【作者】 曾雪嘉；

【导师】 裴炎；

【作者基本信息】 西南农业大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 真菌病害是导致作物减产的重要因素之一，利用广谱抗真菌新资源和新基因培育持久抗病新品种是农业食品安全的重要保证。建立在分子遗传学、分子生物学基础上的基因工程技术为之提供了一条可行的途径。利用转基因技术将抗病基因转入植物，来提高植物的抗病性是目前培育抗病品种的重要策略之一。 本实验以本明烟（Nicotiana benthamiana）的叶盘为受体，通过农杆菌介导法将不同来源的基因（SPCEMA：加有信号肽的CEMA基因，AFP：来源于苜蓿的防御素基因，CHI：来源于苦瓜的几丁质酶基因以及双价SPCEMA-CHI、AFP-CHI、AFP-SPCEMA基因）导入烟草，进行了抗病性检测，并比较了不同转基因植株的抗病效果，从而为它们的进一步利用提供依据。其主要结果如下： 1．抗病检测体系的建立 通过对培养疫霉菌的培养基配方和培养方法的比较，确定疫霉菌在芝麻培养基上生长较好，且获得了一种能产生大量致病力强的游动孢子囊的培养方法，使疫霉菌游动孢子囊的量从5×10⁴个／ml提高到1.5×10⁵个／ml。同时确定了疫霉菌和赤星病菌的接种浓度，疫霉菌浓度为1×10⁴个／ml，赤星病菌浓度为5×10⁵个／ml。 2．转目的基因烟草的获得 取烟草无菌苗叶片，切成0.5cm²大小的叶盘，放入OD₆₀₀值为0.1～0.2的农杆菌菌液中，轻轻振荡侵染5min后，立刻倾去菌液，取出外植体转入表面铺有一层滤纸的共培养基MSB₁（MSB₀+2.0mg／L BA+0.1mg／L NAA）上，24℃，暗处共培养3d。共培养完成后，将外植体直接转入筛选培养基MSB₂（MSB₁+500mg／L Cef+100mg／L Km）中进行分化培养，每3周继代一次。待抗性芽长出，将其切下转入生根培养基MSB₃（MSB₀+200mg／L Cef+150mg／L Km）中筛选生根。 3．转基因植株分子生物学鉴定 首先对筛选到的抗性植株进行GUS组织化学检测，初步筛选确定已转化植株。对GUS西南农业大学硕士学位论文摘要阳性的植株做PCR扩增检测，结果显示每个GUS阳性株系均显示与阳性对照相同的特异带，非转化植株未得到与阳性对照相同的特异带。初步证明6个基因已整合到烟草基因组中，获得的GUS阳性植株是转基因烟草。4.转基因植株抗病性检测4.1转基因植株T。代疫霉菌接种实验 将T0代转基因烟草进行疫霉菌接种实验，结果表明:转基因烟草与对照相比抗性明显增强，6个基因的病情指数从低到高依次为AFP-C川引.67%、APP一SPCEMA5833%、SPCEMA一CH160.87%、AFP63.71%、SPCEMA63.89%、CHI70.54%，而对照的病情指数则为100%。其中三个双价基因烟草的抗病性较好。4.2转基因植株T:代赤星病菌接种实验 将T，代转基因烟草进行赤星病菌接种实验，结果表明:转基因烟草与对照相比抗性明显增强，6个基因的病情指数从低到高依次为AFP一CH137.sl%、AFP一SPCEMA39.69%、SPCEMA一CHI4O%、AFP51.25%、SPCEMA55.63%、CHI56.25%，而对照的病情指数则为100%.说明这六个基因对真菌病都有一定的抗菌作用，并能在后代中稳定遗传与表达。我们又从每个基因中随机选取了四个株系，接种烟草赤星病菌，比较各株系的抗病性。结果表明:不同基因不同株系间抗病效果有差异，以AFP一CHI基因为最好，其各个株系的病情指数均较低。5.转基因植株遗传稳定性鉴定 每个转基因烟草随机选取2个株系，萌发其T:代种子300粒左右，萌动后进行GUS染色，x。2适合性测验结果表明其外源基因的传递符合孟德尔规律，呈3:1单基因显性的孟德尔式分禺。更多还原

