【作者】 朱春燕；

【导师】 俞守义；

【作者基本信息】 南方医科大学 ， 流行病与卫生统计学， 2008， 博士

【摘要】 研究背景和目的心血管疾病是严重威胁人类健康的主要疾病,尤其是发展中国家,承担了全球心血管疾病负担的86%。对发展中国家来说,心血管疾病已经不仅仅是一个威胁健康的问题,还会对社会经济发展产生严重影响。据世界卫生组织报道,2005年全球死于心血管疾病的人数高达1 750万,占全球所有死亡人数的30%,其中超过50%的死亡是由高血脂、高血压及肥胖等导致的冠心病及中风引起的,且80%的死亡发生在发展中国家。到2015年,估计全球每年死于心血管疾病的人数高达2 000万,心血管疾病将成为发展中国家的主要死亡原因。近20年来,我国心血管疾病在总死亡中所占的比例为30%～40%,已逐渐成为城乡居民的主要死亡原因。在我国35～65岁人群中,有2/3的人具有心血管疾病发病的危险因素。目前,对心血管疾病的处理主要是发展和推广普及介入与手术治疗,但它是针对发病后,甚至严重事件发生后进行的干预和补救。要更有效地降低心血管疾病的发病率,必须抓好疾病防治的一级预防措施。白藜芦醇（Resveratrol,Res）是植物为抵抗外界刺激,如紫外线、真菌、病毒感染或机械损伤而分泌的一种植物抗毒素（phytoalexin）。白藜芦醇及白藜芦醇苷的生物学功能主要包括:心血管保护作用、拮抗肿瘤作用、抗炎作用、抗病毒作用、神经保护作用、植物雌激素作用等。近年来,Res的心血管保护作用已成为研究的热点。白藜芦醇及白藜芦醇苷具有抑制血小板聚集,调节血脂代谢,保护血管内皮,拮抗内皮素Ⅰ等作用,从而防止冠心病和动脉粥样硬化等心血管疾病的产生。Res及其糖苷只存在于有限的植物之中,如虎杖、葡萄、花生、松树等。天然植物中存在的Res毕竟是微量的,并且,中药如虎杖等不能长期食用,食物如葡萄、花生等价格偏高、不是普及面广的常用食品。因此,利用基因工程等生物技术生产大量的Res倍受科学家的关注。白藜芦醇合酶（Resveratrol Synthase,RS）是二苯乙烯合酶（stilbene synthase,简称STS）中的一类。它是Res生物合成途径中唯一必需的关键酶,只存在于有Res合成的植物中,因而大多数作物都缺乏RS基因。RS催化Res的前体物质（4-香豆酰辅酶A和丙二酰辅酶A）经一步反应生成Res,而Res生物合成的前体物质普遍存在于植物中。目前对白藜芦醇合酶转基因植物的研究主要集中在Res植物抗毒素方面的定性分析,而对其心血管保护作用等保健功能的研究成功的报道很少;而且存在着外源基因在受体植物中转化率及表达量低等亟待解决的问题。因而应用基因工程技术在异源植物中表达RS基因从而合成白藜芦醇及白藜芦醇苷,将为提高人类的健康水平提供有效的途径。利用植物基因工程的方法,将外源基因转入植物中表达,从而生产价廉且可直接食用的功能性保健食品,这是目前植物基因工程和生物技术研究的热点,也是疾病预防的发展趋势。生菜是一种非常大众化的日常食用蔬菜,且是重要的转基因作物。本研究采用花椰菜花叶病毒35S启动子,构建植物表达载体,把葡萄的RS基因导入生菜中表达Res。同时,应用正交设计和均匀正交设计等统计试验设计方法,从①受体植物（生菜品种、外植体的苗龄、取材部位）、②共培养条件（农杆菌的生长状态、菌液浓度、外植体的预培养时间、浸染时间、共培养时间）、③生境因子及理化因素（生长素、细胞分裂素、AgNO₃、激素配比浓度、乙酰丁香酮等）的角度,筛选出受体植物生菜的最优离体再生体系及农杆菌转化生菜的最优条件,以期望能培育出一种普及面广、使用方便、价格低廉、安全高效的具有预防心血管疾病等功能的保健蔬菜。研究方法（1）含白藜芦醇合酶（RS）基因的植物表达载体的构建提取葡萄基因组总DNA,通过PCR扩增得到RS基因,将此基因连接到克隆载体pGEM-T Vector,得到重组载体pT-RS;经PCR、酶切及序列分析鉴定后,将RS基因克隆到植物表达载体pBI121,得到重组载体pBI-RS,用PCR及双酶切方法进行鉴定。双元表达载体pBI-RS通过冻融法导入根癌农杆菌菌株EHA105,采用PCR及双酶切方法鉴定正确后,保存于-70℃备用。（2）生菜离体再生系统及根癌农杆菌转化条件的优化采用正交设计,研究不同生菜品种、不同消毒时间对培养生菜无菌苗的影响,以及不同浓度的植物生长调节剂、AgNO₃和不同外植体来源对生菜外植体芽直接分化频率及生根成苗的影响,以获得生菜高频率的离体再生体系。采用剂量反应试验,研究不同卡那霉素、羧苄青霉素浓度对生菜再生芽的影响及羧苄青霉素、头孢霉素对分化芽生根的影响。采用均匀正交试验设计,探讨农杆菌的浓度、外植体的预培养时间、共培养时间、浸染时间和乙酰丁香酮对根癌农杆菌转化效率的影响。（3）RS基因在生菜中整合和表达的检测采用PCR、RT-PCR等分子生物学方法检测RS基因在生菜中的整合和表达情况。采用高效液相色谱法（HPLC）和气相色谱-质谱法（GC-MS）定量检测转基因生菜中白藜芦醇及白藜芦醇苷的含量。（4）继代培养分析采集RT-PCR检测阳性的母体植株（T0）的幼嫩叶片,消毒后接种于再生培养基中诱导分化芽。当不定芽长至1～2 cm时,切下不定芽至生根培养基中诱导生根,得到继代培养再生植株。通过PCR方法检测RS基因在转基因生菜继代培养植株中的稳定性。结果（1）DNA序列分析结果表明,葡萄白藜芦醇合酶基因全长1 631 bp,与GeneBank登录号AB046374.1的序列比较,核苷酸同源性达99%,证明已成功得到了葡萄白藜芦醇合酶基因DNA。重组载体pT-RS及pBI-RS的PCR、双酶切及序列分析结果均表明,植物表达载体pBI-RS构建正确。（2）生菜品种“美国大速生”种子用70%乙醇浸泡60 Sec,再用2%次氯酸钠处理15 min,发芽率达到80%,无菌苗生长好。生菜离体培养再生芽的最优分化条件组合为MS附加BA 0.2 mg/L,NAA 0.1 mg/L,外植体为2～3 day的子叶;其中,BA及NAA的交互作用对植株再生有重要的影响。在此条件下,生菜外植体芽的直接分化频率为85.5%。生菜再生芽在含低浓度的生长素（NAA 0.05 mg/L,或IAA0.5 mg/L）的1/2MS培养基中生根情况较好。在农杆菌介导的皱叶生菜子叶（2～3 day苗龄）转化试验中,采用含BA 0.2mg/L,NAA 0.1 mg/L,Kan 100 mg/L,Carb 500 mg/L的MS培养基,最优的转化条件为农杆菌稀释倍数为10倍,预培养时间为1 day,共培养时间为4 day,浸染时间为15 min,不添加乙酰丁香酮。在此条件下,农杆菌的转化率为25.2%。转化再生芽在含NAA 0.05 mg/L,Carb 300 mg/L的1/2MS培养基中生根情况较好。