Study On The Transfer Trichosanthin Gene Into Grapevine By Agrobacterium Tumefaciens

Grape cuItivation has always been an important component in aghcultUreproduction in China, and has been troubled by diseases for a long time. Grape virus isthe most itnportan disease excePt fungi disease. For exarnple, grapevine fan leafvirus and grapevine rolI leaf virus which prevai1 in grapes region alI over the worlddaxnaged grapes grow-th and fruits and caused 25%-30% even 80% reduction in yield.The qua1ity of the fruits was also damaged serious1y. The chemical protection wastednot only time and energ}' but a1so the cost. even pol1uted the environment. Theconventional breeding in disease-resistance is greatly confined because of itscomp]icated genetic background, 1ong 1if e cyc1e and nanow resistant genes. Thedevelopment of plant genetic engineering offer5 a new idea for improving cultivars infrUit trees. It has many advantages 5uch as easy controlling, high frequency, low cost,no pol1uting to environment and wide available gene resources and so on. It is nodoubt that plant genetic engineering provides a new method for plantdisease-resistance breeding.In this stUdy, we constrUcted tWo high expressed plant vectors pBTCS and pBTC,both with lcs gene and transferred them into European grape cultivar "SemiI1ion"mediated by Agrobacterium Tumefaciens. This has not been reported befOre. The highfrequency transforming system was established by investigating the optimal cultUreconditions of plant regeneration, main factors affecting transferring frequency and theoptimal selection method of transformantS. Plenty of positive p1antS of PCR wereobtained. Our research provided not oniy materials for studying \"ide resistance togrape virus of tcs gene, but also theoretical fOundation for get'ting morevirus-resistance transgenic crops. The result was summarized as follo'"stl.Constucted two intermediate vectors(pMTCS, pATCS) and two plant expressionvectors(pBITCS, pBTC)(l)TO transfer tcs gene into grape mediated by Agubacterium TumefQciens, weconstructed plant expression vector pBITCS with plasmids pUTCS, pMM23,pAHC l7 and pBIl21, in which harbors lcs interest gene, ew report gene and nPt IIseIecting marker gene. The resu1t of characterization with endonucleases and PCRanalysis showed the constructed vector is correct.(2)Plant expression vector pBTC was constructed with pIasmids pAHCl7, pBIl21and inter-clone vector pMTCS, in which harbors tcs intere5t gene and npt II selecting3@t$tt&x &#ffi7Y3j2$xfe#ffiH$@3AW$MWA $Jt&&k%marker gene with gus report gene excised to compare itS expression frequency to theinterest gene.(3)There are double 35S protnoters in the tWo plant expression vectors which canimprove expression frequency of the interest gene.2.Optimized plan regeneration system.(1)The optimal medium for germination of somatic embryogenesis is Nitsch basalmedium supplementing 0.lmgh 6-8A and 0.01mg/L NAA. The highest rate ofintact plantS were obtained in this kind of mediurn(67.5%)(2) The most suitable medium at rooting stage is Nitsch basal medium supplementing0.lmg/L IBA and 0.04mgh NAA. In the medium with 0. l mg/L IBA. the mean no.of roots per plant is 5.4 and the average length is 3.3cm without cal1i in the base ofroots, In the medium with 0.04 mgh NAA, the mean no. of rootS per plant is 4.8 andthe average length is 3.9cm without calli in the base of rootS too. There is nosignificant difference in no. of rootS per pIant and mean 1ength betWeen this tWomedium, so both of them can be used as rooting medium.(3) Adding 1g/L active carbon in rootiflg medium can protect root bro'v'ning and therate of browning was reduced by 87.4%.3 .Optimized transformation system.(1)Factors affecting transfOrmation frequency namely explantS of infection, time ofco-cultivation. pH of co-cultivation medium were discussed by 3 factors 3 levelsnormal experiments. Results showed the optimal combination is embryogenic ca1li asinoculated explants. co-cultivating 4 day's and pH 5.2. This...