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

Metabolic engineering of plant secondary metabolites: prospects and its technological challenges

Plants produce a wide range of secondary metabolites that play vital roles for their primary functions such as growth, defence, adaptations or reproduction. Some of the plant secondary metabolites are beneficial to mankind as nutraceuticals and pharmaceuticals. Metabolic pathways and their regulatory mechanism are crucial for targeting metabolite engineering. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated system has been widely applied in genome editing with high accuracy, efficiency, and multiplex targeting ability. Besides its vast application in genetic improvement, the technique also facilitates a comprehensive profiling approach to functional genomics related to gene discovery involved in various plant secondary metabolic pathways. Despite these wide applications, several challenges limit CRISPR/Cas system applicability in genome editing in plants. This review highlights updated applications of CRISPR/Cas system-mediated metabolic engineering of plants and its challenges

Evaluation and Selection of Potential Biomass Sources of North-East India towards Sustainable Bioethanol Production

Vegetation biomass production in North-East India within Indo-Burma biodiversity hotspot is luxuriant and available from April to October to consider their potential for bioethanol production. Potential of six lignocellulosic biomass (LCB) sources; namely, sugarcane bagasse (BG), cassava aerial parts (CS), ficus fruits (Ficus cunia) (FF), “phumdi” (floating biomass), rice straw (RS), and sawdust were investigated for bioethanol production using standard techniques. Morphological and chemical changes were evaluated by Scanning electron microscopy and Fourier transform infrared spectroscopy and quantity of sugars and inhibitors in LCB were determined by High performance liquid chromatography. Hydrothermally treated BG, CS, and FF released 954.54, 1,354.33, and 1,347.94 mg/L glucose and 779.31, 612.27, and 1,570.11 mg/L of xylose, respectively. Inhibitors produced due to effect of hydrothermal pretreatment ranged from 42.8 to 145.78 mg/L acetic acid, below detection level (BDL) to 17.7 µg/L 5-hydroxymethylfurfural, and BDL to 56.78 µg/L furfural. The saccharification efficiency of hydrothermally treated LCB (1.35–28.64%) was significantly higher compared with their native counterparts (0.81–17.97%). Consolidated bioprocessing of the LCB using MTCC 1755 (Fusarium oxysporum) resulted in maximum ethanol concentration of 0.85 g/L and corresponded to 42 mg ethanol per gram of hydrothermally treated BG in 120 h followed by 0.83 g/L corresponding to 41.5 mg/g of untreated CS in 144 h. These ethanol concentrations corresponded to 23.43 and 21.54% of theoretical ethanol yield, respectively. LCB of CS and FF emerged as a suitable material to be subjected to test for enhanced ethanol production in future experiments through efficient fermentative microbial strains, appropriate enzyme loadings, and standardization of other fermentation parameters