Construção de um vetor viral, baseado no DNA-A do Tomato rugose mosaic virus (ToRMV), para a indução de silenciamento gênico em plantas hospedeiras

• Ano de defesa: 2009
• Autor(a) principal: Cardoso, Mariana Santos
• Orientador(a): Barros, Everaldo Gonçalves de
• Banca de defesa: Guimarães, Valéria Monteze , Fietto, Luciano Gomes
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Mestrado em Genética e Melhoramento
• Departamento: Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me
• País: BR
• Palavras-chave em Português: VIGS; Begomovírus; Glycine max; Lycopersicon esculentum
• Palavras-chave em Inglês: VIGS; Begomoviruses; Glycine max; Lycopersicon esculentum
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
• Link de acesso: http://locus.ufv.br/handle/123456789/4685

Begomoviruses belong to the Geminiviridae family which includes viruses with genomes containing one or two single stranded DNA molecules within icosahedral geminate particles. Begomoviruses are transmitted by the whitefly Bemisia tabaci and may cause diseases of economical importance in several crops mainly in tropical and subtropical regions. In Brazil there are several reports of begomovirus causing serious losses to the common bean and tomato crops, and sporadic reports of infection of soybean. Virus-induced gene silencing (VIGS) is an attractive and fast alternative for studying the expression and function of genes because it does not require plant transformation. Viruses with RNA or DNA genomes have been successfully used as vectors to induce gene silencing in plants. However, there are few examples of efficient vectors for inoculation of tomato and soybean. The main goal of this work was to build a viral vector based on the DNA-A of the begomovirus Tomato rugose mosaic virus (ToRMV) to induce gene silencing in plants of soybean, tomato and Nicotiana benthamiana. The viral vector, designated pToR-A1.4ΔCP, was built from an infectious ToRMV-A clone from which the gene encoding the capsid protein was removed and replaced by the multiple cloning site of the plasmid vector pBluescript KS+ (pKS+). Like other begomoviruses, ToRMV does not require the capsid protein to cause a systemic infection. The construction of the viral vector was confirmed by PCR, enzymatic cleavage and DNA sequencing. To be used on gene silencing experiments, fragments from the following genes were PCR amplified from cDNA clones: phytoene desaturase (PDS) from soybean and tomato, myo-inositol-1-phosphate synthase (MIPS) from soybean and stachyose synthase (STS) from soybean. All fragments were amplified with primers contained restriction sites for cloning. After amplification, the fragments were cloned in pGEM-T Easy vector, sequenced and aligned to the corresponding cDNAs to confirm their identity. To clone these fragments in the viral vector they were cleaved out of the pGEM-T Easy vector, purified and cloned into the pToR-A1.4ΔCP vector previously cleaved at the multiple cloning site with the same enzymes used to cleave the plasmid vector. The ligation reaction used T4 DNA ligase and ultracompetent Escherichia coli cells were transformed by heat shock. Transformants colonies were analyzed by PCR and enzymatic cleaved. Systemic infection of soybean, tomato and Nicotiana benthamiana plants with the empty pToR-A1.4ΔCP vector co-inoculated with ToRMV DNA-B was checked by PCR 21 days after inoculation. In the case of N. benthamiana, the viral vector presented 82% infectivity, indicating the high potential of this vector for VIGS studies in this plant species. In the case of soybean and tomato, the viral vector presented low replication efficiency. New infectivity tests need to be done to confirm these results. It is expected that this viral vector will represent an alternative for functional genomic studies and for the analysis of genes involved in several biological processes.