BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF RAFFINOSE FAMILY OLIGOSACCHARIDE (RFO) BIOSYNTHESIS IN LENTIL (Lens culinaris Medik.)
Year: 2014
Type: dissertation
Abstract: Lentil (Lens culinaris Medik.) is an important pulse crop and a balanced source of carbohydrates, proteins, minerals and vitamins.Among the carbohydrates, lentils contain raffinose family oligosaccharides (RFO) or sucrosyl oligosaccharides that upon consumption remain undigested in the lower bowel of humans and monogastric animals causing flatulence and stomach discomfort.The high concentration of RFO in lentils limits human consumption and acceptance worldwide.To develop strategies to reduce RFO concentration in lentils, it is imperative to understand the biochemical and molecular mechanism of RFO deposition in lentil seeds.The main objective of this study was to isolate and characterize RFO biosynthetic genes galactinol synthase (GS or GolS; EC 2.4.1.123),raffinose synthase (RS; EC 2.4.1.82)and stachyose synthase (STS; EC 2.4.1.67).The expression of RFO biosynthetic genes, corresponding enzyme activity and RFO accumulation were also analyzed during lentil seed development.Gene expression studies in developing seeds necessitated an optimized method to extract RNA with high purity in adequate quantities.Previously used RNA isolation procedures, namely (i) Trizol based RNA isolation; (ii) Phenol guanidine based RNA isolation; and (iii) Lysis buffer; were tested with modifications.All three methods did not yield RNA with the required purity and/or in adequate quantity.A CTAB (hexadecyltrimethylammonium bromide) based RNA isolation procedure with lithium chloride precipitation yielded RNA in adequate quantity (210 -260 μg from 200 mg of seeds) and required purity (A260 / 280 ratio of about 2.2).The optimized RNA isolation procedure was used to isolate mRNA to construct a cDNA library from developing lentil seeds and in gene expression studies during seed development.Galactinol synthase (GS or GolS) catalyses the synthesis of galactinol, the first committed step in RFO biosynthesis.Screening a cDNA library prepared from developing lentil seeds identified two cDNA clones LcGolS1 (1336 bp, ORF -1002 bp, 334 amino acids) and LcGolS2 (1324 bp, ORF -975 bp, 325 amino acids).Predicted molecular weights of LcGolS1and LcGolS2 were 38.7 kDa and 37.6 kDa, respectively.Real time quantitative PCR studies revealed that the two GolS alleles were differentially expressed.LcGolS1 increased from 20 days after flowering (DAF), with rapid increase from 24 DAF, and maximum expression was obtained at 32 DAF.The expression of LcGolS2 was observed at 24 DAF and decreased at 26 DAF.Due Ravindra N Chibbar for providing me the opportunity to pursue my PhD under his supervision.
Author: Udhaya Kannan
Language: English
Subfield: Plant Science
Domain: Life Sciences
SDG: Zero hunger
Open Access status: green
OpenAlex ID: W2760054653