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Sequencing of the open reading frame and identification of isoforms of three lignin biosynthesis genes in Panicum maximum and Brachiaria brizantha


Several strategies have been developed to optimize cell wall composition in plants in order to improve its agro-industrial use. When using forages for animal nutrition, cell wall lignifications with the advance in maturity is the main limiting factor for cattle productivity. Notwithstanding its importance for ruminant production in Brazil, there is no published study altering lignin synthesis in tropical forages. Several temperate plants, such as tobacco, Arabidopsis, alfalfa and maize, have been subject to genetic altering the expression of enzymes involved with lignin precursor's synthesis, which resulted in less lignin and higher ruminal digestibility. Recently, expression of the genes caffeoyl O-methyltransferase (COMT), phenilalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) have been identified as correlated with the decrease in cell-wall digestibility of P. maximum stems. Our objectives are to sequence the open reading frame of the genes COMT, PAL and C4H in the grasses P. maximum and B. brizantha; identify the isoforms of these genes and quantify the isoforms expression during development, aiming to generate knowledge for future experiments with genetic engineer aiming to reduce the expression of genes responsible for lignin synthesis in grasses. To do so, the grasses will be harvested with 30, 60 and 90 days of regrowth and total RNA will be utilized in reactions of rapid amplification of cDNA ends (RACE) using specific primers designed based on P. maximum sequences recently deposited in the GeneBank. After amplification, the product of Nested-PCR Will be inserted into competent cells and sequenced. The presence of isoforms for these genes will be verified through the visualization of bands differing in size and also by northern blot of RNA. When existing, the isoforms will be sequenced and its profile of expression in grasses stems will be quantified by real-time PCR. The samples will be characterized regarding its lignin content and cell-wall digestibility, in order to identify which isoform is related to the decline in stem digestibility with advanced maturity. Sequencing of the open reading frame and the identification of the isoforms responsible for the decline in digestibility, will assist in the development of future studies of genetic engineering generating transgenic plants with reduced expression of these genes. The most common method for gene knockout in grasses is the addition of the open reading frame of the gene of interest, in the sense and antisense directions, under control of maize ubiquitin promoter. (AU)

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