（3）120株转基因抗性再生植株中,PCR阳性率为27.5%,RT-PCR阳性率为25.8%。采用高效液体色谱分析和气相色谱-质谱分析法,鉴定转基因生菜中新的化合物分别为白藜芦醇及白藜芦醇苷。转基因生菜叶片中白藜芦醇的含量为0.1330～0.4280μg/g干重,平均含量为0.3050±0.0884μg/g;白藜芦醇苷的含量为0.5528～0.8380μg/g干重,平均含量为0.7200±0.0980μg/g。（4）转基因生菜植株在含Kan 100 mg/L的再生培养基中生长良好,其叶片形状、色泽等形态学特征与非转基因生菜植株没有观察到差异。随机抽取8株继代培养再生植株进行PCR分析,其中6株扩增到RS基因。结论（1）成功构建了含RS基因的植物表达载体pBI-RS。（2）“美国大速生”品种生菜的发芽时间短,发芽率较高（80%以上）,优于意大利耐抽苔生菜。（3）建立了高频率生菜离体再生体系,预测生菜子叶直接芽分化频率为85.5%,重复试验的分化频率达到90%,高于目前国外的转化水平（约84%）。（4）建立了农杆菌转化生菜的优化条件,转化率达25.2%,转化再生植株的PCR阳性率为27.5%,RT-PCR阳性率为25.8%,高于目前国内外的转化水平（约10%）。（5）RS基因在转基因生菜的继代培养中能比较稳定传递,但仍存在一定程度的变异。（6）转基因生菜中白藜芦醇及白藜芦醇苷的平均含量分别为0.3050μg/g、0.7200μg/g,低于虎杖及花生中的含量。含CaMV 35S-35S双启动子的高效植物表达载体正在构建中,希望通过此途径能提高转基因生菜中白藜芦醇及其糖苷的含量。更多还原

【Abstract】 Background & ObjectiveCardiovascular disease（CVD） is the leading risk factor of health status worldwide,and the developing countries account for 86%of the global CVD disease burden.To developing,low and middle-income countries,CVD is not only a risk factor of health status,but a factor of undermining the socioeconomic development. An estimated 17.5 million people died from cardiovascular disease in 2005, representing 30%of all global deaths.Among these deaths,more than 50%were due to coronary heart disease and stroke caused by hyperlipernia,hypertension and obesity,and around 80%of these deaths occurred in low and middle income countries.By 2015,an estimated 20 million people will die from cardiovascular disease every year,and it is estimated that CVD will be the leading cause of death in developing countries.In the recent twenty years,30%～40%of deaths in our country are due to CVD,and CVD has been the leading cause of death.About 2/3 people 35-65 years old in our country have the risk factors of CVD.At present,the main treatments of CVD are intervention treatment and operation,which are the remedy measures aiming directly at the disease,even severe events.In order to effective prevention of CVD,it is necessary to carry out the primary prevention measures of disease prevention and cure.Resveratrol（Res） is a phytoalexin produced in various plants in response to UV irradiation,fungal infection,viral infection or mechanical damage.Resveratrol and resveratrol glucoside have beneficial effects on human health,known as cardioprotection,anticancer effect,antioxidant activity,anti-inflammatory action, antibacterial action,anti-virus action,neuroprotective effect and phytoestrogen effect, et al.For the past few years,the cardioprotection role of Res has been a research hot topic.Resveratrol and resveratrol glucoside could inhibit platelet aggregation,adjust the lipid metabolism,protect the blood vessel endothelium,rivalry endothelin I,and thereby prevent coronary artery disease and artherosclerosis.Resveratrol is a naturally occurring stilbene found in a limited number of unrelated plant species such as