【作者】 李娟；

【导师】 陈克平； 谭小力；

【作者基本信息】 江苏大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 油菜作为世界范围内主要的油料作物,是食用植物油的主要来源,其饼粕是主要的饲料。近年来菜籽油作为生物能源被大量应用于化工、能源等领域,迫使人们通过不断改良油菜品质,以增加油菜产量。涝渍危害严重影响油菜正常生长发育造成减产,涝渍时间过长还会导致植物死亡从而造成绝产。因此,获得一种涝渍抗性油菜提高产量具有十分重要的意义。在微生物工业中供氧不足已成为提高产品产量的主要限制因素之一。运用基因工程改造微生物是解决上述问题的有效策略和手段。血红蛋白在动物体中的生理功能主要是结合氧并协助其运输,从而促进机体中氧的供应、呼吸链电子流量、ATP酶活力等;血红蛋白在微生物体中可以促进菌体的生长、蛋白质的合成、代谢产物的产生等。我们将蓝细菌血红蛋白基因(CHB)分别转入大肠杆菌、酿酒酵母、拟南芥及油菜中鉴定其功能,主要研究内容和结论如下:1.构建了原核表达载体pET30a-CHB,在大肠杆菌中进行了融合表达。Western blotting证明了pET30a-CHB在大肠杆菌BL21中表达的重组蛋白确实为His-CHB融合蛋白。分别在低氧和正常情况下测定转基因菌株和对照菌株的生长曲线,发现CHB基因能够促进大肠杆菌的生长,并能增加其稳定期的菌体量。2.构建了酵母表达载体pYES2-CHB,用RT-PCR鉴定其在酿酒酵母中的表达,测量转基因菌株和对照菌株的生长曲线,发现CHB基因能够促进酵母的生长。用气相色谱和薄层层析测定其脂肪酸含量和成份变化,结果发现转基因菌株的脂肪酸有所下降,但并没有引起磷脂的变化。3.构建了植物表达载体pCAMBIAl300-CaMV35S-CHB转化农杆菌GV3101,然后转化拟南芥,利用抗生素筛选转基因植株,PCR鉴定,得到了T3代纯系种子。在植株生长过程中,转基因植株生长比较旺盛,开花期提前,生长周期缩短。4.将植物表达载体pCAMBIA1300-CaMV35S-CHB转化农杆菌LBA4404,鉴定成功后转化甘蓝型油菜宁油16,使用抗生素浸泡种子法筛选转基因植株,RT-PCR鉴定其在油菜中的表达。CHB的表达能够使转基因植株种子提前萌发,增加耐涝力,并且能使花期提前,缩短油菜的生长周期。更多还原

【Abstract】 Rapeseed is a worldwide main oil crop,its oil is for cook,and its cake is for feeding the animal.In recent years,the oil quality is continually refined and gradually applied in chemical industry,and bio-energy.Submergence stress as a result of flooding for days or longer often compromises plant growth and dramatically decreases the crop yields.Furthermore,a prolonged submergence treatment can lead to the death of plant.Therefore,the plant’s tolerance to flooding/submergence stress is important trait for crop.Oxygen has become one of the major constraint factors to increase yield in the microbial industry.Using genetically engineered microorganisms is an effective strategy to solve these issues.Hemoglobin in animal cell can bound and transport oxygen in order to improve provision of oxygen,fluence of electrons in respiratory chain,vigor of ATPase;and hemoglobin in microbacteria can promote growth of microbacteria,synthesis of protein and production of metabolite.We have transformed CHB gene into Escherichia coli BL21,Saccharomyces cerevisia,Arabidopsis thaliana and Brassica napus and identified the functions.The major results are as follows:1.CHB gene was ligated into the prokaryotic expression vector pET30a and expressed in Escherichia coli BL21.CHB was successfully expressed in E.coli(BL21 strain) as a fusion protein.This protein was further confirmed by Western blot using anti-6 x His monoclonal antibody.The growth curve of Escherichia coli BL21 was measured in both hypoxia and enough oxygen conditions.The results showed that the CHB gene can not only enhance the growth speed of bacteria but also increase the yield of bacteria both in hypoxia and enough oxygen conditions.2.CHB gene was ligated into the yeast expression vector pYES2 and expressed in the protease-A-deficient(pep4) strain of Saccharomyces cerevisia.CHB expression was confirmed by reverse transcriptase-polymerase chain reaction(RT-PCR) using CHB-specific primers.The result of RT-PCR showed that the recombinant plasmid could be transcripted correctly in Saccharomyces cerevisia pep4.The growth curve of Saccharomyces cerevisia pep4 was measured.The result showed that CHB gene can enhance the growth speed of pep4.The expression of CHB decreased the yeast oil content,but did not change the yeast phospholipids.3.The vector pCAMBIA1300-CaMV35S-CHB was transformed into Arabidopsis thaliana mediated by A.tumefactiens strain GV3101.Transgenic plants were selected by antibiotic and identified with PCR.CHB can enhance growth and shorten life span of transgenic plants.4.The vector pCAMBIA1300-CaMV35S-CHB was transformed into Brassica napus mediated by A.tumefactiens strain LBA4404,and transgenic plants were identified with PCR analysis.Transcription of the gene was confirmed by reverse transcriptase-polymerase chain reaction(RT-PCR) using CHB primers.Results showed that the CHB gene was integrated into the genome of the host,transcribed and translated in B.napus.Heterologous expression of CHB reduced germination time, promoted plant growth and shorten life cycle.Furthermore,transgenic plants tolerated waterlogging.更多还原