A good quality RNA is an important prerequisite for any downstream molecular analysis. However, isolation of quality RNA from tea is difficult due to presence of high amount of phenols and polysaccharides. We describe here an efficient protocol for total RNA isolation without liquid nitrogen from apical buds and tender leaves of tea. This protocol yielded RNA 1248+/-23 mu g/g of fresh leaf tissue with high purity. The RNA was also found to be suitable for mRNA purification, cDNA synthesis and cloning through RT-PCR (reverse transcription-polymerase chain reaction) and cDNA library construction.

Tea is the most popular non-alcoholic and healthy beverage across the world. The understanding of the genetic organization and molecular biology of tea plant, which is very poorly understood at present, is required for quantum increase in productivity and efficient use of germplasm for either cultivation or breeding program. Single-pass sequencing of randomly selected cDNA clones is the most widely accepted technique for gene identification and cloning. In the present study, a good quality cDNA library was constructed and preliminary analysis of ESTs was carried out. The titers of unamplified and amplified libraries were 1.4 × 106 pfu/ml and 5.27 × 108 pfu/ml respectively. A total of 210 cDNA clones from the constructed cDNA library were sequenced and analyzed. A total of 84 high quality Expressed Sequence Tags (ESTs) were generated, among which 71 ESTs had significant homology with sequences in NCBI non-redundant protein database by BLAST X analysis. About 80% ESTs had poly (A) tail at 3′ end indicating that the cDNAs were full length. The database-matched ESTs were classified into putative cellular roles, viz. energy-related category (corresponding to 20% of total BLAST X matched ESTs), Transcription (14.2%), protein synthesis (14.2%) cell growth and division (8.6%), cell structure (5.7%), signal transduction (5.7%), transporters (2.9%), disease and defenses (2.9%), secondary metabolism (2.9%) and gene regulation (2.9%). This study provides an overview of the mRNA expression profile and first hand information of gene sequence expressed in tender leaves and apical buds of tea plant.