giant knotweed rhizome,grape,peanut or pine.Its content is microcrystalline in the plants,and giant knotweed rhizome couldn’t be edible for long time,and grape and peanut are expensive.Therefore,the scientists pay close attention to use biotechnology such as gene engineering and fermentation engineering to produce a lot of Res and its glucoside.Resveratrol Synthase（RS） is a kind of stilbene synthase（STS）.The biosynthesis of resveratrol is catalysed by the enzyme resveratrol synthase（RS）,which converts one molecule of 4-coumaroyl-CoA and three molecules of malonyl-CoA into resveratrol.Resveratrol synthase is the key,exclusively necessary enzyme in the resveratrol synthetic pathway and it presents only in the plants which can synthetize resveratrol.Most plants are short of resveratrol synthase gene,while the precursor molecules of resveratrol are present throughout the plant kingdom.At present,the researches about resveratrol synthase transgenic plant are mostly on its phytoalexin role,and few are on its cardioprotection role,and the problems such as the low transformation efficiency of exogenous gene and the low expression are urgently to be solved.Therefore,the introduction of the gene encoding resveratrol synthase for resveratrol production in heterologous plant species using genetic engineering will provide a tempting approach to improve human health.Now,to transfer and express exogenous genes in plants and to produce functionality health-care foods which are cheap and direct-edible,is not only the popular topic of plant genetic engineering and biotechnology,but also the development tendency of disease prevention.Lettuce（Lactuca sativa var.crispa L.） is one of the most popular vegetables that is cultivated worldwide,and is an important transgenic plant.In this study,the plant expression vector pBI-RS carrying the CaMV 35S promoter was constructed and transformed to lettuce,and the biologic activity of transgenic lettuce was evaluated.At the same time,in order to obtain a highfrequency regeneration system in vitro culture of lettuce and the optimal condition of Agrobacterium tumefaciens-mediated transformation of lettuce explants,the acceptor plant（such as lettuce genotype,explants）,the co-culture conditions（such as the density of Agrobacterium cells,the preincubate duration of explants,the co-culture duration and the inoculation time）,habitat factors（such as auxin, cytokinin,AgNO3,cytokinin/auxin molar ratios,acetosyringone） were studied using orthogonal design and uniformly orthogonal design.The objective of this research was to get functional lettuce containing anti-disease or health protection composition, which was popular,convenient,cheap and effective.Methods（1） Construction of plant expression vector containing RS geneThe cloning vector pT-RS was constructed containing RS gene extracted from the