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AFLP Analysis of Genetic Relationships and Diversity of Sri Lankan Oryza sativa Cultivars
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
a suitable mathematical model and also to optimize the cultural conditions for L-Asparaginase production. In order to determine the effect of process variables on the yield of bioproducts, by the way of Response Surface Methodology, an experimental plan (BBD) involving a design matrix of 27 trails was sketched. The design results were fitted with Response surfaces. Empirical quadratic equations were fitted to the experimental data (BBD) relevant to the yields of L-Asparaginase with four chosen variables. The yields predicted by the mathematical equation were found to be in close agreement with experimental values. These Response surface results were compared with the results of artificial intelligence (ANN) model. The ANN model is found to perform better than the BBD model. The optimization is performed using Artificial Intelligence-based evolutionary algorithm of Real coded Genetic Algorithm. doi:10.1016/j.jbiotec.2010.09.884 [PUB-O.24] Expression and purification of recombinant lysozyme from Apostichopus japonicus Tingting Wang ∗ , Yongxin Sun, Liji Jin, Guangyi Wang, Wenjia Liu, Yongping Xu Dalian University of Technology, Dalian, China Keywords: Apostichopus japonicus; Pichia pastoris; lysozyme; Expression; Antibacterial activity Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China. As a crucial biodefense effector in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicas. In the present study, we used a genetic engineering method to produce recombinant Apostichopus japonicus lysozyme (Aj-lysozyme) protein as bioreactor for cheap and mass production, hoping to develop a new substitute for the antibiotics currently used. Aj-lysozyme gene was amplified and inserted into expression vector pPIC9K of Pichia pastoris with the fusion at N terminal. The constructed vector pPIC9K-lys was linearized by digestion with Bgl II and then transformed into Pichia pastoris GS115 (HIS4) competent cells using electropolation. Multicopy transformants were selected on the MM plate and MD plate for phenotype and His+ Muts strains. YPD plates containing 4 mg/ml G418 were used to select the highest expression strain of pPIC9K-lys from His+ Muts phenotype strains. The selected pPIC9K-lys expression strain was then used to express in Pichia pastoris. The Aj-lysozyme was expressed properly and determined by SDS-PAGE and Western-blotting. Express condition was optimized and the highest express level was achieved under the condition of 192 h, 1% methyl alcohol and pH 5, the optimized express level can reach 50 g/ml. The recombined Aj-lysozyme was then purified using Ni-NTA Column. The antibacterial activity of the purified Aj-lysozyme against Micrococcus lysodeikticus was detected and radius of translucent plaque showed that the growth of Micrococcus lysodeikticus was inhibited by the recombinant Aj-lysozyme. doi:10.1016/j.jbiotec.2010.09.885
S539
[PUB-O.25] AFLP Analysis of Genetic Relationships and Diversity of Sri Lankan Oryza sativa Cultivars Gowri Raajkumar 1,∗ , Jegath Weerasena 1 , Kumudu Fernando 2 , Athula Liyanage 3 1
Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka 2 Agricultural Biotechnology Centre, University of Peredeniya, Sri Lanka 3 Plant Genetic Resorce Certre, Sri Lanka Keywords: rice; AFLP; dendrogram; cluster analysis Sri Lanka is considered as a valuable repository of crop germplasm resources and agro biodiversity. When compared to morphological studies and pedigree analysis, molecular markers reveal differences among accessions at the DNA level. There are no records on genetic diversity of Sri Lankan cultivated rice varieties. It was possible to genetically differentiate 28 Sri Lankan local cultivars by Fluorescent Amplified Fragment Length Polymorphism (FAFLP). FAFLP is based on selective amplification by PCR of a subset of restriction fragments from a digest of a whole rice genome. Fragments were separated through a capillary on automated MegaBACE 1000 DNA sequencer. The electropherograms were analyzed by Genetic Profiler software and used for phylogenetic analysis. Ten primer combinations generated a total of 784 fragments sizes ranging from 30 to 500 bp long. Of these 772 were polymorphic (98.4%) and 12 fragments were monomorphic. Principal coordinates analysis was performed to detect the patterns of relationship and genotypes were plotted on first three dimensions. Jaccard’s similarity coefficients varied from 0.139-0.539 and the similarity coefficients matrix was used for the UPGMA cluster and dendrogram was generated. The Mantel’s test for cophenetic correlation r = 0.786 indicated very good fit of the varieties to a group in the cluster analysis. Rice accessions showed four main clusters. One modern rice variety “Taichung Native 1” and a sample from Hygroryza were also included in this study to find out the reliability of data and analysis and these were separated uniquely from others. Two different hybrids BG and BW varieties were separated into two distinct clusters. New improved and old improved varieties also clearly separated. This genetic diversity data provide a more direct, reliable and efficient tool for differentiation and selecting parents for hybridization in order to develop new cultivars with desirable characteristics and useful in conservation of rice genetic resources. This work was supported by National Research Council of Sri Lanka (05/61) doi:10.1016/j.jbiotec.2010.09.886
