Molecular Cloning And Expression Analysis Of CsCAB Gene From 'Navel' Orange And Its Genetic Transformation In Tobacco

Fengjie navel orange (Citurs sinensis L.Osbeck) is the best quality of navel "Washington navel". so, As varieties of citrus in Chongqing, Fengjie navel orange has a strong competitive advantage in domestic and international sales. Efforts have been made to elucidate the triggering factors of peel pitting, but little has been reported about the molecular mechanism.Based on the suppression subtractive hybridization (SSH) library which constructed in order to identify the genes in peel pitting of navel orange with different expression, we choose the EST with significant similarity to CAB gene family as target gene, the full-length cDNA of CsCAB was isolated by RACE. The early results indicate that CsCAB is peel pitting related protein and may play important roles in the development of citrus peel pitting, but little has been reported about the regulatory mechanism.Semi-quantitative RT-PCR was performed to determine the expression pattern of CsCAB in different tissues and different period of peel pitting. It showed that CsCAB was up-regulated during early period of peel pitting and CsCAB was expressed in all tested tissues ( root, leaf, flower and peel). Compared to healthy peel, the expression of CsCAB was enhanced in the pitting peel. The expression of CsCAB and its target genes GAD and ATPase in fruits was induced by wounding in comparison with the control fruit. Peel pitting is a complex network structure, these results indicated that CsCAB protein may play an important role in the process of peel pitting.In this study, the RNA extracted from Citrus sinensis Osbeck peel was used for isolating CsCAB gene by RT-PCR. CsCAB is 621 bp nucleotides encoding a protein of 207 a mino acids. The calculated molecular weight of the CsCAB protein was 22.95 kDa and theoretical isoelectric point was 4.5. Sequence analysis showed that CsCAB protein had a strikingly conserved region of EF-hand, phylogenetic analysis confirmed that CsCAB had high homology to other Ca-binding proteins. Furthermore, its sense and antisense recombinant plasmids, pCAMBIA1301-CsCAB, were successfully constructed, providing a basis to study the function and regulation of CsCAB gene. The two vectors were transferred into tobaccos via Agrobacterium mediated method. Transgenic plants were indentified by PCR detection. The positive transgenic plants containing GUS gene have been detected by GUS histochemical assay and the transgenic lines containing CsCAB gene have been analyzed by RT-PCR expression analysis again. The results showed that some transgenic lines have obtained.