Molecular Cloning，Functional Analysis Of Three Cinnamyl Alcohol Dehydrogenase (CAD) Genes In Relation To Ectropic Obliqua Induction In The Leaves Of Camellia Sinensis

Lignin is a complex polymer formed by Many simple phenylpropane and derivatives which plays a great role in organism. lignin consists of effective physical chemistry barrier that resist the pests and diseases invasion, so it has an important role in pests resistance and diseases invasion. Cinnamyl alcohol dehydrogenase is one of the key genes that plays an important role in the synthesis of the monomeric precursors of lignin. However, the research between the CAD in tea and in pests resistance has not been reported up to now. In this report, we mainly described the molecular characterisation, cDNA cloning and expression of three cinnamyl alcohol dehydrogenase genes in relation to Ectropic obliqua induction in the leaves of Camellia sinensis. The preliminary results as follows:1. The three EST sequence which have the high homology with the CAD genes were isolated from the whole organ transcriptomic library of tea and cDNA SSH after Ectropic obliqua induction in the leaves of Camellia sinensis. The three full length cDNA was obtained by using RACE, one CAD isolated from SSH was named CAD2, another two isolated from transcriptomic were named CAD1and CAD3. The full length CAD2was1574bp, with972bp ORF which encoding324amino acids The full length CAD1was1386bp, with1386bp ORF which encoding382amino acids The full length CAD3was1350bp, with1083bp ORF which encoding361amino acids. The three genes has been submitted to the GenBank and the registration number was HM440158, HQ880207and HQ880208.2. Multiple Sequence Alignment and Protein structure prediction analysis show that CAD1and CAD3has54.2%indentical, however, in contrast to CAD2, Both have lower indentical which have around20%. Only CAD1and CAD3contain the three conserved functional structure domain:zinc1binding sites [GHE（X）2（X）5G（X）2V], zinc2binding sites [GD（X）9.10C（X）2C（X）2C（X）7C] and NADPH Coenzyme binding sites [GXG（X）2G]. however, CAD2has special conserved Epimerase domain, they belong to NADPH dependent epimerase/dehydratase family. Evolutionary analysis show that three cinnamyl alcohol dehydrogenase from the tea plant belong to three categories.3. the results of prokaryotic expression showed that most of CAD2was in supernatant soluble protein, The molecular weight was53kD, however, CAD1and CAD3remained associated with the inclusion bodies, The molecular weight were59kD and56kD respectively. The results of enzymatic activity indicated that the supernatant protein and the denatured inclusion bodies have different catalytic activity. Among that CAD2was the highest with0.429μmol min^-1 mg^-1, the next was CAD3with4.2nmol min^-1 mg^-1, CAD1was the lowest with3.8nmol min^-1 mg^-1. the optimum pH of CAD2was6, the optimum temperature was30℃, the value Km was1.974μmol L^-1 and Vmax was1.7437μmol min^-1 mg^-1.4. Southern blot indicated that there were two copies existing in CAD1and CAD2while CAD3had only single copy in the genomic DNA of tea.5. The expression of three CAD genes was not all the same after Ectropic obliqua induction. The maximum induced of expression did not vary between CAD1and CAD2. However, they have the greater difference in contrast to CAD3There were significant difference in expression of the three genes among varieties, overall that shuchazao was higher than longjing43.also according to induced of expressive regularity, the three genes have difference exists, The expression of CAD1and CAD2were slightly increased with the time going by, the maximum of CAD1was24h while the CAD2was between9h and12h.However, The CAD3was sharply increased to maximum at the beginning and then sharply decreased, The maximum expression was appeared between3h and6h. Under the mechanical injury, The maximum expression difference among three genes were similar to Ectropic obliqua induction,but no significant difference between varieties.According to exogenous chemicals indicated that treatment with MeJA, SA and ABA can up-regulated the three CAD genes. However, The biggest expression among them were different accoing to the type of hormone. under the treatment of MeJA and SA, The maximum expression of CAD1and CAD2were obviously higher than CAD3, while the maximum expression of CAD1and CAD3were obviously higher than CAD2under the treatment of ABA.