【作者】 郑进；

【导师】 洪华珠；

【作者基本信息】 华中师范大学 ， 农药学， 2006， 博士

【摘要】 苏云金杆菌是一类非常重要的昆虫病原体，它能产生特异性的杀虫晶体蛋白，对农业上和生物医学上的许多害虫具有毒杀作用。Bt毒蛋白（或内毒素Bt-toxin）基因是世界范围内广泛使用的抗虫基因，2005年全球转Bt基因作物的种植面积达263,000,000 hm²。虫害是杨树造林生产上面临的一大难题。虽然利用Bt基因转化欧洲黑杨、雄性毛白杨、美洲黑杨、欧美杨等获得了成功，但受多种因素的影响，至今未见转Bt基因抗虫杨用于造林生产的报道，转Bt基因抗虫杨的研究仍是杨树抗虫育种的热点。中嘉8号杨是湖北省重点推广的杨树优良无性系，但虫害发生较严重。本研究通过根癌农杆菌介导的叶盘法，将经过人工改造的苏云金杆菌杀虫晶体蛋白基因BtCrylA成功导入中嘉8号杨，并得到转基因杨树再生植株。经PCR检测、ELISA检测、SDS-PAGE鉴定和MTT法检测证实了BtCrylA基因在中嘉8号杨中的整合与表达。用中嘉8号杨转Bt基因植株叶片饲喂杨扇舟蛾幼虫，结果表明转基因杨植株具有明显的抗虫活性。为探索培育杨树抗虫新品系开辟了一条新途径。本研究的主要结论如下：以水培法萌芽的叶片为外植体，研究了中嘉8号杨组织培养技术。愈伤组织诱导的最佳培养基为：MS+BA1.0 mg／L+2,4-D 0.1 mg／L+蔗糖30g／L+琼脂6.5g／L。经方差分析，2,4-D和BA两因素对愈伤组织分化不定芽的影响都不显著，但2,4-D的影响比BA大。愈伤组织分化不定芽的最佳培养基为：MS+BA0.5mg／L+2,4-D 0.1mg／L+蔗糖30g／L+琼脂6.5g／L，此时不定芽平均分化率为71.1％。叶片直接分化不定芽的最佳培养基为：（1／2N）MS+BA 0.2mg／L+NAA0.02mg／L+蔗糖25g／L+琼脂6.5g／L，此时单个外植体平均产生有效芽3.9个，平均分化率达85％以上。低浓度的KT对杨树组培苗生根具有一定的促进作用，最佳生根培养基为：MS+KT0.05mg／L+NAA0.02mg／L+琼脂6～7g／L+蔗糖30g／L。生根组培苗室内炼苗1～2周，用0.1％ABT生根粉溶液蘸根，用灭菌的园土：山沙（4：1）做基质，生根组培苗移栽成活率可达90％以上。通过几种抗生素对根癌农杆菌菌株LBA4404的抑菌试验和对中嘉8号杨叶片分化不定芽影响的研究，建立了比较有效的中嘉8号杨叶片直接分化芽基因转化受体系统。研究结果表明：噻孢霉素（cef）对农杆菌LBA4404的抑制效果最好，最佳用量为150～200mg／L，此时对农杆菌的校正抑菌率为81.8％～100％，叶片分化不定芽的频率可达73.3％以上。卡那霉素（Km）用量为5～60mg／L时完全抑制愈伤组织和不定芽的发生；Km对芽的伸长生长也有抑制作用，Km=60mg／L时，芽的存活率为0。在筛选转化体时，Km使用浓度对芽苗以30mg／L为宜。生根筛选Km浓度为15mg／L较为合适。链霉素（str）对杨树叶片分化不定芽具有类似生长素的作用效果。用Str10mg／L代替NAA 0.02mg／L与BA配合，不定芽分化率可达71.1％：Str 20 mg／L、NAA 0.02 mg／L与BA0.2 mg／L配合，不定芽分化率高达100％。此外，一定浓度的链霉素影响叶片出芽的部位，在含有链霉素的培养基上，部分叶片在叶柄处分化不定芽，或叶柄叶缘同时分化不定芽，而对照不定芽仅从叶缘发生。研究了影响根癌农杆菌介导BtCry1A基因导入中嘉8号杨的若干因子，建立了中嘉8号杨简单、有效的遗传转化系统。最优转化条件为：预培养12h、菌液(OD₆₀₀=0.3～0.4)浸染外植体10～15min、共培养4d、共培养培养基中附加乙酰丁香酮（AS）150μmol／L及共培养后延迟10d选择，此时Km^r芽平均诱导率最高达到28.9％。本试验讨论的所有影响转化的因子中，选择时间的影响最大，其次是共培养时间。在转化策略上，采用先再生后选择的方式筛选转化体，尽管假转化体多，后期淘汰率高，纯合难度大，但可以克服由于低浓度Km抑制中嘉8号杨叶片诱导愈伤组织和不定芽分化导致转化难以完成的问题。本研究共转化外植体1936个，分化出Km^r芽外植体171个，Km^r芽平均诱导率为8.8％。经不定芽生长及诱导生根阶段卡那霉素连续筛选，获得了17株Km^r植株，平均转化率为0.88％。对中嘉8号杨转BtCry1A基因植株进行了分子检测和抗虫性鉴定。通过分子检测，从17株中嘉8号杨Km^r植株中，共筛选得到转BtCry1A基因的PCR阳性植株6株，占35.3％；酶联免疫测试和SDS-PAGE电泳结果表明BtCry1A基因在PCR阳性植株6个株系中都有表达，Bt毒蛋白含量幅度为3.88～2.36ppb，平均为3.32ppb。检测结果表明，BtCry1A基因已已整合到中嘉8号杨基因组中并得到表达。MTF法测定结果表明，经中嘉8号杨PCR阳性植株蛋白质处理的粉纹夜蛾细胞死亡率较高，所产生的病理变化与Bt毒素蛋白处理粉纹夜蛾细胞的病变类似，证明BtCry1A基因在转基因植株中得到表达，转基因植株对昆虫离体细胞具有毒杀作用。室内抗虫试验结果表明，饲虫第8天，参试的3个转基因株系对杨扇舟蛾幼虫生长的抑制率分别为66.13％、60.94％和46.65％；虫饲第10天，3个转基因株系的幼虫相对死亡率分别为75.99％、36％和55.99％，此时，对照未转基因植株叶片饲喂的存活幼虫全部化蛹，而3个测试株系叶片饲喂的存活幼虫化蛹率分别为33.3％、18.8％、54.5％，且未化蛹的存活幼虫行动迟缓，体型较小，至饲虫第12天仍不能化蛹。室外栽种的PCR阳性植株也表现出了较明显的抗虫性。虫测结果表明，3个PCR阳性株系对杨扇舟蛾幼虫的生长发育都有明显的抑制作用，其中株系X1效果最好。杨扇舟蛾幼虫取食中嘉8号杨转BtCry1A基因植株叶片后，其中肠上皮细胞出现的病理变化具有典型的Bt晶体毒素作用于鳞翅目昆虫的病理学特征。因此，本研究获得的中嘉8号杨转BtCry1A基因植株为抗虫植株，有进一步测试及推广价值。更多还原