leaf tip of Vitis vinifera by PCR amplification and RS sequence was identified by PCR amplification,enzymes digestion and sequence analysis.The plant expression vector containing RS gene was obtained and RS sequence was identified by PCR amplification and double enzymes digestion too.Binary plasmid vector was introduced into Agrobacterium tumefaciens strain EHA105 by freeze-thaw method and was stored at -70℃after identifying by PCR amplification and double enzymes digestion.（2） Optimization of the regeneration system in vitro culture of lettuce and the transformation conditions of Agrobacterium Tumefaciens.To obtain a high-frequency regeneration system in vitro culture of lettuce,the effect of genotype and sterilization time on the sterile seedling of lettuce was studied, and the effect of different plant growth regulators,AgNO₃ and explants on induction of direct adventitious buds and adventitious roots was also studied using orthogonal experiment.The effect of kanamycin（Kan） and carbenicillin（Carb） on regeneration shoots,carbenicillin and cefradium（Cef） on root,were tested using the dose-response tests.To establish the optimal condition of Agrobacterium tumefaciens-mediated transformation of lettuce cotyledons,the density of Agrobacterium cells,the preincubate duration of explants,the co-culture duration, the inoculation time and AS were studied using uniformly orthogonal design.（3） Integration and expression detection of RS in transgenic lettuceThe integration information and expression level of RS in transgenic lettuce were detected by PCR amplification and RT-PCR amplification.The content of Res and glucoside was measured by high performance liquid chromatography（HPLC） and gas chromatogram-mass spectrography（GC-MS）.（4） Subculture analysisThe young leaves from TO plants which were RT-PCR-positive were cultured on regeneration medium after sterilized.The adventitious shoots were carefully excised from the explants when they were 1～2 cm high,and were rooted in the root media and obtained regeneration plants from subculture.RS gene was amplified by PCR in the regeneration plants of subculture.Results（1） The results of sequence analysis showed that the RS gene had a length of 1631 bp.The RS gene sequences were 99%in consistent with the sequences that reported by Gene Bank（accession number:AB046374.1）.The results of PCR and restriction enzyme digestion of the recombinant vector pT-RS and pBI-RS showed that the plant expression vector pBI-RS had expected length of the target fragment.