Correlação entre a expressão dos genes da subunidade α da β-conglicinina e GmFAD2-1A durante o desenvolvimento da semente de soja

• Autor(a) principal: Cunha, Pricila da Silva
• Data de Publicação: 2010
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2418
• topic: Soja; GmFAD2-1A; Expressão de genes; Soybean; GmFAD2-1A; Genes expression; CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOTECNIA::MELHORAMENTO VEGETAL

The increase of the oleic acid content and decrease of the polyunsaturated fatty acids levels (linolenic and linoleic) in the soybean seed may be achieved by reducing of the microsomal ω-6 desaturase enzyme activity through GmFAD2-1A gene silencing which codify this enzyme, resulting in the production of oils with high oxidative stability. The gene silencing must occur specifically in the seed, as it must not alter plant agronomics characteristics and not to interfere negativelly with plant growth and development. For that, the transgene must be under the control of a seed-specific promoter such as the promoter of the β-conglycinin α subunit gene (pBC promoter). The main objective of this work was to correlate the gene expression pattern that codify the β-conglycinin α subunit (BC gene) with the GmFAD2-1A gene expression pattern during the soybean seed development in five soybean cultivars that differ in the protein content ("normal" and "high") and life cycle (early and late maturity). And, through this correlation, to analyse if the pBC promoter presents potential to be used in future experiments of soybean genetic transformation which has as goal to silence the GmFAD2-1A gene. The analysis of the BC and GmFAD2-1A genes expression pattern was done by Real Time PCR (qRT-PCR) using two endogenous controls (GAPDH and EF1b). The β-conglycinin α subunit was quantified bySDS-PAGE/densitometry during seed development in all five cultivars. In general, the β-conglycinin α subunit concentration was lower in initial stages of seed development, increasing in middle stages towards the final of maturation, indicating that there was an accumulation of this polypeptide during the seed filling, and presenting a high and significative concentration in the mature seed. The cultivars of "normal" protein content showed higher α subunit concentrations when compared with their respective isolines of higher protein content, which occurred in middle stages towards the final maturation. The two endogenous genes (GAPDH and EF1b) normalized the BC and GmFAD2-1A genes expression data in a very similar way, with the main differences occurring in the expression patterns of the two targets genes in the middle and final stages of maturation in the early maturity cultivars. However, GAPDH was a better normalizer gene than EF1b, showing a more stable expression among all treatments. In general, the BC gene showed higher expression pattern in initial and middle stages of the seed maturation, and in these stages can be suggested that the pBC promoter reached its maximum capacity to activate transcription of the target gene, with the BC gene expression decreasing towards final stages of the seed development. It can be suggested that this decreasing in BC gene expression is associated with a decreasing in pBC promoter activity, although the pBC promoter still remains sufficiently active to ensure a high level of transgene expression in future experiments of soybean genetic transformation. None significative difference was found in BC gene expression pattern when was compared a soybean cultivar and its respective isoline of higher protein content during seed development. In general, while the α subunit concentration was lower in initial stages of maturation (1st and 2nd), the BC gene showed a higher expression pattern in initial and middle stages of seed development (2nd and 3rd). From the third stage towards the final of maturation, there was an increase in the α subunit concentration while there was a decreasing in BC gene expression in several of these stages. The main difference was found in mature seed of all cultivars, which showed a high polypeptide concentration despite of an almost null expression of the BC gene. In general, it was not found a BC gene expression pattern and of α subunit concentration that allowed to differentiate the group of late maturity cultivars of the group of early maturity cultivars. For the GmFAD2-1A gene, in general, the late maturity cultivars showed highergene expression pattern in middle stages towards the final of seed development, while in early maturity cultivars the GmFAD2-1A gene showed higher expression pattern in initial and middle stages of maturation. In the same manner as for the BC gene, the GmFAD2-1A gene expression was almost null in the mature seed for all cultivars. In general, the correlation between the pattern of BC and GmFAD2-1A genes expression in the late maturity cultivars showed that at stages where there were a higher rate of GmFAD2-1A gene transcription the expression of BC gene decreased after to reach a peak of expression in third stage. However, the BC gene expression was still higher than the GmFAD2-1A gene at these stages. Thus, for the late maturity cultivars, at stages where the GmFAD2-1A gene showed the highest expression values can be suggested that the pBC promoter is active or it is with a maximum capacity to activate the target gene transcription. In early maturity cultivars this correlation showed that the BC and GmFAD2-1A genes presented a similar expression pattern, with high levels of genes expression in initial and middle stages of maturation (2nd and 3rd), decreasing in later stages of seed development. Therefore, it can be suggested that the highest pBC promoter activity occurred in the same stages where the highest values of GmFAD2-1A gene expression were found. Thus, it can be suggested that the promoter of the β-conglycinin α subunit gene presents a high potential to be used in future experiments of genetic transformation which has as goal to silence the GmFAD2-1A gene during the soybean seed development in all cultivars analysed.