Transformação genética de nós cotiledonares de soja visando à co-supressão do gene da lisofosfatidilcolina aciltransferase

• Autor(a) principal: Martins, Polyana Kelly
• Data de Publicação: 2007
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1417
• topic: Ácidos graxos; Biossíntese; Transformação genética vegetal; Agrobacterium tumefaciens; Fatty acids; Biosynthesis; Genetic transformation of plants; Agrobacterium tumefaciens; CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS

The soybean (Glycine max L. Merrill) is one of the most important crops of the world and the biggest in Brazil in cultivated area. Brazil is the second largest world producer of these grain legumes, producing 56.3 million tons in the harvest year 2006/2007 (www.conab.gov.br). Modifications using techniques of genetic engineering can facilitate the fast development of new varieties with agronomic and industrial characteristics of interest. Alterations in the relative proportions of the fatty acids may be reached through genetic transformation of plants by gene silencing. Among these enzymes are desaturases, phosphotransferases and acyltransferases, involved in the fatty acids biosynthesis. The objectives of this work were to isolate the fragment of the soybean lysophosphatidycholine acyltransferase gene (LPCAT), to construct an expression cassette containing this fragment driven by the gene promoter of the α subunit of soybean storage protein β - conglycinine (seed-specific promoter) and to transform soybean cotyledonary node with this construction, seeking to the co-suppression of the LPCAT gene. The RT-PCR analysis showed that the LPCAT gene is expressed in all of the stages of seed development. The cassette of approximately 800 pb containing the seed-specific promoter sequence and of part of the gene LPCAT was cloned in the binary vector pCAMBIA 1304, whose T-DNA presents genes for resistance to the antibiotics kanamicina, for selection in bacteria, and Hygromycin B for selection in plants. The cloning was confirmed through PCR, enzymatic restriction and sequencing. The construction cloned in the vector pCAMBIA 1304 was used for soybean transformation by sonication using Agrobacterium tumefaciens. Mature soybean seeds were sterilized and germinated in B5 medium during, approximately, five days. From a single seedling, two explants were obtained, that were removed the root, followed by most of the hypocotyl and finally separated into two cotyledons. These explants were transformed with bacterial suspension by sonication and acetosyringone addition. After three days of co-cultivation, the cotyledonary node were then transferred to shoot induction medium (SIM) without selective agent, for 14 days. Soon afterwards, it was added to the medium SIM 5 mg/L of Hygromycin B for selection of transformed shoots. The selected explants were transferred to a shoot elongation medium (SEM), still with Hygromycin B, and later for the rooting medium (RM) without the selective agent. Two months after inoculation with A. tumefaciens, multiple shoots were produced in the node area resistant to Hygromycin B that were in direct contact with the medium. Leaf DNA analyses from plants in phase of acclimatization allowed the detection of several transformation events. Seventeen transgenic plants were confirmed by PCR analyses with specific primers for sequences present only in transformed plants.