Objectif Pour établir un modéle cellulaire de l＇ oligonucléotide mutant. Méthodes Par rapport la prise du géne de la protéine de fluorescence verte et agrandisse （ EGFP ） , nous indu... Objectif Pour établir un modéle cellulaire de l＇ oligonucléotide mutant. Méthodes Par rapport la prise du géne de la protéine de fluorescence verte et agrandisse （ EGFP ） , nous induissons une mutation par la technique du point mutant de PCR pour changer du codon de 98éme position du géne egfp GAA au codon d＇ arrét TAA , formant le géne egfp-D de mutant, donc on construit deux vecteurs de plasmides ： pcDNA3-egfp et pcDNA3-egfp-D. Puis les plasmides sont transforms dans les cellules NIH-3T3 par le calcium de phosphate. Résultats Sous le microscope de fluorescence, on n＇a pas vu l＇expression de la EGFP dans tous les cellules transférées par l＇egfp-D de mutant, et en méme temps, les cellules transfgrées par l＇egfp normal peuvent exprimer la EGFP. Ensuite on obtient les cellules stables par le criblage de G418. PCR confirme l＇intégration du géne egfp-D dedans le génome ADN des cellules. RT-PCR et Northern blot analysent les génes egfp et egfp-D peuvent transcrire normalement, il montre les ceUules transferees par le géne egfp-D de mutant n ＇exprimant pas la fluorescence verte est parce que la traduction est terminée en avant. Conclusion Le modéle cellulaire de l＇oligonuclJotide mutant rapporté par le gdne egfp est établi avec succés , ce modéle peut employer en recherche du systéme réparant thérapeutique via le chimeric ARN /ADN oligonucléotide（ RDOs ）.展开更多
Genetic diversity evaluation of mutant lines is essential to facilitate their conservation and utility in breeding programs. Characterization of plant genotypes using morphological markers has limitations which make t...Genetic diversity evaluation of mutant lines is essential to facilitate their conservation and utility in breeding programs. Characterization of plant genotypes using morphological markers has limitations which make the procedure inefficient. Application of molecular tools for characterization and diversity assessment has been found useful to complement phenotypic evaluation of plant population. Therefore genetic diversity of some cowpea mutant lines was studied using simple sequence repeats (SSR) markers. DNA barcoding marker, ribulose-1,5-bisphosphate carboxylase(rbcL) of the chloroplast DNA (cpDNA) was also used for characterization and identification of the mutants to species level. The mean polymorphic information content (0.51) obtained from the microsatellites showed high polymorphism in accessing wide genetic diversity among the mutants and their parents. Dendrogram generated revealed 8 groups with most mutants clustered separately from their parents. Sequence analysis revealed insertions/deletions (InDels) and base substitutions as the two main classes of mutations induced in the plastid DNA of the mutants studied. The nucleotide frequencies were 26.95% (A), 34.43% (T), 24.09% (C) and 14.53% (G). A total of 61.38% AT rich region was identified, while GC rich region was found to be 38.62%. Highest rate of mutations were observed in region 3 - 4 indicating that the region is less conserved in cowpea rbcL gene. The present study proved that SSR markers are useful for the genetic diversity assessment of cowpea mutants. It also proved the efficiency of rbcL markers in mutants’ identification. The results indicate that the mutants are valuable genetic resources that have been developed to widen cowpea genetic base.展开更多
定向诱导基因组局部突变(targeting induced local lesions in genomes,TILLING)技术将化学诱变与高通量突变检测技术相结合,可高效、快速地从突变群体中鉴定出目标基因突变位点。本文在概述TILLING技术应用于水稻、小麦、玉米、大豆等...定向诱导基因组局部突变(targeting induced local lesions in genomes,TILLING)技术将化学诱变与高通量突变检测技术相结合,可高效、快速地从突变群体中鉴定出目标基因突变位点。本文在概述TILLING技术应用于水稻、小麦、玉米、大豆等作物突变研究现状的基础上,重点综述了TILLING分析群体构建与突变位点检测方法的技术改进与发展,探讨了TILLING技术目前存在的问题与前景。展开更多
文摘 Objectif Pour établir un modéle cellulaire de l＇ oligonucléotide mutant. Méthodes Par rapport la prise du géne de la protéine de fluorescence verte et agrandisse （ EGFP ） , nous induissons une mutation par la technique du point mutant de PCR pour changer du codon de 98éme position du géne egfp GAA au codon d＇ arrét TAA , formant le géne egfp-D de mutant, donc on construit deux vecteurs de plasmides ： pcDNA3-egfp et pcDNA3-egfp-D. Puis les plasmides sont transforms dans les cellules NIH-3T3 par le calcium de phosphate. Résultats Sous le microscope de fluorescence, on n＇a pas vu l＇expression de la EGFP dans tous les cellules transférées par l＇egfp-D de mutant, et en méme temps, les cellules transfgrées par l＇egfp normal peuvent exprimer la EGFP. Ensuite on obtient les cellules stables par le criblage de G418. PCR confirme l＇intégration du géne egfp-D dedans le génome ADN des cellules. RT-PCR et Northern blot analysent les génes egfp et egfp-D peuvent transcrire normalement, il montre les ceUules transferees par le géne egfp-D de mutant n ＇exprimant pas la fluorescence verte est parce que la traduction est terminée en avant. Conclusion Le modéle cellulaire de l＇oligonuclJotide mutant rapporté par le gdne egfp est établi avec succés , ce modéle peut employer en recherche du systéme réparant thérapeutique via le chimeric ARN /ADN oligonucléotide（ RDOs ）.
文摘Genetic diversity evaluation of mutant lines is essential to facilitate their conservation and utility in breeding programs. Characterization of plant genotypes using morphological markers has limitations which make the procedure inefficient. Application of molecular tools for characterization and diversity assessment has been found useful to complement phenotypic evaluation of plant population. Therefore genetic diversity of some cowpea mutant lines was studied using simple sequence repeats (SSR) markers. DNA barcoding marker, ribulose-1,5-bisphosphate carboxylase(rbcL) of the chloroplast DNA (cpDNA) was also used for characterization and identification of the mutants to species level. The mean polymorphic information content (0.51) obtained from the microsatellites showed high polymorphism in accessing wide genetic diversity among the mutants and their parents. Dendrogram generated revealed 8 groups with most mutants clustered separately from their parents. Sequence analysis revealed insertions/deletions (InDels) and base substitutions as the two main classes of mutations induced in the plastid DNA of the mutants studied. The nucleotide frequencies were 26.95% (A), 34.43% (T), 24.09% (C) and 14.53% (G). A total of 61.38% AT rich region was identified, while GC rich region was found to be 38.62%. Highest rate of mutations were observed in region 3 - 4 indicating that the region is less conserved in cowpea rbcL gene. The present study proved that SSR markers are useful for the genetic diversity assessment of cowpea mutants. It also proved the efficiency of rbcL markers in mutants’ identification. The results indicate that the mutants are valuable genetic resources that have been developed to widen cowpea genetic base.
文摘定向诱导基因组局部突变(targeting induced local lesions in genomes,TILLING)技术将化学诱变与高通量突变检测技术相结合,可高效、快速地从突变群体中鉴定出目标基因突变位点。本文在概述TILLING技术应用于水稻、小麦、玉米、大豆等作物突变研究现状的基础上,重点综述了TILLING分析群体构建与突变位点检测方法的技术改进与发展,探讨了TILLING技术目前存在的问题与前景。
