Gene Cloning, Vector Constructing Of 3-ketoacyl-CoA Reductase Of Brassica Napus L. And Brassica Napus L. Transgenic Research

Plant oil is a kind of important agricultural products. With only a fewexceptions, the world's major vegetable oils are used primarily as edible oils in foodprocessing and preparation. However, vegetable oils derived from the seeds of cropplants have the potential to serve as renewable and easily extracted resource for avariety of industrial applications. For example, soybean oil, which comprises nearly60% of the world's vegetable oil market, is composed primarily of five fatty acids:plamitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. For plant oilsto be economically viable for industrial applications, they need to be highly enrichedin a single fatty acid. In recent years, considerable effort has been directed towardgenetically improving the industrial properties of oils derived from established crops.Through classic breeding techniques, such as inducing mutation, screening positives,hybridizing, we obtained many crop strains with highly practical value and goodagricultural qualities. In lately years, as the development of molecular biology andgene engineering, searchers are attempting to use biotechnology to produced highlevels of these fatty acids in the seeds of existing crop plants. As a result, cDNA for awide variety of useful fatty acid biosynthetic enzymes have been identified,particularly through the use of expressed sequence tags. And, a particular focus ofthis effort has been the identification and transgenic expression of genes that encodeenzymes involved in the synthesis of fatty acid structures we need. These results doaccelerate the speed of improving rapeseeds oil composition.Erucic acid(C22: 1,Δ13) is a very long chain fatty acid(VLCFA).Wild rape plantsare usually rich in erucic acid(normally 45%-60% in oil). In plant, erucic acid is synthesized by fatty acid synthetase complex. The process of this synthesis containfour successive reactions: condensation of C18-CoA with malonyl-CoA to from a3-ketoacyl-CoA, reduction of the 3-ketoacylCoA, dehydration of the resulting3-hydroxyacyl-CoA and finally reduction of the trans-2, 3-enoyl-CoA. The secondreaction is catalyzed by 3-ketoacyl-CoA reductase. The genetics study of the higherucic acid rape (HEAR) and low erucic acid rape(LEAR) show that the expressionof BnKCR2 gene is different in these two cultivars during seed development.BnKCR2 gene was hardly found in the seeds of LEAR. The metabolic production(erucic acid) was increased by BnKCR2 gene which expressed in the mutantSaccharomyces cerevisiae. So controlling the amount of erucic acid could beachieved by regulating the expression the critical carbon-chain elongation gene.A key component of my study involved inserted the coding sequence of thisgene from high erucic acid Brassica napus, in both sense and anti-sense ways, intothe Agrobacterium-carrying plant expression vector pBI121. Driven by a highefficiency promoter, this transformed the target hypocotyls to create transgenic plants,so as to obtain two low-cost groups of rape plants, either rich in erucic acid or totallynot.Design primers (5'ATG GAG ATC TGC ACT TAC TTC 3'and 5'TTA CTCCTT TTT ACT ACT GGA GTC 3') according to the known sequence of cruciferplants BnKCR2 gene from Genebank. From leaves of Brassica napus L., extract thetotal RNA as the template for RT-PCR. The fragment was amplified with full length960bp and then cloned into pMD18-T vector of Takara Company. The sequencingresult indicated that the fragment maintained significant similarity to knownBnKCR2 genes in plant and contained no intron, which suggests the fragment can beexpressed in E. coli by establishing prokaryotic expression vectors.We inserted the fragment of BnKCR2 into pBluescript-SK plasmid in antisenseways and obtained the pSK recombinants with BnKCR2 sequence in antisensedirection.We inserted the fragment of BnKCR2 from aforementioned recombined pMD18and pSK into pBI121 plasmid in both sense and antisense ways and obtained two kinds of pBI121 recombinants with BnKCR2 sequence in different directions, whichcan be used to transformed plants through Agrobacterium.Then we transferred above-mentioned recombined pBI121 vectors and theunmodified pBI121 as a negative control into competent Agrobacterium EHA105and did transgenic research of Brassica napus by tissus culture method to furtherinvestigate the function of BnKCR2.Design primers including EcoR I site at 5' end of the BnKCR2 sequence.Thenthe sequence was amplified by Pfu polymerase, digested by EcoR I and inserted intothe prokaryotic expression GTK vector. Induced by IPTG. Procaryotic expressedvestor was gained.