Cloning,Expression Analysis And Function Identification Of Cinnamyl Alcohol Dehydrogenase Gene From Chimonanthus Praecox (Linn.) Link

Most lignin distributes in cell wall of plant lignification mechanical tissue and vascular tissue.It can increase the cell wall intensity and enhance the ability of the cell wall watertightness and stem’s mechanical strength,as well as the liquid-transporting ability.Moreover,it also plays an important role in the cell resistant by effective physical and chemical barrier mechanism.Cinnamyl alcohol dehydrogenase (CAD) is one of the key enzyme in controlling lignin biosynthetic pathway.In this paper, one gene encoding the cinnamyl alcohol dehydrogenase was cloned from Chimonanthus praecox (linn.). based on the cDNA library from Chimonanthus praecox,named CpCAD. bioinformatics analysis, real time quantitative PCR. over-expression in tobacco was used to study the function of CpCAD gene. The main results in our research are as follows:1.The full length of CpCAD cDNA sequence was1432bp,no introns.with an open frame(ORF) of936bp encoding a putative polypeptide of311amino acid residues. Blastn and blastp analysis on NCBI showed that the amino acid sequence of the protein encoding by CpCAD gene shares a high similarity with other reported CAD.especially with CAD from Medicago truncatula, Camellia sinensis and Vitis vinifera (95%,90%and86%identity respectively). The bioinformatics analysis showed that the CpCAD protein belong to the CAD1family and contained the typical conservative domain structure of alcohol dehydrogenase in N terminal.such as NAD(P)binding site.catalytic Zn binding site and structural Zn binding site;putative substrate binding site; and a dimer interface.Secondary structure prediction showed that CpCAD protein was composed of α-helix (21.86%), extended strand (22.19%)and random coil (55.95%). There were no signal peptide and trans-membrane domain in the deduced amino acid sequence of the CpCAD protein. The subcellular localization prediction showed that the CpCAD may localized in nucleus, microbody(peroxidase),mitochondrial matrix and lysosome (lumen).There are one threonine phosphorylation site. two tyrosine phosphorylation sites and fourteen serine phosphorylation sites in the amino acid of CpCAD.The protein was a dimer composed of two subunit, with the typical Rossmann fold (β-α-β) structural domain,which was the main combination of coenzyme area by tertiary structure prediction.2.Gene expression studies by real-time quantitative PCR showed that The CpCAD gene exhibited different transcription levels in different tissues and had a significantly higher expression in flowers than other tissues. In the bloomed flower, the expression of CpCAD has the maximum at the outer petal, the CpCAD gene in the flower buds gradually increased in the early stages of flower development from sprouting stage to initiating bloom period stage and presented a peak at blooming stage. We also studied the gene expression of the CpCAD under CuSO4and ABA stress treatment.The results showed that the CpCAD genes were general up-regulated by the CuSO4treatment and had the maximum expression at0.25h after the treatment and then decreased,and then up at6h to12h. the CpCAD genes were general down-regulated by ABA treatment,3. The plant over-expression vector of CpCAD named pCAMBIA2301g-CpCAD was constructed and transformed into tobacco plants using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens GV3101(including pCAMBIA2301g-CpCAD recombinant plasmid). twenty-eight transgenic tobacco plantlets were obtained, in which the transgenic lines were identified by GUS((3-glucuronidase)histochemical staining and PCR analysis4.Some physiological indexes.including chloroplast pigment content, superoxide dismutase (SOD) activity, malondial dehyde (MDA) content of the transgenic tobacco.which were treated by CuSO4was measured.The result showed that the chlorophyll a, chlorophyll b.total chlorophyll content and superoxide dismutase (SOD) activity were higher than wildtype tobacco.while the malondial dehyde (MDA) content was significantly lower than wildtype tobacco. The content of lignin in leaves of transgenic tobacco plant increased significantly, in which, the highest increased18.7%comparing to the wild type tobacco.