Sulfur Amino Acids,Lignin Metabolic Regulation And Molecular Breeding Research Of Alfalfa (Medicago Sativa L.)

Alfalfa (Medicago sativa.L) is one of the most important forages to feed livestock such as cows and sheep,spreading all over the world. And alfalfa is called as "the king of forages". Sulfur amino acids (SAAs) and lignin is very important index of its nutritional value. We genetically modified SAAs and lignin contents of alfalfa in the molecular level, and created a new germplasm containing high SAAs or cellulose.SAAs could improve the quality and production of milk and wool. Aspartate kinase (AK) and Adenylylsulfate reductase (APR) are the key enzymes of aspartate family biosynthesis and sulfate assimilation pathway respectively. Former researches indicated over-expressing single gene can enhance the SAAs levels, whether the synergy of carbon and sulfur resource metabolic pathways can increase SAAs contents is an important issue. This essay performed AK and APR genes from bacteria and co-expressed them in alfalfa through RMDAP. Several transgenic plants had been analyzed by PCR,Southern Blot, RT-PCR, RT-qPCR and Western Blot, results indicated AK and APR integrated in transgenic plants genome, and inherited to Ti progeny stably. Transgenic plants had no significant changes in phenotype and agricultural traits. The expression levels of three key genes: CGS, SAT, MS increased in different amounts. Amino acids analysis revealed that Cys increased by 28.6%, Met increased by 58.8%, Asp increased by 30.7%-40.4% and Lys increased by 38.7%-51.6%, but Pro decreased by 35.6%-40.0% ,Ser decreased by about 10% and OAS increased by 21.0%-47.5% in the T0 transgenic plants. There is a similar result in T1 transgenic progeny. And, changes of amino acids contents connected with TCA circle through oxaloacetate and a-ketoglutarate, the increase of Asp family accompanies with the declined of Glu family. Additionally, an increase in cysteine was likely at the expense of serine, the carbon skeleton donor of cysteine.Lignin can affect the digestibility and palatability of forages. Recently, alfalfa has also been proposed as a next-generation feedstock for biofuel because it is rich in lignocellulose biomass, but lignin is an obstacle in the conversion from cellulose to alcohol. To reduce the lignin content of alfalfa,we constructed the two key genes (HCT and C3H) interfered cassettes in a transformed vector through RMDAP. And transformed alfalfa, several transgenic plants had been confirmed by PCR and the expression levels of HCT and C3H were down-regulated by RT-qPCR. Through the analysis of paraffin section and lignin content, compared to wild-type plants, the lignin content of Linell reduced by about 38% and cellulose increased by 86.1%, saccharification efficiency of Line8 increased by 20.9%significantly, and the degradation rate of Linell is also enhanced by 13.0%.Through regulating SAAs and lignin biosynthesis pathway by multi-gene transformation technology, the new germplasm of high SAAs or high cellulose was created. These results provided new ideas and methods to plant molecular breeding, had an important significance for the development of prataculture and animal husbandry.