2017-09-252017-09-252014-112014-12-12November 2http://hdl.handle.net/10388/ETD-2014-11-1826http://hdl.handle.net/10388/8137Lentil (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 LcGolS1 and 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 to similarity in nucleotide sequences, RNA gel blot analyses could not distinguish transcript accumulation due to two LcGolS alleles. LcGolS transcript accumulation corresponded to the GS enzyme activity that was highest at 26 and 28 DAF and gradually decreased as the seed matured. For raffinose synthase (RS), a full length LcRS sequence of 2379 bp, coding for 792 amino acids and predicted molecular weight of 88.2 kDa was isolated. Gene expression of LcRS during seed development showed maximum expression at 24 - 26 DAF. Raffinose synthase enzyme activity correlated with gene expression with maximum activity obtained at 24 DAF. Raffinose concentration during seed development showed rapid increase from 20 DAF with maximum accumulation at 28 DAF. Raffinose accumulation correlated with accumulation of its precursors, sucrose and galactinol during seed development. LcRS gene expression and RS enzyme activity correlated with oligosaccharide accumulation, suggesting that the isolated gene contributed to raffinose accumulation in seeds. In lentil seeds the predominant RFO is stachyose, synthesized by stachyose synthase (STS) which transfers a galactosyl residue from galactinol to raffinose. A cDNA clone LcSTS was isolated that is 2669 bp long (5'UTR - 30bp, ORF - 2559, 3'UTR 80 bp followed by polyA) coding for 853 amino acids with predicted molecular weight of 96 kDa. RNA gel blot and qPCR analyses showed maximum accumulation of LcSTS transcript at 26 and 24 DAF respectively. Stachyose and verbascose concentrations increased during seed development with maximum concentration at 32 DAF. STS enzyme activity correlated with LcSTS expression resulting in maximum activity at 26 DAF. The gene coding for verbascose synthase (VS) was not isolated in this study, however enzyme activity for VS was studied during seed development. Maximum VS enzyme activity was observed at 26 DAF. RFO can also be synthesized by galactinol independent enzyme (Galactan:galactan galactosyl transferase, GGT) and its activity was also detected in developing lentil seeds. Maximum GGT activity was observed from 26 - 32 DAF. Throughout the seed developmental stages, oligosaccharide accumulation correlated well with transcript accumulation and enzyme activity of RFO biosynthetic genes. In conclusion, genes for three key enzymes in RFO biosynthesis were identified and characterized by DNA sequencing. During lentil seed development, transcript accumulation of the three genes correlated with respective enzyme activity and corresponding RFO accumulation. The gene sequences for these three key RFO biosynthetic genes can be used in the near future to study allelic variation to understand their role in influencing total RFO or its constituent concentrations in lentils.application/pdfRaffinose, OligosaccharideThesis2017-09-25