栝楼RTBs胚胎模式的新型转基因体系研究
毕业论文关键词：栝楼；RTB；转基因体系
The Study of Novel Transformation System on Trichosanthes kirilowii Base on RTBs (Rhizoid Tubers) Model
Abstract: The novel transformation system of Trichosanthes kirilowii base on RTBs (Rhizoid tubers) model was established through screening aseptic seedlings cultivation, different explants and hormone combination condition for RTBs formatin in this study. The root, stem and leaves of Trichosanthes kirilowii were taken as eaplants were placed on MS medium supplemented with 1.0mg/L NAA for rhizoid induction under darkness, then the RTBs were induction from the tip of rhizoids on MS medium supplement with 20.0mg/L TDZ under light. The plasmid transformation was operated through vacuum extraction in vial after analysis extraction number, time and extraction interval. The extracted RTBs were transferred onto MS supplement with 5.0mg/L 6-BA for resistant buds. The results showed that, the beat extraction transformation method was three times with 10 minutes interval (each extraction until no bubble), and the worst was the one time. And the transformation efficiency were respectively 15.63, 38.63 and 61.30%. The novel transformation system on Trichosanthes kirilowii base on RTBs was successfully constructed by analyzing the GUS dyeing and PCR of RTB.
Key words: Trichosanthes kirilowii; RTB; Transformation system
目 录
摘 要 1
Abstract 2
引言 2
1 材料与方法 2
1.1 材料 3
1.2 培养准备 3
1.2.1 无菌苗培养 3
1.2.2 RTB的诱导 3
1.2.3 RTB的胚萌诱导 3
1.2.4 RTB转化渗透介质 3
1.2.5 转化质粒 3
1.3 方法 3
1.3.1 无菌苗获得 3
1.3.2 外植体选择 4
1.3.3 类根体的诱导 4
1.3.4 RTB的诱导 4
1.3.5 RTB的转基因体系构建 4
1.3.6 RTB的再生诱导 4
1.3.7 RTB的转化鉴定 4
1.3.8 统计分析 4
2 结果与分析 4
2.1 诱导结果 5
2.2 RTB的胚性鉴定与形态学分析 5
2.3 RTB胚状体的内起源和发生发育过程 7
2.4 RTB的发育和再生 8
2.5 RTB的转化鉴定 9
3 讨论 10
3.1 外植体和激素浓度配比对RTB诱导效率的影响 10
3.2 抽提时间与抽提次数对RTB转化效率的影响 11
参考文献 12
致谢 14
栝楼RTBs胚胎模式的新型转基因体系研究引言
栝楼（Trichosanthes kirilowii Maxim）是多年生葫芦科栝楼属药用植物，约有80多种[1-3]，雄栝楼根为著名中药“天花粉”，一直用于医治异位妊娠和抗臃肿等病变[4]。其有效成分天花粉蛋白（Trichosanthin, TCS）属于I型单链核糖体失活蛋白（Ribosome inactivating protein, RIP）[5]，一类专一作用于真核细胞核糖体，抑制蛋白质合成的细胞毒蛋白[6,7]。对治疗早、中期妊娠引产率可达90%以上，针剂被广泛用于糖尿病、高血压、高血脂、高血黏度、偏头痛、宫外孕、葡萄胎和异位妊娠等难杂病症[8-12]，及恶性肿瘤、绒毛上皮癌、前列腺癌、宫颈癌等瘤癌的前期治疗[13-15]。同时，TCS具有广谱的抗病毒作用，它对乙型肝炎病毒、麻疹病毒、乙型脑炎、腺病毒Ⅲ型、柯萨奇B2、单纯疱疹病毒与人类免疫缺陷病毒（HIV）等7种病毒的复制均有明显的抑制作用[16-18]；TCS蛋白是单链毒蛋白的代表，除了能使蛋白质合成受阻，特别是TCS能在不影响细胞本身蛋白质合成的情况下，强效抑制艾滋病病毒HIV在感染的免疫细胞内的繁殖[19]，这说明TCS蛋白有可能直接对病毒的RNA起作用。利用TCS的翻译抑制活性，常被作为“弹头”制备免疫毒素，并已在一些癌症和自身免疫疾病的治疗中取得了较好疗效。此外，还能作为免疫导向药物的效应剂，是目前最有希望的特异性抗癌与抗病毒药物。
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