【作者】 沈奇；

【导师】 赵德刚；

【作者基本信息】 贵州大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 油菜是重要的油料经济作物，利用基因工程手段调控其脂肪酸代谢途径，改善油脂膳食营养，提高植株抗逆性是目前油料作物改良的重点。拟南芥脂肪酸脱饱和酶FAD8基因编码叶绿体ω—3脂肪酸脱饱和酶，该酶在低温下催化二烯酸迅速脱饱和产生三烯酸，该酶可显著提高植物中不饱和脂肪酸含量，增强种子油脂营养价值及植株对低温的适应力。本研究建立了油菜高效转化体系，利用本实验室克隆的拟南芥FAD8基因构建正义和反义表达载体遗传转化油菜。筛选转基因植株研究了FAD8基因脂肪酸代谢和抗寒性的影响，获得较好的结果。1、以甘蓝型油菜特选4号下胚轴及子叶为转化受体材料，研究基因型、苗龄、激素组合及浓度、取材时间、培养时间、外源基因等因素对其转化效率的影响，确定最适的分化培养基为：MS+6-3mg／L BA+0.1mg几NAA。在添加0.1mg／L2，4-D的MS培养基中预培养3d，重悬5倍的菌液中浸染转化（下胚轴30s；及子叶5min），转化后再经2d共培养、6d脱菌培养后进行筛选培养转化效率较高。2、采用农杆菌介导法将FAD8基因（载体为pSH-FAD8、Psh-anti-FAD8）遗传转化油菜下胚轴、子叶、子叶柄等外植体，并获得转基因植株。经徒手切片GUS染色检验出阳性植株，再经PCR及RT-PCR鉴定，进一步检测出7株转化正义FAD8基因植株，9株转化反义FAD8基因植株。3、提取油菜脂肪酸进行反相薄层层析及气相色谱分析，结果表明转化反义FAD8基因叶片中亚麻酸降低，油酸及亚油酸均有不同程度提高，转化正义FAD8基因叶片中仅有1株亚麻酸提高。转化正义及反义FAD8基因F1代种子均获得60％以上的高油酸含量，可做为油菜品种改良的新材料。4、叶片离体抗寒实验结果表明，转基因植株抗寒性明显高于对照。筛选出两个抗寒能力最强的转基因油菜单株，其F₁代植株进行抗寒性鉴定：叶绿素含量、超氧化物岐化酶、过氧化氢酶、过氧化物酶、丙二醛含量、脯氨酸含量等均反映出转化FAD8基因植株的抗寒性显著增强。5、转基因植株在田间可正常生长并开花结实，但转FAD8植株现蕾及抽薹期比对照推迟70d，转anti-FAD8植株比对照推迟40d。转基因植株叶型、叶片组织、株高、株型均有改变，部分薹茎自然扭曲。转基因植株还出现雄性不育的性状。更多还原

【Abstract】 Rapeseed is an important oil economic crop.The mainly developed focus of oil crop is regulate the pathway of fatty acid metabolism, improve the oil nutrition and prompte the plant to endure stress by Genetic engineering . Arabidopsisω-3 fatty acid desaturase FAD8 is an enzyme which catalyze that the dienoic acid rapidly unsaturase to produce trienoic acid in cold condition., which can increase the content of unsaturased fatty acid in the seeds and plants, and improve the plant oil nutrition and provide plants obviously resistance in cold condition. In our research, set up better regenerated and transformed system , used agrobacteria -medidated method transformed Arabidopsis FAD8 gene into the rapeseed, research fatty acid metabolism and cold-resistance in the transgenic rapeseeds, and got better results.1、the Texuan 4 hypocotyls and cotyledon be used as the recipient, studied the various influence factors of transformed frequency, contending genotype,seedling age, hormone compose , cultural time et al. And found that the 6-day hypocotyls segments had better differentiated ability. Using MS+6-BA(3mg/L)+NAA(0.1mg/L) as differetiated medium could got higher induced frequence. The hypocotyl segments were precultured on preculture medium for 3 days, then submerged into 5-fold diluted Agrobacterium suspension (hypocotyls for 30 s and cotyledon for 5min), and placed on co-cultivation medium for 2 days could result in the highest transformation efficiency.2、Used agrobacteria-medidated method transformed the FAD8genes (the plasmid is pSH-FAD8 and pSH-anti-FAD8) into the rapeseed hypocotyls、cotyledon and petiole of cotyledon. Identified transgenic rapeseed plants by GUS staining、genomic PCR and RT-PCR, finally acquired 7 rapeseed plants of transformed the pSH- FAD8 gene and 9 plants of transformed the pSH-anti-FAD8 gene.3、Analysed the fatty acid content in the transgenic rapeseed by the reverse phase chromatography and gas chromatography method and showed descreased linolenic acid contents, increased oleic acid and linoleic acid contents in transformed the pSH-anti-FAD8 gene plant leaves. Only 1 plants in transformed pSH-FAD8 rapeseed had increased linoleic acid content. Analysed F1 seeds fatty acid contents by HPLC and found the oleic acid could reach to up 60%, which plants can made a new material in rapeseed breeding.4、Rapeseed leaves anti-cold experiment showed that obviously implove cold-resisted ability of transgenic rapeseed. Choosed two plants of transgenic pSH-FAD8 and pSH-anti-FAD8 plants, and study the cold-resisted ability of the F1 plants:the Chlorophyll、SOD、POD、CTA、proline content and malondialdehyde all showed that cold tolerance enhanced obviously in transgenic gene rapeseeds plants.5、The transgenic rapeseed plants in cropland can grow well and can bloom and harvest seeds, but the bud—differentiated and stem-growing period of transgenic pSH-FAD8 plants are latter 70days than control, transgenic pSH-anti-FAD8 plants are latter 40days . the Leaves type , leave organization , plant height, plant type in transgenic plants all have changed, the part of stem have naturally warping。Some of transgenic plants showed the phenomenon of male sterile.更多还原