Lignin Biosynthesis Regulated By Antisense 4CL Gene In Medicago Stativa L.

Medicago stativa L,a perennial legume,is the most widespread cultivated forage of the world and has been planted in China for over 2000 years,mainly in the North,Northwest,and Northeast of China.This forage,known as “king of grass”,well adapts to various environments,gives high yield,and is very nutritious,making it a very important forage for animal husbandry of China.However,it's relatively hard to digest,which directly diminishes its economic value,so it becomes an important task for genetic breeding to improve its digestibility.Lignin is an important component of plant cell wall beside cellulose and hemicellulose,which enhances mechanical strength of the plant,and plays an important role in normal life activity of plant,such as transport of water and response to external invasion.But lignin can't be digested by domestic animals.Current studies have shown that digestibility of Medicago sativa is negatively correlated with its lignin content.So,it would be possible to improve its digestibility and nutritional features by lowering its lignin content through genetic approaches.4-coumaric acid coenzyme A ligase(4CL)is a key enzyme in lignin biosynthesis,which catalyzes thioesterification of hydroxy phenyl acetic acids into corresponding thioesters and regulates synthesis of lignin.Studies have shown that 4CL-suppression mutations decreased lignin synthesis in Arabidopsis,Neosinocalamus,and poplar to different degrees.The research results are as follows:1)Based on the general procedure of Agrobacterium mediated transformation,conditions of genetic transformation were optimized,the best age is 10d;the best pre culture time is 3d;the optimal infection time is 20min;the optimal co culture time was 3d;the optimal concentration for screening is Kan 50mg/L;the best antibacterial concentration is Cef 400mg/L.2)Antisense 4CL-expressing vector was introduced into Medicago sativa via Agrobacterium-mediated transduction,which was confirmed by PCR and sequencingof target fragment.The first generation of plants were planted in field,flowered and reproduced offsprings after conventional management.The second generation of plants were tested by PCR,RT-PCR,and sequencing of the target fragment,and the results indicated that the antisense gene successfully integrated into genome DNA,and stably passed onto offsprings.3)Detection of physiological and biochemical indexes of transgenic plants showed that T0 and T1 generation plant lignin content decreased by 45.77% and31.97%,at the same time plant height,plant weight had no significant difference.However,in the outdoor environment,the growth state of the transgenic plants is different from the wild type plants,such as shading on leaf surface and low rate of emergence,which may be related to lignin content decreased.At the same time,this situation could be significantly improved in second years.