【作者】 徐建；

【导师】 李名扬；

【作者基本信息】 西南大学 ， 花卉学， 2007， 硕士

【摘要】 花色（flower color）是指显花植物的整个生殖器官的颜色，狭义上是指花瓣的颜色，广义上是指花的器官：花萼、雄蕊甚至苞片发育成花瓣的颜色。花色素主要由类胡萝卜素（carotenoids）、类黄酮（flavonoids）、生物碱（alkaloids）三类物质所组成，其中类黄酮类物质具有产生蓝色的效果。在类黄酮类物质的代谢中，F3’5’H基因（即蓝色基因）编码关键酶，决定无色二氢黄酮醇羟化的位置和程度，能使花色素苷生物合成途径趋向于产生蓝色的翠雀素-3-葡糖苷，从而使花趋向于蓝色。虽然自然界中的花色种类繁多，但是玫瑰、康乃馨、郁金香等重要的花卉，却很少有蓝色和紫色系。花色改良是花卉改良育种的一个重要部分，长期以来，传统育种及定向选择技术虽然对花色改良作出了巨大贡献，但难于打破生物物种的生殖隔离，育种周期长，效果并不理想。而植物基因工程的发展为改良和修饰花卉性状开辟另一条崭新的途径，改良形状有目的性、周期短、检测方便等优点，使基因工程和分子育种成为花卉改良研究的大热点。采用基因工程能有目的地改良植株，并通过目测或少量辅助手段即可判定改良效果。因此，本试验便是将F3’5’H基因转入菊花中，以期获得新的菊花品种。F3’5’H基因作为蓝色花卉基因的重要组成部分之一，确定F3’5’H基因在不同物种间的表达差异，可以为改造其他名贵花卉的研究打下基础。但是同时也要认识到，蓝色花卉的产生所需要的条件很多，其中各因素的相互作用也十分复杂，F3’5’H基因虽然对植株产生蓝色花朵有很大的促进作用，但是效果还远远不够。真正蓝色花卉的产生，还有待于对其他更多因素的共同研究和综合作用。菊花（Dendranthema morifolium）是多年来经过人工培育和天然种间杂交而来，杂交造成的种质渗入以及多倍化和种下变异机制是菊花品种丰富性的来源。菊花的品种多，叶、花型、瓣型变化很大，是色、香、姿、韵俱佳的我国传统名花，与梅、兰、竹被共称为花中“四君子”，菊花不仅受国人的珍惜和推崇，而且也受到世界各国的关注和喜爱。多年来常规育种方法在菊花育种中已经取得巨大成就，但存在着不能利用其它物种基因资源的局限性。随着菊花遗传转化体系的建立发展、外源基因在细胞中的表达及转化方法的进步等，定向修饰它的某些目标性状并保留原有优良性状，使其在花色、花期、花型、抗性等方面都得到了极大的改观和发展。由于国内外菊花品种分类标准存在的差异，国外文献报道的一些菊花品种的遗传转化的体系并不适合我国的一些传统菊花品种，因此研究适合我国菊花品种的遗传转化体系有十分重要的意义。论文研究内容与结果如下：1、四种菊花高频再生体系的优化本研究以菊花‘黄冠’、‘久米荣’、‘山城之光’、‘冬桔’四个品种为试验材料，研究了不同基因型，不同激素浓度，不同预培养时间对菊花愈伤组织诱导、不定芽发生的影响；优化了菊花的再生条件，获得了菊花叶片的高效再生体系并得出了四个品种的叶片的最适分化培养基，分别为：冬桔MS+6-BA2.0 mg/L+IAA2.0 mg/L；久米荣MS+6-BA1.0mg/L+IAA2.0 mg/L；山城之光MS+6-BA2.0 mg/L+IAA5.0 mg/L；黄冠MS+6-BA1.0mg/L+IAA3.0 mg/L。其中，菊花品种山城之光的最高再生频率，能达到100.0％，冬桔、久米荣、黄冠分别为86.7％、85.3％和81.2％。所以后期操作选择了山城之光叶片为外植体的体系作为遗传转化的操作体系。2、构建F3’5’H基因的植物正向表达载体pC2301-F3’5’H及农杆菌菌株的转化用KpnⅠ和BamHⅠ同时双酶切含有目的基因的克隆载体pT-F3’5’H和表达载体质粒pC2301，然后回收、连接目的片段与载体，构建成正向植物表达载体pC2301-F3’5’H。将pC2301-F3’5’H转入农杆菌菌株LBA4404中，经检测，得到含有F3’5’H基因的农杆菌LBA4404-F5’3’H。3、根癌农杆菌介导的F3’5’H基因对菊花的遗传转化切取大小为6mm见方的菊花品种山城之光的无菌苗叶片，以OD₆₀₀=0.5的农杆菌LBA4404-F5’3’H菌液感染20min，用无菌滤纸吸干多余菌液，然后接入培养基MSG：MS+6-BA2.0mg/L+IAA 5.0mg/L中进行暗培养3d后，在延迟培养基MSY：MS+6-BA 3.0 mg，L+IAA 5.0mg/L+Cef 200 mg/L中延迟筛选4d，再转入筛选培养基MSS1：MS+6-BA 2.0mg/L+IAA 0.5mg/L+Km 20 mg/L+Cef 100 mg/L中进行筛选分化培养。待培养基中生长的芽长到长约1cm后，将其切下接入培养基MSS：1/2MS+Km 10 mg/L中进行生根筛选，筛选出能够正常生根的转化苗，并做进一步的鉴定。4、转化植株的PCR鉴定根据35s启动子、F3’5’H基因序列分别设计特异引物，对转化植株进行PCR检测，所得阳性转化株系命名为Y^F3’5’H，未转化的菊花对照用Y⁰表示。把经抗性筛选得到转基因植株通过PCR扩增，得到与阳性对照相同的条带，筛选得到15株阳性植株，初步鉴定目的基因已经成功插入到菊花品种山城之光的基因组中，总转化率为3.85％。更多还原

