Molecular cloning of key genes involved in piperine biosynthesis pathway from piper sp.

Abstract

The study entitled “Molecular cloning of key genes involved in piperine biosynthesis pathway from Piper sp.” was carried out at Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani during the year 2022-2023. The objective of the study was molecular cloning and sequence comparison of piperic acid CoA ligase and piperine synthase genes involved in piperine biosynthesis from selected Piper sp. Bush pepper plants of Piper nigrum varieties Panniyur 1 and Panniyur 4; Piper longum and Piper colubrinum were used for the study. The total RNA was isolated from the leaf and immature fruits of Panniyur 1, Panniyur 4, P. longum and P. colubrinum plants. Piperine synthase and piperic acid CoA ligase were amplified from each cDNA samples using designed primers of Piperine synthase and piperic acid CoA ligase gene. Amplicons of size approximately 1000 bp and 150 bp were obtained for leaves of Panniyur-1, Panniyur-4 and Piper colubrinum, whereas only 150 bp amplicon was obtained for leaves and immature fruit of Piper longum and spikes of Panniyur-1, Panniyur-4 and Piper colubrinum. Amplicon of approximately 1000 bp size was obtained from the leaves of Panniyur-1, and Panniyur-4. No bands were obtained for the leaves and immature fruits of Piper colubrinum and Piper longum and spikes of Panniyur-1, Panniyur-4 and Piper longum. Since intact bands of expected size was not obtained on the amplification of cDNA from Piper longum using designed primers, it is concluded that, there could be variation in the primer binding site of Piper longum genomic sequence, which was later confirmed by analysing the transcript sequence. PCR product was cloned into pGEM®-T vector and sequenced. The sequence obtained was subjected for in silico analysis. Nucleotide sequence of piperine synthase and piperic acid CoA ligase was found to contain 1018 bp and 970 bp respectively. The BLAST analysis of piperine synthase nucleotide sequence showed 99% similarity with piperine synthase reference gene in NCBI database. Amino acid sequence from Panniyur 1, Panniyur 4 and P. colubrinum was subjected for BLAST analysis and showed a similarity of 48 % with benzyl alcohol O-benzoyltransferase in Coffea arabica. BLAST analysis of amino acid sequence from Panniyur 1 and Panniyur 4 showed 65% similarity with acyl-CoA ligase of Macadamia integrifolia. Eventhough protein sequences of reference gene and query sequence of piperine synthase and piperic acid CoA ligase are similar, there is a substitution of amino acids at distinct regions. Meanwhile, P. colubrinum showed distinct sequence variation at the active sites of piperine synthase gene. The phylogenetic analysis of piperine synthase gene from different Piper sp. showed relation with Zingiber sp. and Rhodamnia sp. Phylogenetic tree with piperic acid CoA ligase from Piper nigrum varieties formed a separate lineage from CoA ligase enzyme from Camellia sp, Telopia sp and Macadamia sp. with a common branch point. Tertiary structure predicted were similar for Panniyur 1 and Panniyur 4, whereas differed for P. colubrinum due to sequence variation. Piperic acid CoA ligase protein from Panniyur 1 and Panniyur 4 were found to be similar. The stability of the predicted tertiary structure was determined using Ramachandran plot, which was found to be stable. The present study revealed the sequence information of piperine synthase gene from two cultivated varieties Panniyur 1, Panniyur 4 and a wild variety, P. colubrinum and piperic acid CoA ligase gene from Panniyur 1 and Panniyur 4. Although the sequences are similar to the reference sequence of Indonesian black pepper cultivar, distinct coding variants and single nucleotide polymorphisms that lead to substitution of certain amino acids was detected in both Indian cultivars and wild sp. As these variations affects the protein function and activity, future work will be directed towards the recombinant expression and further validation of the protein.