| Lotus corniculatus Linn,a perennial herbaceous plant of Leguminosae,is aboriginal at temperate zone in Europe.and Asia. Lotus corniculatus Linn is with high adaptability,high production and the richest nourishment in leguminous grass that the protein is about 17.4% after maturation harvest It has fine quality and is proved a dainty pasture by feeding it to the cattles and the birds.It has rich root ,can fertilize the soil ,keep the soil and water effectively.The whole plant can be made use of for medicinal material.It is good nectar source too.It is growed in Europe, North America, Australia, New Zealand,and Japan,has wild species in Guizhou,Yunnan,Hubei,Hunan and Shanxi of China,growed in south China, southwest China, northwest China and north China,is a promising excellent pasture at temperate and humid zone in China.Rabbit Hemorrhagic Disease is a acute, high infectant deadly disease that is result from Rabbit Hemorrhagic Disease virus,and is mostly characterized by edema, gore and bleeding of physical organ in whole body.the incidence and mortality of RHD are very high,is destructive infectious disease of rabbit.The dease firstly broke out in Wuxi and Jiangyin and so on in Jiangsu of china in1984,then happened continuously at rabbit breeding area in world, caused high economic losing to rabbit breeding. Capsid Protein VP60 of Rabbit Hemorrhagic Disease Virus is directly correlative with inducing immunoreaction of antivirus,is protective antigen of viral immunity. Otherwise RHDV has only a serotype. Injecting vaccine is the most efficient technique to defend RHD,but because RHDV has not adapted well to any cell culture up to now, inactivated tissue vaccine is only made from liver and spleen of rabbit died being infected RHDV,this method has lots of abuse.then this is a new way that sub-unit vaccine of RHD is made from VP60.The experiment studied a series of technologies for production of RHDV edible vaccine with Lotus corniculatus Linn as plant bioreactors ,which established plant regeneration system , constructed vector and established genetics transformation system.This system is the elementary technology for production of vaccine of RHD including other kinds of vaccine , especially mammal edible vaccine, with transgenic Lotus corniculatus Linn as plant bioreactorsAt present ,the internal study on tissue culture of Lotus corniculatus Linn is little.The experiment studied plant regeneration system of Lotus corniculatus Linn so completely and exactly on the base of the former scientists'work with orthogonal test that the feasibility and efficiency of the system is enhanced. MS was selected as the base culture medium. Cotyledons, hypocotyls,stems,and leaves was selected as explants. The experiment analysed effects of the different phytohormone combination on the inducement of callus , bud and root through adusting the varieties and concentration of hormones. The result showed that the medium of MS+2,4-D 2.0mg·L-1+KT2.0mg·L-1 could efficiently induce calluses,and the calluses were bottle-green,growed well, the construction was tight,which had regeneratine capacity with hypocotyls as explants. Hypocotyls were the best explants. the medium of MS+6-BA1.0mg·L-1+NAA0.1mg·L-1 is the best culture medium for bud induction which could quickly induce bud of leaf.And the number of buds differentiated in the single explant in this culture medium is more than in other culture medium. The medium of MS +NAA 0.1 mg/L induced the regeneration of roots quickly, and the regenerated plant formed.Plant expression vector VP60-pBI121 was constructed based on the vector pBI121.The vector pBI121 and the pGEM-T easy(including VP60 gene) were digested by the restriction enzymes Xba I and Sac I.The ling segment of pBI121 and segment of VP60 gene were recycled,and then were connected together through a night.The positive clone was got after genetics transformation and selection.It was proved that the VP60 had been connected with the vector pBI121 by PCR,enzyme digestion and sequencing.Agrobacterium tumefaciens LBA4404(including plasmid VP60-pBI121) and Agrobacterium tumefaciens EHA105(including plasmid VP60-pBI121) infected Lotus corniculatus Linn based on the regeneration system that had been established to search the high efficiency transformation system of Lotus corniculatus Linn.The regenerated resistant plants could be obtained by infecting the 3-day pre-culturing hypocotyls with Agrobacterium tumefaciens LBA4404 suspension (OD600≈0.6) for 20 min , after a 3-day co-cultivation period transferring to the callus inducement media containing 300mg/L Carbenicillin and 50 mg/L Kanamycin for 20d. There aren't the reports about research of genetics transformation system with Lotus corniculatus Linn as transformation recipient of transgenic plant vaccines production. |
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