【Abstract】 Fungal diseases are one of the major factors limiting crops production worldwide.The important guarantee of agriculture foodstuff safety is to breed new disease resistance variety by using new antifungal resource and genes.It is offered a feasible approach with gene engineering technique basing on molecular genetics and molecular biology.At present, one of the important strategies to breed disease resistance variety is transfered disease resistence genes into plant to enhance resistance.In this paper, we selected the leaf dish of Nicotiana benthamiana as the explants and introduced six different genes (SPCEMA, AFP, CHI,SPCEMA-CHI, AFP-CH1, AFP-SPCEMA)into tobacco by Agrobacterium mediated transformation, we compared the resistant effect of different foreign genes in transgenic plants. The results are as following:1. Development of examination method of disease resistanceFrom experiment,it was shown that sesame agar can be used to culture Phytophthora nicatianae which grew well on it.At the same time, a new culture method which can produce a lot of zoosporangia was obstained.The outputs of zoosporangia were increased from 5×104conidia/ml to 1.5×105conidia/ml.The inoculative concentration of Phytophthora nicatianae and Alternaria alternate were 1 ×104conidia/ml and 5×105 个/ml.respectively.2. Acquirement of transgenic tobacco of different genesSterile tobacco leaves sliced into leaf dishes (0.5cm2/tablet) were immerged into theAgrobacterium suspension of OD600 0.1-0.2 for 5min, and the leaf dishes were co-cultured on MSB1(MSB0+ 2.0mg/L BA +0.1mg/L NAA) medium covered with a sterile filter paper disc for 3d indarkness at 24℃. After co-culture was over, the exolants were directlv transferred to the selectionMedium MSB2 (MSB1+Cef500mg/L+Km100mg/L) for three weeks. The explants were selected twice. After green buds appeared, they were sliced and transferred to radication medium MSB3 (MSB|+ Cef500mg/L+Kml50mg/L). Finally, green seedlings were selected for further assays.3. Molecular assays of transgenic plantExplants from the Kanamycin-resistant regenerated plants were stained for assaying GUS activity using a protocol derived from Jefferson (1987). Expression of the GUS gene confirmed that the T-DNA had been transferred and integrated into the regenerated plants. At the same time, the introduction of the target genes were confirmed by PCR analysis of the DNA samples isolated from the GUS positive regenerated plants.4. Resistance analysis4.1 Inoculation with Phytophthora nicatianae of TO- transgenic plantsTo-transgenic plants of different genes were inoculated with Phytophthora nicatianae in greenhouse. The results showed that the resistance of transgenic plants containing different g enes was strengthened distinctively, compared with control. The disease index of the six gen es were AFP-CHI41.67%, AFP-SPCEMA58.33%, SPCEMA-CHI60.87%, AFP63.71%,SPCEM A63.89%,CHI70.54%.4.2 Inoculation with Alternaria alternata of T1-transgenic plantsT1-transgenic plants of different genes were inoculated with Alternaria alternat in green house.The results manifested that the resistance of transgenic plants containing different gene s was strengthened distinctively, compared with control.The disease index of the six genes w ere AFP-CHI37.81%, AFP-SPCEMA39.69%, SPCEMA-CHI40.00%, AFP51.25%,SPCEMA55.6 3%,CHI56.25%.From the above,we can see that all of the six genes had antifungal effect an d can be transmitted and expressed in offspring.5. Assaying the stability of descendiblity in transgenic plantswe randomly selected two plants from transgenic plants of every gene. After germinated, they were stained for assaying GUS activity. The x c2 aptness workout indicated that the heritability of foreign gene accorded with the law of segregation.更多还原