a suitable mathematical model and also to optimize the cultural conditions for L-Asparaginase production. In order to determine the effect of process variables on the yield of bioproducts, by the way of Response Surface Methodology, an experimental plan (BBD) involving a design matrix of 27 trails was sketched. The design results were fitted with Response surfaces. Empirical quadratic equations were fitted to the experimental data (BBD) relevant to the yields of L-Asparaginase with four chosen variables. The yields predicted by the mathematical equation were found to be in close agreement with experimental values. These Response surface results were compared with the results of artificial intelligence (ANN) model. The ANN model is found to perform better than the BBD model. The optimization is performed using Artificial Intelligence-based evolutionary algorithm of Real coded Genetic Algorithm. doi:10.1016/j.jbiotec.2010.09.884 [PUB-O.24] Expression and purification of recombinant lysozyme from Apostichopus japonicus Tingting Wang ∗ , Yongxin Sun, Liji Jin, Guangyi Wang, Wenjia Liu, Yongping Xu Dalian University of Technology, Dalian, China Keywords: Apostichopus japonicus; Pichia pastoris; lysozyme; Expression; Antibacterial activity Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China. As a crucial biodefense effector in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicas. In the present study, we used a genetic engineering method to produce recombinant Apostichopus japonicus lysozyme (Aj-lysozyme) protein as bioreactor for cheap and mass production, hoping to develop a new substitute for the antibiotics currently used. Aj-lysozyme gene was amplified and inserted into expression vector pPIC9K of Pichia pastoris with the fusion at N terminal. The constructed vector pPIC9K-lys was linearized by digestion with Bgl II and then transformed into Pichia pastoris GS115 (HIS4) competent cells using electropolation. Multicopy transformants were selected on the MM plate and MD plate for phenotype and His+ Muts strains. YPD plates containing 4 mg/ml G418 were used to select the highest expression strain of pPIC9K-lys from His+ Muts phenotype strains. The selected pPIC9K-lys expression strain was then used to express in Pichia pastoris. The Aj-lysozyme was expressed properly and determined by SDS-PAGE and Western-blotting. Express condition was optimized and the highest express level was achieved under the condition of 192 h, 1% methyl alcohol and pH 5, the optimized express level can reach 50 g/ml. The recombined Aj-lysozyme was then purified using Ni-NTA Column. The antibacterial activity of the purified Aj-lysozyme against Micrococcus lysodeikticus was detected and radius of translucent plaque showed that the growth of Micrococcus lysodeikticus was inhibited by the recombinant Aj-lysozyme. doi:10.1016/j.jbiotec.2010.09.885
S539
[PUB-O.25] AFLP Analysis of Genetic Relationships and Diversity of Sri Lankan Oryza sativa Cultivars Gowri Raajkumar 1,∗ , Jegath Weerasena 1 , Kumudu Fernando 2 , Athula Liyanage 3 1
Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka 2 Agricultural Biotechnology Centre, University of Peredeniya, Sri Lanka 3 Plant Genetic Resorce Certre, Sri Lanka Keywords: rice; AFLP; dendrogram; cluster analysis Sri Lanka is considered as a valuable repository of crop germplasm resources and agro biodiversity. When compared to morphological studies and pedigree analysis, molecular markers reveal differences among accessions at the DNA level. There are no records on genetic diversity of Sri Lankan cultivated rice varieties. It was possible to genetically differentiate 28 Sri Lankan local cultivars by Fluorescent Amplified Fragment Length Polymorphism (FAFLP). FAFLP is based on selective amplification by PCR of a subset of restriction fragments from a digest of a whole rice genome. Fragments were separated through a capillary on automated MegaBACE 1000 DNA sequencer. The electropherograms were analyzed by Genetic Profiler software and used for phylogenetic analysis. Ten primer combinations generated a total of 784 fragments sizes ranging from 30 to 500 bp long. Of these 772 were polymorphic (98.4%) and 12 fragments were monomorphic. Principal coordinates analysis was performed to detect the patterns of relationship and genotypes were plotted on first three dimensions. Jaccard’s similarity coefficients varied from 0.139-0.539 and the similarity coefficients matrix was used for the UPGMA cluster and dendrogram was generated. The Mantel’s test for cophenetic correlation r = 0.786 indicated very good fit of the varieties to a group in the cluster analysis. Rice accessions showed four main clusters. One modern rice variety “Taichung Native 1” and a sample from Hygroryza were also included in this study to find out the reliability of data and analysis and these were separated uniquely from others. Two different hybrids BG and BW varieties were separated into two distinct clusters. New improved and old improved varieties also clearly separated. This genetic diversity data provide a more direct, reliable and efficient tool for differentiation and selecting parents for hybridization in order to develop new cultivars with desirable characteristics and useful in conservation of rice genetic resources. This work was supported by National Research Council of Sri Lanka (05/61) doi:10.1016/j.jbiotec.2010.09.886