Identification and characterization of flowering T (FT) gene in black pepper (Piper nigrum L.)

Abstract

The study entitled “Identification and characterization of Flowering Locus T (FT) gene in black pepper (Piper nigrum L.)”, was carried out at the Integrated Biotechnology Block, College of Agriculture, Vellayani during 2015-2016. The main objective of the study was the isolation, cloning and sequencing of genes homologous to FT, a floral integrator gene from black pepper and functional characterization of the gene by studying its temporal and spatial expression patterns. The sample material used for this study was black pepper (Karimunda variety). Black pepper samples at different stages were collected from the Instructional Farm, College of Agriculture, Vellayani and RNA and DNA were extracted. cDNA was prepared from RNA by reverse transcription. PCR (Polymerase chain reaction) was done using both genomic DNA and cDNA with different degenerate primers and showed amplifications in nested reactions with expected size ~141 bp and ~256 bp. The amplicons were sequenced after elution and purification. Further sequence analysis using tBLASTx, NCBI conserved Domain Search and Clustal Phylogeny program showed that the sequence of the amplicons obtained around 124 bp and 230 bp were the partial fragments of flowering time and floral integrator gene named FT and inflorescence meristem identity gene named TERMINAL FLOWER 1 (TFL1) from black pepper. NCBI conserved Domain Search programme, showed that both the FT and TFL1 sequences belonged to PEBP (Phosphatidyl Ethanolamine- Binding Protein) super family and phylogenetic tree created from Clustal Phylogeny program also showed that the FT (124 bp) sequence was closer to FT mRNA of Cymbidium faberi and Oryza sativa. TFL1 (230 bp) sequence was closer to TFL1 mRNA of Solanum tuberosum. Semi quantitative analysis with FT primers showed that the FT gene was expressed in the mature spike with berries, spike with young berries, mature leaves, spikes with flowers, immature spikes and plagiotropic shoots. The expression of TFL1 gene was observed in mature leaves and slightly in spike with flowers. Microscopy studies were done including fixation of pepper spike tissues in the fixative FAA (Formaldehyde acetic acid alcohol) solution and Carnoy’s fluid, followed by sectioning of the tissues and staining with safranin and fast green to see the changes occurring in different development stages of spikes ranging from immature spike to mature spike with berries. Sequence analysis and expression studies confirmed that the isolated sequences were partial orthologues of PEBP genes from black pepper and this study has identified the flowering time and floral integrator gene (FT) and the inflorescence meristem identity gene (TFL1) from black pepper for the very first time.