【Abstract】 Flower colors are mean the colors of the entire genitalia of phanerogam which meaning petalines’ colors in narrow sense ,but generalized mean the colors of flower apparatus including calyces、stamen even the petaline from the bud .Flower pigment mostly being composed of three kinds substances—carotenoids, flavonoids and alkaloids. Flavonoids can produce blue color. F3’5’H gene which coded key enzyme decides the location and degree of achromaticity DFR; it can make biosynthesis of anthocyanin tending to produce blue flower. Flower color is a one of the important qualitative characters and economic parameters for the ornamental plants. Because of the colors for many focal flowers are limited, for example, the roses, carnations, and tulips and so on, which flowers are lack of the color for blue and violet in nature. And the reforms for the ornamental plants colors are provided with the advantages such as the visual arts, and the large economic potential. Gene engineering technology is superior to the Cross breeding and Directive breeding technology with its short cycle , low cost and high benefit. Though traditional breeding technology has been used for a long time.So the F3’5’H gene is transferred into Chrysanthemum to breeding new variety Chrysanthemum.Making sure the F3’5’H gene which one of the most important blue flower genes express in different species can be benefit of breeding precious flowers else.Chrysanthemum is from human cultivation and natural hybridization for many years, the abundance sources of chrysanthemum are high hybridization, multiple and species variation mechanism. Chrysanthemum is the famous flower in China; pass for four flowers man of hornour with plum, orchid, bamboo. It is not only cherished and canonize by Chinese, but also attentioned and favored by all the countries around the world. Conventional breeding methods have obtainted great achievement in chrysanthemum breeding for many years already; however it has localization which couldn’t utilize gene resources from other species. Along with establishing and improving chrysanthemum genetic transformation system, expressing exogenous gene in cell, improving of transformation systems ect, directional decorating certain targeting properties and keeping it inhere propertyes, changing and developing in design and color, florescence, model, resistance. The different standards in classifing genetic transformation systems of chrysanthemum reported on broad are not disagreed with some traditional varieties in home. So it has been significant in researching genetic transformation system which adapt to chrysanthemum cultivars in China.The main experiment and results of this paper are such as follows:1、optimizte of efficient regeneration system of four chrysanthemum varietiesFour varieties of chrysanthemum researched in this paper are ’Dong ju’, ’Jiu mi rong’, ’shan cheng zhi guang’, ’Huang guan’. The result describes an efficient regeneration system of four chrysanthemum varieties with differentiation medium, several factors such as different genotypes, combinations of hormone, pre-culture, explant affected callus, shoot inducement of chrysanthemum. A rapid and efficient regeneration system of chrysanthemum has been established. The results shown that the optimal differentiation medium are: ’ Dong ju’ MS +6-BA 2.0 mg/L+IAA 2.0 mg/L; ’ Jiu mi rong’ MS+6-BA 1.0mg/L+IAA2.0mg/L; ’Shan cheng zhi guang’ MS+6-BA2.0mg/L +IAA 5.0 mg/L; ’Huang guan’ MS+6-BA 1.0 mg/L+IAA 3.0 mg/L. Compared to regeneration of four chrysanthemum varieties that the highest regeneration frequence of Shan cheng zhi guang can reach 100.0%. Dong ju , Jiu mi rong, Huang guan are 86.7%、85.3% and 81.2%.Therefore we have choosed the system of explant of Shan cheng zhi guang leaves been transformation in this research subsequently.2、Construction of plant expressional vector pC2301-F3’5’H and transffere into LBA4404.pT-F3’5’H and pC2301 both digested with Kpn I and BamH I were constructed for plant sense expressional vector pC2301- F3’5’H of F3’5’H gene. Then we have transffered the plant expressional vector pC2301-F3’5’H into agrobaterium LBA4404.3、Introduction of F3’5’H gene into chrysanthemum by Agrobacterium tumefaciens.Young leaves were excised from asepsis chrysanthemum Shan cheng zhi guang, cut into6×6mm² , dipped into Agrobaterium (OD₆₀₀ adjusted to 0.5) for 20min. After patted dry redundant agrobaterium on the sterile filter paper, been transferred into MSG: MS+6-BA 2.0 mg/L+IAA 5.0 mg/L medium and co-cultured for 3 days in darkness. Subsequently, the explants were transferred to MSY:MS+6-BA 3.0 mg/L+IAA 5.0 mg/L+Cef 200 mg/L for 4 days to delay selection culture before formal selection （MSS1:MS+6-BA 2.0 mg/L+IAA 0.5 mg/L+ Km 20 mg/L +Cef 100 mg/L）. When the resistant shoots were cut down until 1cm and then been rooted on MSS:1/2MS+ Km 10 mg/L. The transferred the plantlets with normal roots were used for further identify analysis.4、The accidence identification analyse of transformed plants by PCR.35s promoter and F3’5’H gene were amplified respectively by common PCR to identify transformed plants. The PCR results suggested that transgenic has 15 positive plantlets named Y^F3’5’H while untransgenic named y⁰. Integration of F3’5’H gene into chrysanthemum was confirmed by using PCR amplification.The transgenetic were obtained the same strip with the marker. We principle identify that the target gene has been translated into genome of Chrysanthemum varieties Shan chenng zhi guang successfully.更多还原