Molecular cloning, expression and computational analysis of a water stress inducible copper-containing amine oxidase gene (CuAO) from tea plant [Camellia sinensis (L.) O. Kuntze]

Copper-containing amine oxidase (CuAO) is the enzyme known to play diversity of function in plant responses to environmental stresses through its reaction products. Here, for the first time we report full length cDNA encoding CuAO protein from a drought tolerant tea cultivar. It was found to be 785 bp long with a 70 bp 5.-UTR, 193 bp 3.-UTR, 522 bp mORF and a polyA adenylational signal. It codes for a poly-peptide of 173 amino acids having predicted molecular weight and isoelectric point of 19 KDa and 7.75 respectively. Heterologous expression and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the protein in Escherichia coli revealed similar size as predicted by in silico analysis. Blastp analysis and template based homology modeling in Phyre2 has identified a copper amine oxidase domain with ligand binding site for copper at residue 123 (Histidine) which suggests its probable role in plant responses to environmental stresses. Interestingly, no signal peptide sequence was detected in the predicted protein which is in contrast to the CuAO so far reported in plants. Although, in slico analysis of the protein have indicated its probable structure and functions, further functional characterization is needed to better understand its role during drought and other environmental stresses in tea.Key words: Camellia sinensis, copper amine oxidase, homology modeling, molecular cloning

Identification and validation of stable reference genes in camellia species

We aimed at finding and validating a stable reference gene in Camellia sinensis and Camellia assamica from a set of four putative housekeeping genes in various samples exposed to different experimental conditions mainly biotic and abiotic stresses. Variation in gene expression across Camellia sinensis leaf tissues exposed to nine different kind of experimental sets was studied. The suitability of 18S rRNA, 26s rRNA, rubisco bis phosphatase (RuBP) and camellia actin (Act) as reference genes were validated by geNorm and BestKeeper programs and revealed 18S rRNA and RuBP to be the most stably expressing housekeeping gene. We therefore recommend use of RuBP as a stable reference gene for normalisation of transcripts abundance experiments in tea leaf samples

Rubisco-bis-phosphate oxygenase (RuBP)- A potential housekeeping gene for qPCR assays in tea

The present experiment is an effort to find a stable reference gene in Camellia sinensis and Camellia assamica under different biotic and abiotic stresses. This study evaluate the variation in gene expression across tea leaf tissues in nine experiments. The suitability of 18S rRNA, 26S rRNA, rubiscobis- phosphatase oxygenase (RuBP) and Camellia tubulin (CaT) as reference genes were validated by geNorm and BestKeeper programs. The finding reveals 18S rRNA and RuBP to be the most stably expressed housekeeping genes, the latter being the first report of its kind in tea. The finding paves the way for their application in accurate quantification of trait specific gene expression and other genomic studies in tea.Keywords: Camellia sinensis, Camellia assamica, qPCR, BestKeeper, geNorm, housekeeping gen