous ABA was transient. Otherwise, no significant changes was showed in the expression of CrNCED3 under low temperature of 4℃and salt stress. It indicated that the NCED gene family had different expression patterns and mechanisms under different stress.3. Constructed binary vector of 35S::CrNCED3 and transformed the target gene into tobacco with agrobacterium tumefaciens-mediated transformation.17 transgenetic lines were identified by PCR. 4. The the results of relative water loss, relative electrolyte leakage and NBT, DAB dye staining between transformed and non-transformed tobacco under dehydration showed the transformed tobacco had better water status and less damage to the membrane. The NBT and DAB dye staining indicated the less ROS level in the transformed plant. In a word, the overexpression of CrNCED3 in tobacco enhanced the tolerance to drought.