Cloning and expression of delayed bitterness gene encoding limonoid glucosyltransferase from Kinnow mandarin (Citrus reticulata Blanco)
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Date
2016
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Punjab Agricultural University, Ludhiana
Abstract
Kinnow mandarin (Citrus reticulata Blanco) is the major citrus fruit crop of north western India and it occupies maximum fruit area in Punjab. But citrus juice processing industry has been suffering from delayed bitterness problem which is mainly because of limonoid aglycones such as limonin content in citrus juices. In order to study citrus limonoid metabolism, limonoid glucosyltransferase (LGT) gene which encodes a natural debittering enzyme was isolated from the fruit tissues of Kinnow mandarin. After confirmation and characterization, this full length gene sequence (1533 bp) was submitted on NCBI. BLASTn and BLASTp analysis of this isolated LGT showed 98% nucleotide sequence identity and 94-99% identity with the amino acids of LGT from different Citrus spp. respectively. The transcript expression of LGT was evaluated in different tissues such as young leaf (YL), flavedo (F), albedo (A), sac covering (Sc) and seed (S) of Kinnow mandarin during early (90-120 days after flowering (DAF)), mid (150-210DAF) and late (240-270DAF) fruit developmental stages using semi-quantitative method. Its expression was highest in flavedo among other tissues. The LGT expression level increases with each fruit developmental stage but in F only, which signifies that delayed bitterness level which is inversely related to LGT transcript level, decreases till the late fruit developmental stage. Further, a prokaryotic overexpression construct was generated by cloning LGT gene into pET-28a vector. This pET28a-LGT construct plasmid was transformed into DH5alpha, and BL21 (DE3) Escherichia coli strain for its protein expression study in future.
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economics, biological phenomena, resins, productivity, technological changes, exhibitions, developmental stages, marketing, biological development, selection