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Open Access
MAP BASED CLONING OF BACTERIAL BLIGHT RESISTANCE GENE Xa30(t) IN RICE
(Punjab Agricultural University, Ludhiana, 2010) Bhasin, Hemal
Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a widespread disease in tropical Asia, contained largely through the deployment of race-specific resistance genes. Cloning of R genes not only helps in elucidating molecular mechanism of resistance specificity based on structure and function, but also in understanding evolutionary process of complex R gene loci. The present study aimed at cloning of bacterial blight resistance gene Xa30(t) identified from Oryza nivara and transferred to Oryza sativa cv. PR114. Xa30(t) was mapped to a 38.4 kb region on chromosome 4L between markers LOC_Os04g53060 and LOC_Os04g53120 covering the BAC clone OSJNBb0085C12. Thirty primers were designed, in several rounds, to amplify the three candidate genes LOC_OS04g53030, LOC_OS04g53050 and LOC_OS04g53060 present in the BAC clone OSJNBb0085C12. Full length sequence data could be obtained only for LOC_Os04g53030. LOC_Os04g53060 didn’t amplify in O. nivara. LOC_Os04g53050 also showed either non-specific amplification as was evident after sequencing results or no amplification at all. However, sequence data of a nearly 1kb region that successfully amplified the targeted region on LOC_Os04g53050 showed evidence of significant rearrangements in O. nivara as compared to PR114 and Nipponbare with 116 SNPs and 81 gaps. The sequence data for LOC_Os04g53030 was obtained using primer walking strategy. O. nivara and PR114(Xa30) completely resembled at sequence level for LOC_Os04g53030 as well as for part of region from LOC_Os04g53050. Gene prediction results suggested LOC_Os04g53030 in O. nivara to be a 5044 nucleotide long NBS-LRR gene with CDS length of 4482 bp and predicted protein length of 1493 amino acids. It has four predicted exons separated by three introns which are 121bp, 237bp and 204bp in length. The predicted O. nivara protein harboured several conserved motifs typical of NBS-LRR proteins and showed evidence of many non-conservative substitutions as compared to PR114. Results obtained provide clues to evolution of these R gene loci with evidence of significant rearrangements. Predicted O. nivara protein sequence at Xa30(t) locus was obtained which can be confirmed later using gene expression and RNA interference studies. Recognition of a 48bp deletion between O. nivara acc 81825 and PR114, led to development of an STS marker, which co-segregates with Xa30(t) and can be used for marker-assisted selection.
Open Access
Designing, generating anti-fungal gene cassettes and engineering of Citrus cells for transient expression
(Punjab Agricultural University, 2010) Gumber, Hardeep Kaur; Sandhu, Jagdeep Singh
In the present study two anti-fungal gene constructs were generated comprising CaMV35SChitinase- NOS and CaMV35S-Glucanase-NOS using 905 bp and 1764bp Open Reading Frames (ORFs) respectively from the indigenously cloned chitinase (Accession no. GU187942) and glucanase (Accession no. GU300766) genes. ORFs were PCR amplified using specifically designed primers containing overhanging restriction sites and ligated into pBI121 cleaved by corresponding restriction enzymes. The gene constructs were further subcloned into high copy number plasmids pUC18 and pGEM 9Zf(-) respectively for particle bombardment of citrus cells for transient expression analysis. The gene constructs were bombarded individually as well as in combination. PCR analysis demonstrated insertion of gene construct in 78% of calli bombarded with chitinase gene construct, in 28% of the calli bombarded with glucanase gene construct and in 9.3% of the calli co-bombarded with both the gene constructs. Keywords: Sub-cloning, chitinase, glucanase