摘要榄香烯（β-elemene）脂质体是从姜科植物（Zingiberaceae）温郁金块根当中提取出的抗癌活性有效成分。以榄香烯为基础的联合用药（β-elemene -based combination therapies，ACTs）是治疗肿瘤的特效药。吉马烯A合酶基因 (Germacrene A synthase gene, GAS)是榄香烯生物合成途径上游的一个关键酶基因，催化生成吉马烯A，吉马烯A在高温条件下会自动发生Cope分子重排变成β-榄香烯，在紫茎泽兰毛状根中异源表达莴苣来源的吉马烯A合酶基因 (Germacrene A synthase gene, GAS)，增加榄香烯生物合成途径中的代谢流量。随后通过激素诱导生成再生植株，得到含莴苣来源的吉马烯A合酶基因的紫茎泽兰新品种。【目的】建立紫茎泽兰无菌苗及毛状根诱导方法，得到紫茎泽兰GAS转基因毛状根。并探索紫茎泽兰毛状根培养基中的激素培养条件，诱导紫茎泽兰毛状根生成再生植株，为紫茎泽兰毛状根再生植株的诱导提供技术参考和借鉴。【方法】根据目的基因GAS的DNA序列，由上海捷瑞生物工程有限公司通过全基因合成技术合成GAS，并通过双酶切连至植物高效表达载体pCAMBIA1301，得到重组质粒；将重组质粒转入农杆菌C58C1的感受态细胞中，构建重组菌株，并通过聚合酶链式反应（Polymerase Chain Reaction, PCR）进行验证；将活化后的重组菌液用来侵染紫茎泽兰无菌苗的叶片，通过发根农杆菌介导法诱导紫茎泽兰生成毛状根，用一步裂解法获得毛状根基因组，PCR验证是否为紫茎泽兰GAS转基因毛状根。在培养基中添加激素，探索诱导紫茎泽兰毛状根生成再生植株的激素条件。【结果】成功获得发根农杆菌C58C1-GAS-P1301重组菌株；紫茎泽兰叶片在诱导10天以后长出毛状根；将长出的毛状根PCR验证，琼脂糖凝胶电泳显示 rol B、GAS均含有条带，说明目的基因GAS已成功整合到紫茎泽兰毛状根基因组中。探索诱导紫茎泽兰毛状根再生植株的激素条件，结果在激素浓度6-BA2。0mg/L+IAA1。0mg/L的条件下成功诱导生成了GAS转基因紫茎泽兰再生植株。【结论】PCR结果证实目的基因GAS已经整合到毛状根基因组中，建立了紫茎泽兰毛状根转基因体系。并成功探索出了紫茎泽兰毛状根再生植株的诱导条件，为进一步利用代谢工程技术培育转基因植株提供了参考和借鉴。89459

毕业论文关键词：榄香烯；紫茎泽兰；转基因；吉马烯A合酶基因

Abstract  β-elemene is the effective anticancer component extracted from Zingiberaceae plants among the root tuber of Curcuma tulip。 Combination therapy based on elemene is an effective treatment for cancer。 Germacrene A synthase gene is a key enzyme gene of elemene biosynthesis。 Germacrene A will automatically generate Cope rearrangement into β-elemene on conditions of high temperature。 Heterologous expression of germacrene A synthase gene (GAS) in Eupatorium adenophorum hairy roots, was performed to increase the metabolic flux in the biosynthesis pathway of elemene, and to obtain a high yield anticancer active ingredient in the hairy root of Eupatorium adenophorum。 A new type of Eupatorium adenophorum with high activity of anticancer activity was obtained by inducing regenerated plants。    [Objective] the aim was to establish a method for inducing the sterilized seedlings and hairy roots of Eupatorium adenophorum for further heterologous expression of GAS，The conditions of hormone culture in the hairy roots of Eupatorium adenophorum were studied。 It was suggested that the hairy roots of Eupatorium adenophorum could be regenerated to provide the technical reference for the regeneration of other hairy roots。 [Method] according to the DNA sequence of the target gene GAS, Shanghai Jierui Biotechnology Co。 Ltd。 performed the synthesis of GAS gene, and the recombinant plasmid was obtained with the GAS gene cloned into plant expression vector pCAMBIA1301。 The recombinant plasmid was transferred into the competent cells of Agrobacterium C58C1, and the recombinant strain was constructed successfully and confirmed by PCR product electrophoresis。 After the recombinant bacteria solution was used to infect the leaves of the sterile seedlings of Eupatorium adenophorum, coculture and dark culture were used to induce the hairy roots of Eupatorium adenophorum。 After the hairy roots were grown, the hairy root gene was extracted by one-step method, and PCR was used to determine the transgenic Eupatorium adenophorum hairy root。 Hormone was added into the culture medium to explore the hormone conditions for inducing the regeneration of plants from hairy roots of Eupatorium adenophorum。 [Result] The target gene GAS was successfully transferred into Agrobacterium tumefaciens C58C1。 The hairy roots were induced to grow successfully after 10 days of dark culture。 The results showed that the rol B, Hygr and GAS bands were extracted from the extracted hairy root DNA, which indicated that the target gene GAS was successfully integrated into the hairy roots of Eupatorium adenophorum。 Under the conditions of 6-BA 2。0mg/L + IAA 1。0mg/L, the GAS transgenic plants were successfully induced by hormone conditions。 [Conclusion] the results of PCR showed that the target gene GAS had been integrated into the hairy root genome, and the transgenic system of hairy root was established。 The conditions for the regeneration of the hairy roots of Eupatorium adenophorum were studied, which provided a reference for the further use of metabolic engineering to cultivate transgenic plants。源Q于W优E尔A论S文R网wwW.yOueRw.com 原文+QQ75201,8766