| In plant genetic engineering, selectable marker is needed to distinguish transformant. As the commercialization of transgenic plants, their safety are paid more and more great attention, among which mainly refers to the safety of selectable marker. At present, genes encoding proteins that are antibiotic resistance or herbicide resistance are usually used as selectable marker in plant genetic engineering, these genes have problems of unsafety, In order to increase the safety of transgenic plants, biologists began to search for selectable marker with biosafety.In this research, two genes that encode key enzymes in the synthesis of methionine and lysine, cystathionine synthase and dihydrodipicolinate synthase, were cloned from Arabidopsis thaliana and Nicotiana tabacum L.cv respectively, then a molecular reform were made in vitro to relief the feedback suppression or inhibition, and a research about the possibility of using the mutate genes as selectable markers was made, analog of methionine or lysine as selectable reagent. Du to the feedback mechanism in the pathway of amino synthesis. Non-transgenetic plants expired for stoping synthesizing corresponding amino acid under the pressure of amino acid anolog, transgenetic plants can synthesize amino acid and therefore can survival for free of feedback effect. The purpose of the research was to find new types of selectable marker with biosafety. The main results were summarized as follows:1.Cloning of genes CGS1 and DHDPSGene sequences were searched from Genbank, the Genank locus are U43709 and X79675 respectively. genes CGS1 and DHDPS were cloned from Arabidopsis thaliana and Nicotiana tabacum L.cv respectively using RT-PCR.2. Molecular reform of genes CGS1 and DHDPS in vitroOn the basis of past reports of documents, structural domain was analyzed. Genes CGS1 and DHDPS were reformed by means of site-specific mutation, PCR primers were designed and amplified bidirection, then the whole gene was obtained by means of overlap extension.3. Construction of plant expression vectorsFive plant expression vector were constructed, incuding pBIC,pBICMI,pBICMII,pBID,pBIDM. There are regulated by promoter CaMV35S, and traditional selectable marker nptII was used for the sake of contrasting analysis.4. Transformation mediated by Agrobacterium TumefaciensThe five plant expression vector above-mentioned were transferred into tobacco "Longjiang 911" mediated by Agrobacterium Tumefaciens. At the stage of screening, two categorys of selectable reagent wre added in screening medium respectively, kanamycin and analog of amino acids. Some resistant plants were obtained via screening.5. Identification of resistent seedlingsKm-resistent seedlings with transformation of CGS1 gene were identified with PCR, 4 PCRpositive plants were obtained out of 5.Km-resistent seedlings with transformation of CMI gene were identified with PCR, 4 PCR positiveplants were obtained out of 5.ETH-resistent seedlings with transformation of CMI gene were identified with PCR, 1 PCRpositive plants were obtained out of 3.Km-resistent seedlings with transformation of DHDPS gene were Identified with PCR, 6 PCRpositive plants were obtained out of 8.Km-resistent seedlings with transformation of DM gene were identified with PCR, 2 PCR positiveplants were obtained out of 5.AEC-resistent seedlings with transformation of DM gene were identified with PCR, 1 PCRpositive plants were obtained out of 2.Km-resistent seedlings with transformation of CGS1,CM,DHDPS,DM genes were identified withRea-Time PCR,alI of the 15 plants were overexpression.6. Feasibility analysis of the new-type selectable markersThrough genetic transformation of tobacco, and corresp nalogs was used for selection, some PCR positive tobacco seddlings were obtained overexpressing exogenous genes. During differentiation stage, differentiation ratio using nalogs of amino acid was obviously high than Km did, additionally these genes' superiority in increasing the nutritive value of transgenic plants, genes that encode key enzymes in the synthesis of amino acid have great applied perspective, As the penetration of research, the negative effect of these organic selectable marker bring about will be rationally soved,... |
