Abstract
The Indian pepper ‘Guijiangwang’ (Capsicum frutescens L.), one of the world’s hottest chili peppers, is rich in capsaicinoids. The accumulation of the alkaloid capsaicin and its analogs in the epidermal cells of the placenta contribute to the pungency of Capsicum fruits. To identify putative genes involved in capsaicin biosynthesis, RNA-Seq was used to analyze the pepper’s expression profiles over five developmental stages. Five cDNA libraries were constructed from the total RNA of placental tissue and sequenced using an Illumina HiSeq 2000. More than 19 million clean reads were obtained from each library, and greater than 50% of the reads were assignable to reference genes. Digital gene expression (DGE) profile analysis using Solexa sequencing was performed at five fruit developmental stages and resulted in the identification of 135 genes of known function; their expression patterns were compared to the capsaicin accumulation pattern. Ten genes of known function were identified as most likely to be involved in regulating capsaicin synthesis. Additionally, 20 new candidate genes were identified related to capsaicin synthesis. We use a combination of RNA-Seq and DGE analyses to contribute to the understanding of the biosynthetic regulatory mechanism(s) of secondary metabolites in a nonmodel plant and to identify candidate enzyme-encoding genes.
Highlights
8-methyl-6-nonenoyl-CoA, which are produced as two branched chains, are converted to capsaicin by acyl-transferase (AT)[7]
The results showed limited (0.08 mg/g) capsaicin accumulation in 10 days after pollination (DAP) fruit, 1.06 mg/g at 20 DAP, and 13.82 mg/g at 30 DAP
To identify new genes that could contribute to capsaicin accumulation, we examined differences in differentially expressed genes (DEGs) expression patterns by comparing various combinations of samples (10 vs. 20, 20 vs. 30, 30 vs. 40, 40 vs. 50, 10 vs. 30 and 20 vs. 40 DAP)
Summary
8-methyl-6-nonenoyl-CoA, which are produced as two branched chains, are converted to capsaicin by acyl-transferase (AT)[7]. Inherited traits that control developmental processes in pepper are influenced by many genes with small effects[10] Their direct participation in the capsaicinoid biosynthesis pathway has not been demonstrated[12]. A comparison of cDNA libraries prepared from the placenta of chili fruit indicated that PAL, Ca4H, CCoAOMT, Kas, and pAMT are differentially expressed. A BLAST search determined that these genes had high homology with pAMT, Kas, Acyltransferase (AT), and FAO13 They are expressed in placental tissue and are accompanied by capsaicin accumulation. The RNA-Seq platform[15,16] was used to analyze the expression profiles of putative capsaicin biosynthetic genes in placental tissue, during five developmental stages of pepper fruit. The identified candidate genes could help to elucidate the molecular basis of pungency
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