【作者】 梁燕；

【导师】 王鸣； 陈杭；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2002， 博士

【摘要】 茄红素对人类健康的特殊作用近年来才被发现，然而植物中茄红素含量很低，人体自身又不能合成。因此，如何提高植物中茄红素含量并使茄红素生产规模化、商品化，是当前急待研究的课题。本研究对胡萝卜（Daucus carota L．）茄红素合成的基因调控以及细胞培养条件下茄红素合成代谢的激活因子进行了研究，取得了如下结果： 采用反义RNA技术，成功地构建了CaMV35S启动子驱动的胡萝卜茄红素p-环化酶cDNA 3′端306bp、5′端397bp及中间段739bp反义RNA植物表达载体，为利用这些基因对植物茄红素代谢进行调控奠定了基础。 用构建的三种载体通过叶盘法对两个烟草品种（SRI和Xanthin）进行了遗传转化，得到了转基因植株。转基因植株的Southern杂交以及茄红素含量分析结果表明，目的基因已整合到烟草基因组中，转基因烟草叶片中茄红素含量高于阴性对照，证明目的基因已在烟草中表达，载体构建正确，载体在对农杆菌的转化过程中保持了完整的反义基因结构。 用经烟草转化验证的三个载体采用下胚轴浸染法，转化两个胡萝卜品种（华育二号和新透心红），得到了转基因植株和种子。Southern结果证明目的基因已整合到胡萝卜基因组中，Northern杂交显示转基因植株中由35S启动子驱动的反义茄红素-β-环化酶基因得到了正常表达，转录出对茄红素-β-环化酶基因特异性的反义RNA。茄红素含量分析表明，转基因植株叶片中茄红素含量高于阴性对照，说明反义基因抑制了内源基因的表达。通过对不同反义基因片段转化的植株叶片、愈伤组织及悬浮细胞中茄红素含量的分析发现，3′端反义RNA对茄红素β-环化酶的抑制作用较5′端强，中间段居中。 通过对胡萝卜转化体系研究，认为B₅培养基体系较MS培养基体系更适于转化胡萝卜愈伤组织的诱导与生长，细胞悬浮培养较固体培养诱导再生植株效率更高。这一结果的取得，迈出了植物茄红素代谢基因调控的第一步，为进一步深化研究奠定了技术和材料基础。 细胞培养是生产次生代谢物质的有效途径，为了使茄红素生产以及高茄红素含量材料能尽快用于茄红素的商业化生产，以橙色品种TEBE和紫色品种新透心红细胞系为材料，采用二段式培养法，研究了培养条件、培养基种类、蔗糖浓度。植物生长调节剂以及无机离子等对胡萝卜悬浮细胞茄红素合成代谢的影响及其作用机制。结果表明，培养条件对不同细胞系茄红素代谢的影响不同。光照和适当低温有利于新透心红茄红素合成，而暗培养利于TEBE茄红素合成，温度对TEBE茄红素合成的影响不显著。新透心红细胞中茄红素含量最大值出现在培养25～30天，而TEBE含量最大值出现在培养15天左右。 培养基和蔗糖浓度对不同细胞系茄红素合成影响趋势基本一致。MG。培养基和高浓度蔗糖对新透心红和TEBE茄红素的合成具有促进作用。 无机离子对茄红素合成代谢的影响不同。PO。’－浓度增加，新透心红茄红素合成增强，浓度愈高茄红素合成代谢愈旺盛，当浓度高于 10InM时，茄红素合成极显著增强，NO。－为25mM利于茄红素合成。而PO。’－对TEBE茄红素合成代谢无显著影响，NO。－浓度高于54mM，TEBE茄红素合成增强。 植物生长调节剂对不同细胞系的影响不同。1．omg／L 2，4－D，3．omg／L NAA促进新透心红茄红素合成。而NAA对TEBE茄红素合成无显著影响，5．omg儿 2，a｛使TEBE茄红素合成极显著增强。 以八氢番茄红素合成酶（PSY），zeta－胡萝卜素脱饱和酶（ZDS）以及茄红素p－环化酶（LYC亿）基因为探针，对各处理细胞中相应酶mRNA的转录活性进行了检测，发现不同因子对不同细胞系茄红素合成相关酶mRNA的转录量影响不同。对TEBE茄红素合成的促进，主要是通过茄红素合成上游酶表达量的整体提高，增加茄红素合成前体物来提高合成量。而对新透心红茄红素合成的促进，主要通过对茄红素－8－环化酶基因转录的抑制，减少茄红素向p－胡萝h素的转化来增加茄红素含量。 所有因子只影响TEBE茄红素合成代谢的水平高低，茄红素含量提高，D－胡萝卜素的含量也同步提高，而不改变TEBE细胞中原茄红素与p－胡萝卜素含量的比例。PO。’一、温度和培养时间对新透心红茄红素合成的影响则不同，高浓度PO；‘。适当低温（20 C）、较长的培养时间。高蔗糖浓度不但使茄红素含量提高，而且使茄红素含量高于户－胡萝卜素含量，使茄红素与巳－胡萝卜素含量的比值由小于1变为大于1。所以这些因子更有利于茄红素的合成与生产。 茄红素影响因于及其机制的研究，特别是对茄红素合成代谢具有特异作用的影响因子的发现，为茄红素的工厂化生产以及生产体系的进一步优化奠定了技术和理论基础。 胡萝卜茄红素代谢的基因调控研究，证明了利用反义RNA技术对植物次生代谢进行调控的可行性和有效性。本试验创造了高茄红素含量材料，发现胡萝卜茄红素－8－环化酶基因不同反义片段对内源茄红素寸－环化酶基因表达的抑制效果不同，为对茄红素寸－环化酶基因表达的有效调控提供了依据。对茄红素代谢影响因子的研究提出了适合不同细胞系茄红素合?更多还原