（2） The germination rate reached 80%and the sterile seedlings grew well when the lettuce seeds were treated for 60 sec with 70%ethanol,and then with 2%sodium hypochlorite for 15 min.The optimal condition of regeneration system in vitro culture of lettuce was Murashige and Skoog’s medium（MS） supplemented with 0.2 mg/L 6-benzylaminopurine（BA）,0.1 mg/Lα-naphthaleneacetic acid（NAA）,and using the cotyledons of seedling with 2～3 days-old as explants.The interaction of BA×NAA played an important role in the expression of regeneration responses. Under this condition,the predicted frequency of shoots regeneration was 85.5%.The adventitious roots grew well in half-strength basal MS（1/2 MS） medium containing 0.05 mg/L NAA or 0.5 mg/L indole-3-acetic acid（IAA）.The greatest efficiency in Agrobacterium-mediated transformation of lettuce cotyledons（2～3 days-old） was obtained by 10-time dilution of Agrobacterium cells, 15-min inoculation time,1-day pre-culture,4-day co-culture and without AS using the MS（Murashige and Skoog’s,1962） medium supplemented with 0.2 mg/L 6-benzylaminopurine（BA）,0.1 mg/Lα-naphthaleneacetic acid（NAA）,100 mg/L Kan and 500 mg/L Carb,.Under this condition,the shoot rate of transformation explants was 25.2%.Roots of transformation regeneration shoots grown well on 1/2 MS medium supplemented with 0.05mg/L NAA and 300 mg/L Carb.（3） PCR-positive rate was 27.5%and RT-PCR-positive rate was 25.8%among 120 transgenic Kan-resistant regeneration plants.In transgenic lettuce plants,two additional compounds were identified as resveratrol and glucoside by HPLC and GC-MS.Quantitative analysis showed that the average content ofresveratrol reached 0.3050±0.0884μg/g leaf dry weight,and glucoside was 0.7200±0.0980μg/g leaf dry weight.（4） The transgenic lettuce plants could grow in regeneration medium containing 100 mg/L Kan,no difference was observed between transgenic versus non-transgenic lettuce seedlings in morphological assays.RS gene was found in 6 among 8 randomly selected regeneration plants of subculture.Conclusions（1） The plant expression vector pBI-RS containing RS gene was successfully constructed.（2） The germination capacity of Lactuca sativa var.crispa L.,cultivar "MGDSS" had an advantage on cultivar "Italian Yearly Very Late Bolt Lettuce".（3） The high-frequency regeneration system in vitro culture of lettuce was established and the predicted frequency of shoots regeneration was 85.5%.（4） The optimization transformation condition of Agrobacterium-mediated transformation of lettuce explants was obtained,and the shoot rate of transformation explants was 25.2%,PCR-positive rate was 27.5%and RT-PCR-positive rate was 25.8%,which was higher than that of other studies（about 10%）.（5） RS gene could transmit in subculture of the transgenic lettuces,but the gene variation appeared.（6） In transgenic lettuce plants,the average content of resveratrol and Rglucoside were 0.3050 +/- 0.0884μg/g and 0.7200 +/- 0.0980μg/g leaf dry weight, respectively.The effective plant expression vector carrying CaMV 35S-35S double promoter is being constructed,which is expected to improve the content of resveratrol and R- glucoside in transgenic lettuce.更多还原