Study On 1-SST Gene Transfered Into Sugar Beet And Production Of Fructan

Object:Fructan can be used to prevent and regulate a variety of human diseases.And it doesn’t exist completely in the side effects of human body and is considered to be of nutritional health food products.Many countries have been approved to use widely fruction in food,cosmetic,fodder etc.Besides,fructan,as an important osmotic adjustment substances,is considered to closely relate to the resistance of plants.Therefore,in order to lay the foundation to use high yield plant as the bioreactor to produce functional polysaccharide at an easy rate,the fructan content was improved in sugar beet by importing foreign fructose transferase genes,geting a high yield sugar beet strain.Methods : With the p BI121 initial carrier,restructuring the Allium cepa of sucrose:suerose-1-fruetosy ltransreras SST and root specific promoter MLL gene,constructed the plant expression vector p BI121-MLL-SST.Transforming the sugar beet varieties(Beta.vulgaris)through the method of agrobacterium mediation,STFD4,identifying transformed plants using PCR,detecting SST gene expression by RT-PCR technique,determining relative water content,MDA content,relative conductivity,PSII relative amount of leaf and the sugar index in transgenic beet lines and wild beet.Results :(1)Roots cell-specific promoter MLL and SST gene were cloned successfully,and the plant expression vector p BI121-MLL-SST was built,and was transformed into the sugar beet through the method of agrobacterium mediation.The sugar beet was identified transformed plants lines by PCR and RT-PCR,and was obtained transgenic beet lines.(2)useing Sugarbeet petiole as the explant,established and optimized plant regeneration system of sugar beet petiole in vitro.The results indicated that the best way to sterilization was that seeds were treat with 10% H2O2 for 10 mins,then 0.1% Hg Cl2 for 10 mins in sequence after testa broken.MS+6-BA1.0 mg·m L-1+NAA0.2mg·m L-1 was the suitable medium for explants differentiated number;The 1/2MS+NAA1.0 mg·m L-1+IBA0.2 mg·m L-1 was the best medium for rooting,with the rooting rate more than 95%;After rooted seedings were transplanting into greenhouse,the survival rate were over 96%.(3)It was found that drought stress could delay wilted leaves of transgenic lines,but they recovered faster and more completely after rewatering as compared with WT plants.Transgenic lines also showed lower reduction of relative water content and relative quantum yield of PSII and smaller increasion of relative conductivity and malondialdehyde content as compared with WT plants.Under drought conditions,compared with the contrast,each strain of transformed plants can reduce the loss of the water in the plants,improving the activity of antioxidant enzymes in living cells more effectively,making the transformed beet strain to obtain higher drought resistance.So driven by the MLL gene(promoter of the root specific expression),transformed beet strain get a further rise in drought resistance.(4)Determining the change of the concentration of carbohydrates in the root,The results showed that the concentration of carbohydrates in transgenic lines was changed compared with the control,and the concentration of kestose,glucose,fructose(mg · m L-1)was higher than that of wild beets.But,the concentration of sucrose(mg ·m L-1)was lower than that of wild-beet.And increase ratio of Fructan concentration was at 28.57%~74.34%.conclusion:The concentration of low poly fructose in the beet could be improved effectively using the method of genetic engineering,getting a high yield sugar beet strain.These results will lay a solid foundation on openning industry regarding sugar beet as bioreactor to produce low poly fructose.