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Authors: Basha, P. Osman
Keywords: CLONING
Issue Date: 2008
Abstract: Insertional mutagenesis is one of the most powerful tools for annotating gene functions. Basmati 370 insertional mutants, Oligo culm (Osoc), Seedling lethal (O.ssl) and Small grain (Ossg) mutants were isolated using Agrobacterium tumefaciens mediated transformation with binary plasmid containing HmRD.s-. The Oligo culm (Osoc) mutant had 60-70% reduced tillering capacity and retarded growth of seminal roots. The Seedling lethal (Ossl) mutant showed 5-15% of albino plants which died soon after germination while among non-albino 10-20% died slowly without tillering after transplantation. The Small grain (Ossg) mutant had 10-15% reduced weight and length of grains. Southern hybridization of Oligo culm and Seedling lethal mutants indicated single T-DNA insertions. The Oligo culm mutant (Osoc) was crossed with indica fine rice cultivar PR106 to develop F2 mapping populations. Histogram for plant height and days to flowering phenotypic data showed nearly normal distribution while for tiller number it showed skewed distribution towards Oligo culm mutant. The observed %2 value for segregation of hygromycin resistance and hpt amplification in F2 population gave a good fit to 3:1 segregation ratio indicating a single T-DNA insertion. The linkage between T-DNA insertion and Oligo culm mutant phenotype was confirmed in F2 population of O.voc/PR106. Bulk Segregant Analysis (BSA) was performed to identify chromosomal location of Osoc. Uniformly distributed rice SSR markers on twelve chromosomes were selected. Out of 209 SSR markers used, 98 were found to be polymorphic between Basmati 370 and PR106. Polymorphic SSR markers RM 279 (6.37cM), RM236 (2.45cM) and RM12413 (1.47cM) mapped on rice chromosome 2 were found linked to xv Oligo culm (Osoc) mutant and the T-DNA insertion was flanked by RM236 and RM12413 markers. The T-DNA flanking sequences of Oligo culm (Osoc), Seedling lethal (Ossl) and Small grain (Ossg) mutants identified using Genome walking and TAIL-PCR, showed their T-DNA insertions on chromosome 2, 11 and 11, respectively. The positions of insertions were confirmed by designing T-DNA and rice genome specific primers on chromosome 2 and 11 for Oligo culm (Osoc) and Seedling lethal (Ossl), respectively. In Oligo culm mutant the insertion was present within the exonic region of the putative zinc-binding protein. The function of this protein has been predicted to be like that of a transcription elongation factor (Elf1) in Oiyza sativa. The gene starts at base 1819090 and stops at base 1816334 (complementary sequence) of chromosome number 2 with UTR regions, 4 exons and 4 introns. The length of the gene is 2,757bp with 875bp mRNA. One paralog was identified on rice chromosome 7 and the alignment results of paralogs using CLUSTALW program, showed high conservation between them. Orthologs of Elfl were found throughout the living kingdom. This family of short proteins contains a putative zinc binding domain with four conserved cysteines. Oligo culm (Osoc) gene transcription analysis studies were performed using RTPCR. Total RNA was extracted from roots and shoots of Basmati 370 and Oligo culm after six and twelve days of gennination and the cDNA was synthesized. The results of amplification using Oligo culm gene specific and actinl (reference gene) primers confirmed that the Osoc was a knockout mutant phenotype. Oligo culm (Osoc) EST database suggested that the gene was expressing more in roots followed by callus, panicle xvi and stem. Five FSTs were found for Osoc gene but no phenotypic descriptions are available for these FSTs. In the Seedling lethal (Ossl) mutant the T-DNA is inserted on chromosome number 11 where no gene has been predicted in japonica and indica rice. Basmati 370 might have an important gene at the insertion site, knocking out of which probably gave the seedling lethal phenotype. In Small grain (Ossg) mutant the T-DNA was inserted in the intronic region of the Pectin methyl esterase gene also on chromosome 11. The total length of the gene was 2,185bp with five exons and two introns. The length of mRNA was 1749bp which coded for protein with 423 amino acid residues. The gene Pectin methyl esterase contains a putative pectinesterase domain. The EST database suggests that the Small grain (Ossg) gene is expressed in panicles and flowers. The Oligo culm (Osoc) gene of Basmati 370 has been cloned using series of PCR primers based on Nipponbare sequence. Alignment results of Basmati 370 and japonica Osoc gene showed 23 SNPs at nucleotide level and 3 amino acid differences at protein level which coded for a protein with the predicted function of transcription elongation factor. The techniques Bulk Segregation Analysis, Genome Walking, TAIL-PCR and RT-PCR techniques and bioinformatics tools led to the cloning of Oligo culm (Osoc) gene controlling tillering in rice. Detailed morphological and molecular analyses of Seedling lethal (Ossl) and characterization of Small grain (Ossg) mutants has been done. The cloned and identified genes with respective morphology can be validated by genetic complementation or RNAi approaches.
Other Identifiers: Ph.D
Appears in Collections:DOCTORAL THESES (Bio.)

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