【作者】 张斌；

【导师】 张兴；

【作者基本信息】 西北农林科技大学 ， 农药学， 2015， 硕士

【摘要】 雷公藤(Tripterygium wilfordii Hook.f.)是我国一种传统的杀虫植物,体内含有多种重要的农用活性物质,但由于该种植物资源稀缺、活性物质含量低、化学合成难度大等因素,严重制约着其在农药研究领域中的广泛应用。稳定高产离体培养体系的优化与放大以及通过代谢途径工程调控活性物质生物合成是解决上述问题的有效途径之一。在前期研究中,我们已经获得稳定的雷公藤发状根遗传转化体系,并通过雷公藤抑制差减杂交cDNA文库成功富集了与Me JA诱导相关的基因,但这些基因的功能有待验证。HMGR和DXR被认为是萜类合成途径关键酶,因此,这两个酶的基因表达将会直接影响萜类物质的生物合成。RNA干涉(RNAi)是基因表达调控的一种重要机制,是由双链RNA诱导产生的转录后水平的同源基因沉默现象。RNA介导的基因沉默具有特异性强、效率高等特点,近年来逐渐发展成为适于大规模研究基因功能的反向遗传学手段。本研究通过Gateway Cloning System构建了雷公藤HMGR和DXR基因的RNA干扰表达载体,并经发根农杆菌介导的遗传转化获得了雷公藤发状根。然后对干扰株系的表型,基因表达情况以及次生代谢产物含量变化进行较为详细的分析,进一步探讨HMGR和DXR基因对雷公藤萜类物质次生代谢的调控作用。主要结果如下:1、本试验通过Gateway克隆技术构建了雷公藤萜类化合物生物合成关键酶基因HMGR和DXR的RNA干扰表达载体,并转入发根农杆菌中。2、通过农杆菌介导法,成功将干扰载体转入到雷公藤基因组中,并通过抗性筛选,放大培养,PCR鉴定,获得HMGR和DXR阳性发状根根系。3、通过倒置显微镜荧光检测,检测到转基因发状根根系具有红色荧光,因此确认干扰载体转入发状根中,并得到表达。4、通过对转基因发状根体系进行荧光实时定量,结果显示,HMGR-RNA干扰体系中,TwHMGR基因的表达量相对于对照组都有明显的降低。DXR-RNA干扰体系TwDXR表达量也明显降低。干扰效率可达20%-60%,说明,RNAi影响了目的基因的表达。5、通过对转基因发状根体系次生代谢产物含量进行液相检测,H-RNAi-1和D-RNAi-3干扰系的内酯醇,吉碱,次碱的含量均明显降低。D-RNAi-3的内酯醇,吉碱,次碱的含量分别为13.24μg/gDW(对照的34.61%),243.83μg/gDW(对照的51.30%),81.36μg/gDW(对照的63.42%);H-RNAi-1的内酯醇,吉碱,次碱的含量分别为17.81μg/gDW(对照的46.55%),157.59μg/gDW(对照的33.15%),40.84μg/gDW(对照的31.83%)。而且在H-RNAi-1发状根体系中,吉碱和次碱的含量下降趋势明显要高于雷公藤内酯醇;与此相反,D-RNAi-3发状根体系中,雷公藤内酯醇含量下降趋势明显要高于吉碱和次碱,表明,在雷公藤中,HMGR基因对倍半萜生物碱的合成相对影响较大,而DXR对二萜类物质的生物合成影响较大。研究结果证明,利用GatewayCloningSystem大规模构建RNAi载体并转化植株,可以实现高效的基因沉默,并引起次级代谢产物含量的变化。本研究利用RNAi揭示了雷公藤HMGR和DXR基因对萜类化合物生物合成的调控作用。一方面提供了一种研究雷公藤基因功能的方法,另一方面也可以帮助揭示雷公藤萜类次生代谢产物的生物合成机理。更多还原

【Abstract】 As a traditional pesticidal plant, Tripterygium wilfordii Hook.f. contains many secondary metabolites having agricultural activities. However, the extremely low contents and high level of difficulty by chemical synthesis severely constrain its widely application. Optimization and scale up of stable and high-production culture system and metabolic engeering of metabolic pathways provide an alternative strategy for this problem. In previous research,the hairy root culture has been established. A suppression subtractive hybridization(SSH) c DNA library was also successfully established with enriched genes related with Me JA elicitation and secondary metabolites biosynthesis and modulation,but the function of these genes remains to be validated.HMGR and DXR were considered as two key enzymes in the terpenoid biosynthesis pathway, so the genes expression of these two enzymes may directly affect the biosynthesis of terpenoids. RNA interference(RNAi) is exensively used for repressing of genes in functional genomies research. The principle of RNAi is that the homology-dependent mechanism is induced by ds RNA and results in the knockdown of gene function through the degradation of m RNA transcrips.Currently, RNAi becomes the reverse genetic technique for large-scale gene functional assays due to the specificity and efficiency.This reserch constucted the RNAi interfence expression vectors of Tw HMGR and Tw DXR by Gateway Cloning System and obtained the transgenic hairy roots of Tripterygium wilfordii Hook.f. through agrobaeterium-mediated transformation. The transgenic hairy roots phenotypes, the expressions of target genes and the contents of terpenoids were tested and analyzed to further validate the gene function and regulatory mechanism of Tw HMGR and Tw DXR. The mainresults are as follows:1,This experiment constructed the RNAi interfence expression vectors of Tw HMGR and Tw DXR by Gateway Cloning System and transferred them into Agrobacterium rhizogenes 15834.2,The hairy roots induced through agrobaeterium mediated method, and resistance selection,cultivation and PCR identification were used to screened the positive transgenic hairy root lines. The hairy roots appeared fast growth without adding any phytohormone into the medium and had a lot of branches and root hairs, both of which are classic characteristics of hairy root.3,Red fluorescence were detected in the transgenic hairy roots by fluorescent inverted microscope which therefore confirmed that the interference vectors were transferred into hairy roots and expressed.4,The results of Real-time PCR detecting showed that expression level of Tw HMGR in the three HMGR-RNAi transgenic lines were significantly reduced than contol.Same as the Tw DXR in the DXR-RNAi transgenic lines.The interference efficiency could reach 20%- 60%, which indicated that the RNAi effected the expression of target gene.5,The results of HPLC showed that the contents of triptolide, wilforgine, wilforine were decreased significantly in the transgenic hairy roots H-RNAi-1 and D-RNAi-3 compared with control.The contents of triptolide, wilforgine, wilforine in D-RNAi-3 were 13.24μg/g DW(34.61% of control), 243.83μg/g DW(51.30% of control), 81.36 μg/g DW(63.42% of control), respectively.The contents of triptolide, wilforgine, wilforine in H-RNAi-1 were17.81μg/g DW(46.55% of control),157.59μg/g DW(33.15% of control),40.84μg/g DW(31.83% of control). And in H-RNAi-1,the downtrend of the content of wilforgine and wilforine is higher than that of triptolide. Therefore it stated that the HMGR has a greater influence on the the synthesis of sesquiterpenes than diterpenes,and the DXR exhibited the opposite function.This research confirms the feasibility of large-scale ihp-RNA construction by Gateway Cloning System for gene silencing. This study revealed the regulations of HMGR and DXR genes on the bilsynthesis of terpenoids. This could not only supplied a reliable method for studying the functions of related genes in T. wilfordii Hook.f.,but also helped understanding the biosynthesis mechanism of sesquiterpene pyridine alkaloids.更多还原