Abstract
Alfalfa (Medicago sativa L.) is one of the most important forage crops used to feed livestock, such as cattle and sheep, and the sulfur amino acid (SAA) content of alfalfa is used as an index of its nutritional value. Aspartate kinase (AK) catalyzes the phosphorylation of aspartate to Asp-phosphate, the first step in the aspartate family biosynthesis pathway, and adenylylsulfate reductase (APR) catalyzes the conversion of activated sulfate to sulfite, providing reduced sulfur for the synthesis of cysteine, methionine, and other essential metabolites and secondary compounds. To reduce the feedback inhibition of other metabolites, we cloned bacterial AK and APR genes, modified AK, and introduced them into alfalfa. Compared to the wild-type alfalfa, the content of cysteine increased by 30% and that of methionine increased substantially by 60%. In addition, a substantial increase in the abundance of essential amino acids (EAAs), such as aspartate and lysine, was found. The results also indicated a close connection between amino acid metabolism and the tricarboxylic acid (TCA) cycle. The total amino acid content and the forage biomass tested showed no significant changes in the transgenic plants. This approach provides a new method for increasing SAAs and allows for the development of new genetically modified crops with enhanced nutritional value.
Highlights
Alfalfa (Medicago sativa L.) is one of the most important forage crops and is grown throughout the world
Through site-directed mutagenesis, Aspartate kinase (AK) was modified to reduce feedback inhibition from downstream metabolites, and the altered AK was subcloned into vector pOSB108 containing a chloroplast transit peptide sequence. pOSB108-AK was co-integrated with the recipient vector pDES200 in vivo using Cre/loxP-mediated recombination
There are two main ways to enhance the expression of sulfur amino acid (SAA) according to the amino acid metabolic pathway (Fig 8)
Summary
Alfalfa (Medicago sativa L.) is one of the most important forage crops and is grown throughout the world. As fodder, this crop provides proteins, vitamins, and other minerals to such ruminants as cattle and sheep. Sulfur amino acids (SAAs), mainly methionine and cysteine, are important EAAs, and previous studies have recognized the importance of sulfur in alfalfa [1]. The effects of amino acids in ruminants have been studied [2], and it has been reported that low amounts of SAAs in forage limit wool growth in sheep and milk production and meat quality in cattle [3,4]. Quality breeding, to increase the content of SAAs, is the most important aspect of alfalfa engineering
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