Agrobacterium-mediated Transformation Of CCoAOMT Gene Expression Interference Vector And LePT1 Gene In Lotus Corniculatus L.

Lotus corniculatus also known as horns flowers,five-leaf grass,is a perennial legume LotuVarious livestock like to eat Lotus corniculatus due to its high nutritional value Various livestock like to eat Lotus corniculatus due to its high nutritional value and low saponin content.Although Lotus corniculatus has many advantages,but because of its slow growth of the seedling stage and the high rate of hard seed,the development of breeding industrial production is slowly.Genetic engineering techniques can speed up the breeding process and is an important mean to cultivate high-yield,high-quality new varieties of Lotus corniculatu.Lignin is a major component of the plant cell walls,there is widespread among plants,located between the cellulose fibers,with the function of compression,support and resistance to pests and diseases and so on.But because of the interference of lignin to the cellulose digestive absorption of animal seriously affect the economic value of the forage plants.Therefore,the use of genetic engineering methods to reduce the lignin content of the plant has a very important significance.Coffee acyl coenzyme A-O-methyltransferase(CCoAOMT)is a key enzyme in lignin biosynthesis pathway by inhibition of CCoAOMT activity can reduce the synthesis of lignin effectively,which can reduce the content of plant lignin.The purpose of the experiment is to improve the quality of the Lotus comiculatus,transferring the CCoAOMT interference vector to genome of the Lotus corniculatus by Agrobacterium-mediated gene interference,reducing the lignin content in Lotus corniculatus plants,created the foundation of low lignin content Lotus corniculatus breeding material.Phosphorus is one of the important plant growth nutrients that regulate metabolic processes of plants.However,the phosphorus content that can supply plants to absorb in soil is very low and can not meet the requirements of the plant,hindering the growth and development of plants.Phosphate transporter protein is mainly responsible for the transport and absorption of phosphorus and phosphorus absorption in plants is a very complex process.In a low-phosphate environment phosphorus content,the plant will effectively increase the amount of phosphate transporter gene expression,and thus to promote root and root symbiotic mycorrhizal hyphae plant phosphorus uptake and transport.Therefore,increasing the expression of phosphate transporter protein can enhance plant uptake of phosphorus in the soil.In this experiment,with the purpose of enhancing the ability to adapt to the environment and increasing phosphorus uptake and utilization,we translated the exogenous LePT1 gene into Lotus corniculatus genome.The hypocotyl of the Lotus corniculatus was taken as the explants and studied the effects of different hormone compositions on callus induction,plantlet regeneration and plant roots.This research built the best regeneration system that had laid a sound foundation for the subsequent regeneration of transgenic research.The experiment created a constructed a high-frequency regeneration system,putting CCoAOMT genes and LePTl genes expression vector into Lotus corniculatus respectively disturbance by Agrobacterium-mediated method.Resistant plants were detected by PCR and Sequencing on the nucleic acid level,getting the first generation of plants.The first generation of plants will be transplanted,after surviving through conventional management,flowering plants,fruitify.Through the analysis of PCR and RT-PCR to the second generation,the interference gene was integrated into the transgenic plants and expressed.The detection of physiological and biochemical indexes of transgenic plants showed that the lignin content was reduced and maintained at a suitable range.For the determination of lignin content in transgenic plants,the results showed that lignin content decreased by 34.5%,to observe its appearance,the plants grew normally.