【Abstract】 Bacillus thuringiensis （Bt） is a very important entomopathogen which can produce specifically toxic crystal proteins against many agriculturally and biomedically detrimental pests. Bt（or endotoxin Bt-toxin） gene is a gene that is wildly used for controlling the pests in the world. In 2005, the areas of transgenic crops have increased to 2630 ha. Poplars are seriously attacked by insects in many areas and insect-resistant poplars are expected in forest production. Bt gene has been successfully transformed into Populus alba, Populus nigra, Populus tomentosa, POpulus simonii, Populus deltoides. However, it is little known about the plantation of transgenic insect-resistant poplars to date. Further researching of transgenic poplars with Bt gene is fundamental for poplars insect-resistant breeding. In the present studies, the modified BtCrylA gene was transformed to the explants of Populus deltoides （I-63×I-69） mediated by Agrobacterium tumefaciens. PCR amplification of the chromosomal DNA demonstrated that the BtCrylA gene was detected in the transformed plants. ELISA, SDS-PAGE and MTT analysis indicated that the insecticidal protein was efficiently expressed in P. deltoides （I-63×I-69）. The anti-insect experiments showed that the transformed poplar plants obtained evidently the resistance to the larvae of Clostera anachoreta （Fabricius）. These results suggested that transgenic plants would offer a new way for protecting the poplars against attacking of insect pests. The following are results:Water-cultured method was used in this experiment, the optimal induction medium is MS + 1.0 mg/L BA + 0.1 mg/L 2,4-D + 30g/L sucrose + 6.5g/L agar. Among the callus culture of bud differentiation, the differentiation rate can reach 71.1% when cultured on the medium of MS + 0.5mg/L BA + 0.1mg/L 2,4-D + 30g/L sucrose + 6.5g/L agar. The best medium for buds differentiation of the leave directly is （1/2N）MS + 0.2mg/L BA + 0.02mg/L NAA + 25g/L sucrose +7g/L agar, with the bud differentiation rate of 85%. KT has the stimulation to the plants rooting of P. deltoides （I-63×I-69）, the better rooting medium is MS + 0.05mg/L KT + 0.02 mg/L NAA + 6-7 g/L agar + 30g/L sucrose, with the rooting rate of 