| Alfalfa(Medicago sativa Linn)Alfalfa is the oldest and most widely distributed perennial leguminous grass in the world.High nutritional value and good palatability,It plays an important role in animal husbandry production.With the serious degradation of grassland and the destruction of ecological environment,Yields of Alfalfa drop.Due to the increasing demand for grass,it is urgent to cultivate a large number of high-quality alfalfa to supplement the growing demand for Animal husbandry.Efficient and stable regeneration system is the key to the utilization of alfalfa transgenic engineering.The regeneration of alfalfa is highly influenced by genotype and environmental conditions.Alfalfa can?t regenerate high-frequency stablely has always been a bottleneck in the development of alfalfa gene engineering.Water stress can induce multiple gene expression.The dehydrin gene was abundant in plants during drought,dehydration and response to various environmental stresses and ABA hormones.Dehydration is the family of LEA – II,it is a protect protein expressed in plant stress.In this experiment,we using the bermudagrass ?C299? as material,using RACE method,a dehydration gene DHNS was obtained from drought treated ?C299?,then analysis sequence and amino acid coding sequence,protein structure analysis of DHNS gene,DHNS expression in leaves of ?C299? under drought stress,salt tolerance and acid-base energy analysis of DHNS,drought tolerance analysis.At the same time,screen cultivars with good reproductive performance out of 11 alfalfa cultivars.Then study the effects of different explants,sterilization time,hormone type and different medium on callus induction and differentiation,Explore the optimal regeneration system experimental program,Establish a stable and high-frequency regeneration system of alfalfa,lay the foundation for further construction of Alfalfa transgenic system.Then,based on the optimized regeneration system,to explore Agrobacterium-mediated transgenic system of alfalfa,study Agrobacterium tumefaciens inhibition rate of explants,callus induction rate and Callus differentiation rate in different concentrations of bacteria,infection time,co-culture time,concentration of cleaning solution,different types and concentrations of screening antibiotics in culture medium,construction of efficient alfalfa transgenic system.The research results include the following aspects:1.The dehydrin gene have the length of 495 bp,which encoding 164 amino acid residues.Sequence analysis showed that the amino acid sequences have the characteristics of dehydrin proteins.Dehydrins encoded by the gene belongs to YSK2 dehydrin and were formed by two K fragments,one S and one Y fragment.The result of Circular Dichrosim show that DHNS is a typical disordered protein.In addition,overexpression of ?C299? dehydrin genes in E.coli and Arabidopsis enhanced tolerance in drought,500 m M Na Cl,500 m M KCl,p H=4 and p H=9.It is shown that the dehydrin gene DHNS has strong and broad-spectrum stress-resistant physiological function.2.The best explant for callus induction was the hypocotyls from sterile seedlings of alfalfa grown for 5-7 days.Alfalfa seeds treated with 75%alcohol for 1min first,and then treated with 0.2%Na Cl O for 8-12 min,in favor of the growth and differentiation of callus.7d dark culture of explants contributes to cell proliferation and organ differentiation.During the induction of callus culture,select modified SH1 with 2mg/L 2,4-D and 0.2mg/L KT,callus induction rate is high,and the cycle is short.The best medium for callus differentiation was modified SH2,The medium of seedling formation was l/2MS.3.The optimum concentration of Agrobacterium tumefaciens for explant infection is OD600=0.5,the infection time 30 min,the co culture time is 2-3 days,the liquid for cleaning use MS with 500mg/L Cef.In the period of callus culture,use modified SH1 with 2mg/L 2,4-D,0.2mg/L KT,250mg/L carb and 5mg/L Hyg,the callus differentiation medium was modified SH2 with 250mg/L carb and 5mg/L Hyg. |
PDF Full Text Request