Tea 

Catechins and their derivatives are responsible for astringency and bitterness of tea and taste. These are also important pharmaceutical compounds being strong antioxidants. While the biochemistry of catechin biosynthesis in tea is known, the control points and molecular aspects have not been considered. To engineer the pathway for its desired modulation in the clone or the plant of choice or to synthesize the flavonoids in vitro for use in pharmaceutical and otherrelevant industry, it is implicit to clone the niche-pathway genes from tea. Differential display reactions were done several ESTs were identified. Several of these ESTs did not show homology with reported sequences and those depicted homology could be grouped into 3 majorcategories. One group depicted genes possibly involved in the signaling pathway such as Ca++ dependent protein kinase, lectin like protein etc. The second group had genes those could possibly be associated with high metabolic activity. These included the genes for ATP synthase, cytochrome oxidase and so on. The third group had genes for catechin biosynthesis. It encompassed genes such as chalcone synthase, chalcone isomerase, flavonoid 3’5’ hydroxylase, dihydroflavonol reductase,  leucoanthocyanidin dioxygenase, coumaryl coA ligase and anthocyanin reductase. Having a series of sequence information generated through differential display of mRNA, this year 5’ and 3’ RACE was performed to clone full- length cDNAs. RACE generated fragments were cloned, sequenced and analysed. Data generated was used to clone full-length cDNAs. To maintain the  priority, these genes were deposited in the gene databank. In total, 7 full-length cDNAs of the pathway were cloned. Importantly, this institution was the first to deposit all 7 genes of the flavonoid pathway from tea. Fig. 4.13 shows the gels of various reactions of RACE that allowed cloning of full-length genes of catechin biosynthesis pathway.

ACTIVITY ASSAY OF F3H, DFR AND LAR WITH THE OVEREXPRESSED AND PURIFIED RECOMBINANT PROTEINS AND PREPARATION OF THESE GENE CONSTRUCTS FOR PLANT TRANSFORMATION 

Functional analysis of isolated catechin pathway genes, viz. flavone-3-hydroxylase (F-3-H), dihydroflavanone reductase (DFR) and lecoanthocyanidin reductase (LAR) were cloned in to bacterial expression vectors and overexpressed. The activity of these enzymes was measured after purifying with Ni-NTA column. Recombinant proteins after purification showed consistency in their activity up to five days. Activity of DFR, F3H and ANR was presented in (Table4.3.)  

Table  4.3. Enzyme activities of three recombinant proteins expressed in E .coli and purified on Ni-NTA column. The data is the average of three independent determinations ± SD.  

These genes were cloned into plant transformation vector pCAMBIA1302. Agro-mediated transformation of tobacco was carried out to study the influence of individual gene upon overexpression in tobacco on the catechins content and other physiological processes.       

Figure 4.7. Confirmation of the Agrobacterium transformants with colony PCR.  pCAM-F-3-H (A),  pCAM-DFR (B) and  pCAM-LAR (C)