Author(s): Zongyong Tong, Can Xie, Lei Ma, Liping Liu, Yongsheng Jin, Jiangli Dong, Tao Wang *
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. Due to the lack of a biosynthetic pathway for methionine, cysteine, lysine, and other essential amino acids (EAAs) in vivo , animals must obtain these EAAs from their diet. Sulfur amino acids (SAAs), mainly methionine and cysteine, are particularly important EAAs, and previous studies have recognized the importance of sulfur in alfalfa . The effects of amino acids in ruminants have been studied , 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 , . Indeed, quality breeding, particularly to increase the content of SAAs, is the most important aspect of alfalfa engineering.
Compared to traditional breeding methods, transgenic techniques are more efficient for directly manipulating the characteristics of plants, and the transfer of a few sulfur-rich protein genes into plants has been reported using engineering technology -. The SAA levels in transgenic alfalfa were increased via the transformation of the sunflower seed albumin (SSA ) gene, which led to the production of an additional 40 mg of SAAs in the leaves; when these leaves were fed daily to sheep, their wool growth rates were significantly increased . It was also reported that overexpressing sulfur amino acid-rich protein (HNP)  and Arabidopsis cystathionine [gamma]-synthase (At CGS)  could significantly increase SAAs in transgenic alfalfa. Although this method has led to some success, most of these high-SAA proteins were found to be unstable in the plants -; furthermore, the increase in SAA in the transgenic plants occurred at the expense of other endogenous sulfur-rich proteins or compounds , .
There is an increasing demand for high-protein forage due to the rapid development of the animal industry, and there is a constant need for more effective genes that can enhance desirable characteristics in livestock. Aspartate kinase (AK) catalyzes the phosphorylation of aspartate, forming Asp-phosphate. This phosphorylation is the first step in the aspartate family biosynthesis pathway and is regulated by several feedback inhibition loops in plants . Threonine feedback inhibits the activity of AK-homoserine dehydrogenase , whereas lysine negatively regulates the activity of the monofunctional AK isozymes , , . However, site-directed mutagenesis of AKIII from Escherichia coli was performed to substitute an isoleucine for a threonine at amino acid 352, which reduced the feedback inhibition of AK by lysine . Overexpression of a bacterial feedback-insensitive AK in transgenic plants led to an increase in threonine that was accompanied by a reduction in both aspartate and glutamate; whereas the level of methionine, which diverges from this branch, was not significantly altered , . Additionally, the seed-specific expression of a bacterial AK was shown to increase the threonine and methionine levels in the seeds...