Genome walking for putative phytic acid (InsP6)Unigene in Black pepper (Piper nigrum L.)
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Date
2017
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Centre for Plant Biotechnology and Molecurar Biology, College of Horticulture, Vellanikkara
Abstract
Black pepper (Piper nigrum L.), is an important spice crop contributing a major share of country’s foreign exchange. However the genomic information of this crop is limited. Expressed sequence tags (EST) data obtained by next generation sequencing technology in a previous study to understand the genes expressed during berry development in black pepper showed a candidate unigene (Pnc135- 995 bp) coding for inositol pentakisphosphate 2-kinase (ipk1). This is an enzyme involved in the biosynthesis of Myo-inositol hexakisphosphate (InsP6) or commonly called as phytic acid. The reports of ipk1 gene sequences from other crops showed that the size of ipk1 gene varied widely between crops in the range of 3- 6 kb. Apart from helping in phosphate storage, phytic acid plays diversified role in plants such as a signalling molecule during drought stress, key role in plant defence mechanism, cofactor in auxin mediated gene expression of the auxin receptor, safe binding site for Fe during Fe transport in cytosol etc (Graf et al., 1984; Lemtiri- Chlien et al., 2003; Murphy et al., 2008).
Being a commercially important crop and considering the important biological roles played by phytic acid in the plant systems, a study on ‘Genome walking for putative phytic acid (InsP6) unigene in black pepper (Piper nigrum L.)’ was taken up to sequence the flanking regions of unigene (Pnc135) and to detect the presence of InsP6 using polyacrylamide gel electrophoresis (PAGE) in black pepper.
Genomic DNA was isolated from var.Panniyur1 and used for the PCR amplification of the unigene sequence Pnc135. As a preliminary step of the study, two sets of primers (PNC F1/PNC R1, PNC F2/PNC R2) were designed based on the unigene sequence for the confirmation of sequence information and two primer sets (PNC F3/PNC R3, PNC F4/PNC R4a, PNCR4b) were designed based on the pairwise alignment of selected sequences from BLAST search using unigene for the amplification of flanking regions of unigene. Three sets of primers (PNC
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F5/PNCR5, PNC F6/PNC R6, PNC F7/PNC R7) were designed based on the multiple sequence alignment of unigene with the selected sequences of 28 different crops and PCR amplification was carried out with the different primer combinations. Among the six PCR products sequenced (F1R1, F3R3, F4R4a, F6R5, F7R6 and F7R7), the product F6R5 obtained by primer set PNC F6/ PNC R5 designed based on the consensus regions of Pnc135 with ipk1 gene sequence reported in 28 other crops using multiple sequence alignment gave an amplicon of 394 bp. This PCR product (F6R5) showing similarity to ipk1 gene was used for performing genome walking for the flanking regions.
Genome walking is a method to sequence the flanking regions of a known sequence. The genome walking protocol by Reddy et al. (2008) which make use of the strand displacement amplification of phi29 DNA polymerase was used in the present study. The walker adapter primers (WA1, WA2, WA3, WA4) and the walker primer (WP1) and nested walker primer (WP2) used in the study were same as described in the protocol by Reddy et al. (2008). The forward and reverse locus specific primers (LSP F1, LSP F2, LSP R1, LSP R2) and the corresponding nested primers (NLSP F1, NLSP F2, NLSP R1, NLSP R2) were designed based on the sequence of F6R5 for genome walking. From the nested PCR amplification, four PCR products A3F1 and A2F1 (flanking regions obtained towards the 3’end of F6R5) and A4R1 and A4R2 (flanking regions obtained towards the 5’end of F6R5) were sequenced and homology search was done for similarity with ipk1 gene. Among these, only one PCR product viz., A3F1 of 933 bp showed similarity to ipk1. Based on overlapping regions, this was assembled with the F6R5 sequence (394bp) to get a total length of 1072 bp (Pnipk1).
The assembled sequence (Pnipk1) was analysed for the coding region (Open Reading Frame) and was found to have 522 bp coding for 173 amino acids. The nucleotide sequence and amino acid sequence were used for the phylogenetic analysis using ipk1 sequences of other crops showing similarity in BLAST search.
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The analysis showed that Pnipk1 was evolutionarily closer to sequences from plants like Phaelenopsis equistris, Musa acuminata, and Phoenix dactylifera.
Polyacrylamide gel electrophoresis (PAGE) analysis was carried out to detect the presence of InsP6 in Panniyur 1 leaf sample. The leaf extract as well as phytate standards were loaded onto gels and the corresponding band intensities of each concentration of phytate standard and samples were recorded using the software GelQuant.NET. The quantity of phytate in the sample was calculated from the standard curve drawn by plotting band intensities of phytate standards on the y- axis against quantity of phytate standards (nmoles) on x-axis. Quantity of phytate in the sample was estimated to be 620 nmoles/g fresh weight in the leaf tissues.
The study has given the confirmation of the presence of ipk1 gene in black pepper and was able to decipher a total sequence length of 1072 bp by genome walking towards the 3’end of the 394 bp amplicon obtained from var. Panniyur1. Identification of the sequence towards the 5’end to get the full length gene will help to understand the role played by the gene in biotic and abiotic stress resistance in black pepper.
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