【Abstract】 It becomes very imminent to improve lycopene content in plant and to make lycopene production industrialized with the special functions of lycopene on human health discovered recently, and the gene regulation and effectors on lycopene metabolism in carrot (Daucus carota L.) were studied in this experiment.The main results of gene regulation on lycopene metabolism by anti-sense RNA of lycopene b-cyclase (LYC-b) are as follows:On the basis of anti-sense RNA technology, plant expression vectors with anti-sense 306bp at 3’ end, 397bp at 5’ end and 739bp in the middle of lycopene b-cyclase cDNA from carrot were constructed by using CaMV 35S as a promoter, and named pBIL-3, pBIL-5 and pBIL-M. respectively.Three vectors were transformed into Agrobacterium tumefaciens GV3101 by tri-parent hybridization. In order to certify the vectors integrity during the transformation, and the GV3101 strains with different vectors were used to transform two tobacco varieties (SRI and Xanthi) by infecting tobacco leaf plates. Southern blot and lycopene analysis of leaves from transformed tobacco plants showed that the transformed genes had not only integrated into tobacco genome but also expressed.Two carrot varieties were transformed by infecting hypocotyl with the GV3101 strains checked by tobacco transformation respectively. Southern blot demonstrated the integration of anti-sense gene fragments into carrot genome, and Northern dot showed that the transformed gene had transcripted out anti-sense RNA special to internal LYC-b in carrot, and the analysis of lycopene content indicated that the expression of anti-sense RNA suppressed the expression of internal Z7C-6,which led to the contents of lycopene in transgenic plants higher than the contents in untransgenic plants. The comparison of lycopene contents among transgenic plants, callus and suspension cells with pBIL-3 pBIL-5 and pBIL-M exhibited that the influences of different fragments on lycopene content were not identical. The effect of the anti-sense of 3’ end on lycopene content was the strongest, that of 5’ end was the weakest, and the middle fragment was in the middle.The influences and the action mechanisms of culture condition, medium, sucrose concentration, plant growth regulators and inorganic ions on lycopene synthesis in suspension systems were studied by employing TEBE and Xin Touxinhong suspension systems as materials and two-stage culture as a method. The results are as follows:The effects of culture conditions on two cell systems were different. Lighting and proper low temperature (20 癈) were favorite to lycopene synthesis in Xin Touxinhong, whereas darkness was good for lycopene synthesis in TEBE , and temperature had no significant influence on lycopene synthesis in TEBE. The maxium value of lycopene content in Xin Touxinhong appeared during 25~30 days after inoculation, and that in TEBE appeared about 15 days.The tendence of influences of medium and sucrose concentration on lycopene synthesis in two cell systems was same. Both MG$ medium and high sucrose concentration enhanced lycopene synthesis in cells of TEBE and Xin Touxinhong suspensions.The actions of plant growth regulators on lycopene synthesis in two cell lines were not same. 3.0mg/L NAA and l.Omg/L 2,4-D improved the lycopene synthesis in Xin Touxinhong, while NAA had no significant effect, and 5.0mg/L 2,4-D significantly improved synthesis in TEBE system.Inorganic ions had different effects on lycopene synthesis in two lines. 25mM NOs" and more than lOmM P04 " most significantly improved lycopene synthesis in Xin Touxinhong , and more than 54mM NOs" enhanced lycopene synthesis in TEBE, PC>43~ had no most significant effect on it.The action mechanisms of various factors for two cell systems were different. Northern dot of mRNAs of PSY, ZDS, LYC-b indicated that the improvement oflycopene synthesis in TEBE was mainly through improving the expressions of up-stream genes including PSY and ZDS, and the enhancement of lycop更多还原