Study On Agrobacterium Mediated Zein Gene Transformation Into Medicago Sativa L.

Alfalfa is the most important cultivated forage crop in the world, considering as"the king of forage". However, legume proteins are deficient in sulfur-containing amino acids. It has been shown that wool growth in sheep, milk production in dairy animals and meat production is limited by the availability of sulfur-containing amino acids.So the forage rich in sulfur- containing amino acids is benefit to the wool growth,the increase of sulfur containing in wool,the enhancement of yield of milk.The genetic engineering method was used to transfer the sulfur- protein gene into Alfalfa to improve the sulfur-containing amino acids level in forage protein. The 10kDδ-zein have proportionately high levels of sulfur-containing amino acids.The zein is characterized by its ruminal stable and intestines digestible, so it can be the candidate gene to improve the quality of Alfalfa. Inserting the ER retention signal KDEL followed by the stop code, the stability of the zein in transgenic plant will be remarkably enhanced .In this paper, 10KDzein:KDEL was transferd into alfalfa mediated by Agrobacterium tumefaciens.According to the conservative sequence of the 10KDzein, primers were designed and the ER retention signal KDEL was inserted followed by the stop code. ORF was cloned from the genomic DNA of maize seedling by polymerase chain reaction. The gene was 465bp in length ,encoding 154 amino acid residues , of which S-amino acids were 24.03% in total. The plant expression vector(pCAMBIA13011) was constructed to enhance the quantity of S-amino acids in alfalfa.The regeneration system of alfalfa was achieved. Cotyledons and hypocotyls were used as explants . The explants were cultured on SH with KT 1.5 mg/L and NAA3.0 mg/L to form shoot by direct–differentiation .The expression vector pCAMBIA13011 containing the10KDzein:KDEL gene was introduced into Agrobacterium tumefaciens EHA105 by freeze-thaw method. The Agrobacterium mediated gene transformation procedure was established. Carb strength was 200mg/L and Hrg was 10mg/L. Transgenic plants were screened by PCR.10KD zein gene was transferd into alfalfa and the transgenic plants were gained. S-amino acids content was increased in alfalfa. The fundamental research was established to improve the quality of alfalfa and ruminant nutritional structure.