82.2%.Several antibiotics were used to eliminate A .tumefaciens LBA4404 during plant transformation. Their effects on inhibition of A. tumefaciens LBA4404 and bud differentiation of leaf from P. deltoides （I-63×I-69） were analyzed. Proliferation of A. tumefaciens LBA4404 was completely suppressed in the medium containing 150-200mg/L cefotaxime with the buds differentiation rate of 73.3%. Analyzing from many angles, the lowest limit concentration of kanamycin used for selection of transformation seedings was 30mg/L when taking buds as acceptor; at rooting stage, lower limit concentration was 15mg/L. The experiment also proved that streptomycin（str） has the same effect as NAA on the bud regeneration. The combination of 10 mg/L str and 0.2 mg/L BA with the differentiation rate of 71.1%, and 20mg/L str+0.02 mg/L NAA+0.2 mg/L BA with the differentiation rate of 100%.Several factors affecting BtCry1A gene transformation of P. deltoides （Ⅰ-63×Ⅰ-69） mediated by A. tumefaciens were studied, and a simple and effective protocol with optimized condition for transformation of P. deltoides （Ⅰ-63×Ⅰ-69） was established. The results demonstrated that the highest frequency of the transformation was 28.9% under these conditions: the 12 h of pre-eulture time, explants infected by A. tumefaciens (OD₆₀₀=0.3～0.4), co-culture time（4d）, acetosyringone （150μmol/L） was added during co-culture and selection was delayed after 10 days of co-culture. Through successive selection in buds growth and root induction stage at high concentration of kanamycin presence we obtained 17 kanamycin-msistance rooted plants.PCR analysis showed that 6 of these 17 kanamycin-resistant rooted plants were BtCry1A gene positive. These 6 PCR positive plants were further confirmed by ELISA and SDS-PAGE analysis. Our results indicated that BtCry1A gene was efficiently expressed and the quantity of Bt insecticidal protein is about 3.88～2.36ppb, MTr analysis showed that the protein of PCR positive plant named X1 was strikingly insecticidal to cultured Trichoplusia ni Hubner cells in vitro. Bioassay results that X1 was significantly resistant to feeding by first larvae of Clostera anachoreta（Fabricins）, compared with the untranaformed control plant. The significant decrease of leaf consumption by larvae, a slow weight gain of larvae and a higher larvae mortality rate of Clostera anachoreta （Fabricius） were observed.更多还原