RESUMO
Oryza officinalis has proven to be a natural gene reservoir for the improvement of domesticated rice as it carries many desirable traits; however, the transfer of elite genes to cultivated rice by conventional hybridization has been a challenge for rice breeders. In this study, the conserved sequence of plant stress-related NAC transcription factors was selected as a probe to screen the O. officinalis genomic transformation-competent artificial chromosome library by Southern blot; 11 positive transformation-competent artificial chromosome clones were subsequently detected. By Agrobacterium-mediated transformation, an indica rice variety, Huajingxian 74 (HJX74), was transformed with a TAC clone harboring a NAC gene-positive genomic fragment from O. officinalis. Molecular analysis revealed that the O. officinalis genomic fragment was integrated into the genome of HJX74. The transgenic lines exhibited high tolerance to drought stress. Our results demonstrate that the introduction of stress-related transformation-competent artificial chromosome clones, coupled with a transgenic validation approach, is an effective method of transferring agronomically important genes from O. officinalis to cultivated rice.
Assuntos
Cromossomos Artificiais , Secas , Oryza/genética , Estresse Fisiológico , Transformação Genética , Adaptação Biológica , Oryza/crescimento & desenvolvimento , Fenótipo , Plantas Geneticamente Modificadas , Característica Quantitativa HerdávelRESUMO
The identification of new targets for vaccine and drug development for the treatment of Chagas' disease is dependent on deepening our understanding of the parasite genome. Vectors for genetic manipulation in Trypanosoma cruzi basically include those that remain as circular episomes and those that integrate into the parasite's genome. Artificial chromosomes are alternative vectors to overcome problematic transgene expression often occurring with conventional vectors in this parasite. We have constructed a series of vectors named pTACs (Trypanosome Artificial Chromosomes), all of them carrying telomeric and subtelomeric sequences and genes conferring resistance to different selection drugs. In addition, one pTAC harbours a modified GFP gene (pTAC-gfp), and another one carries the ornithine decarboxilase gene from Crithidia fasciculata (pTAC-odc). We have encountered artificial chromosomes generated from pTACs in transformed T. cruzi epimastigotes for every version of the designed vectors. These extragenomic elements, in approximately 6-8 copies per cell, remained as linear episomes, contained telomeres and persisted after 150 and 60 generations with or without selection drugs, respectively. The linear molecules remained stable through the different T. cruzi developmental forms. Furthermore, derived artificial chromosomes from pTAC-odc could complement the auxotrophy of T. cruzi for polyamines. Our results show that pTACs constitute useful tools for reverse functional genetics in T. cruzi that will contribute to a better understanding of T. cruzi biology.
Assuntos
Clonagem Molecular , Regulação da Expressão Gênica/fisiologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Animais , Cromossomos Artificiais , Camundongos , Organismos Geneticamente ModificadosRESUMO
The centromere is a specialized region of eukaryotic chromosomes, the site of kinetochore formation, spindle attachment and regulation of chromosome segregation during mitotic and meiotic cell divisions. To identify sequences which increase mitotic stability and/or act as potential centromeres in Leishmania major, we first generated libraries of Leishmania linear artificial chromosomes (LACs) bearing 30 kb inserts of randomly selected genomic DNAs. These were introduced into parasites, and then their stability was assessed following a period of 10 passages of growth in the absence of selective pressure. Approximately 80% of the 108 transfectants tested lost their LACs promptly and only 20% of the recombinants were retained; of these six showed strong but partial stability (maintained in 30-46% of cells). Mapping and sequencing of one clone (cSC10), which confers the highest degree of maintenance, revealed the presence of a sequence that was found within another stable episome, and which is dispersed in the genome of L. major. The implications of these data to the possible mechanisms of chromosomal maintenance are discussed.