【作者】 韩科厅；

【导师】 戴思兰；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2010， 博士

【摘要】 菊花是我国传统名花之一,是全球著名的切花和盆花,是重要的出口创汇花卉。通过传统育种的方法,已经培育出除蓝色系以外的所有色系。但由于基因资源的限制,不能通过传统的育种手段获得蓝色的新异色系。而转基因技术则可以突破物种限制,可以往菊花的基因库中导入新的基因,从而使蓝色花的培育成为可能。但菊花花色形成机理、花色素化学分析和花色转基因育种等研究较为初步,同时菊花品种特异性强、遗传转化率低等问题也没有得到有效解决。因此,研究参与菊花花青素苷生物合成途径的关键结构基因的表达,建立高效的菊花遗传转化体系,分析影响菊花蓝色花形成的关键结构基因对菊花花色和基因表达的影响,对菊花蓝色花育种具有重要的理论意义和实际意义。本研究建立了菊花节间横切薄层(tTCLs)不定芽再生体系。结果表明,以节间tTCLs为外植体,不定芽再生并未表现出明显的品种特异性。研究还发现,不同的取材部位会影响不定芽的发生率,温度和琼脂浓度也会影响不定芽发生率和不定芽的玻璃化率。不同外植体的不定芽再生和遗传转化评价研究发现,叶柄tTCLs是菊花进行遗传转化的最佳外植体,MS+6-BA 3.0+NAA 0.5mg／L为最适的不定芽分化培养基,而MS+6-BA 1.0+2,4-D0.1+Kan 15+Carb 400mg／L是农杆菌侵染后抗性细胞分化的最佳培养基。对‘日切桃红’(‘DF-3’)头状花序的9个不同发育时期的舌状花进行花青素含量和花青素苷合成关键基因表达分析,结果表明,由于菊花体内的F3′H表达舌状花中只能积累矢车菊素。花青素苷积累和结构基因表达模式相似,但花青素苷的积累表现出一定的滞后性,且CmDFR、CmANS是花器官特异表达基因。此外,转录因子失活会使多个花青素苷合成相关的结构基因表达受到抑制,从而不能积累花青素苷,花色变白。通过农杆菌介导的方法将瓜叶菊F3′5′H同源基因SCFH导入‘DF-3’中,优化了不定芽分化和不定芽生根的选择压浓度。共试验了865个外植体,最后获得8个独立的抗性株系,经PCR检测,其中6个为SCFH阳性苗,转化率为0.69%。转化苗中,有5个株系检测到了SCFH的转录本,但对内源F3′H的表达未产生影响。转化苗花色较对照更深,花色的红度和蓝度增加。采用HPLC的方法测定转化苗舌状花中的花青素苷含量和成分,结果表明花青素苷含量有显著增加,但没有检测出新的色素成分。通过RT-PCR和RACE的方法从菊花‘DF-3’舌状花中分离了菊花DFR的同源基因CmDFR cDNA全长。在菊花‘DF-3’中过表达CmDFR,能使花青素苷含量增加,花色加深。利用RNA干扰技术抑制内源CmDFR的表达,使花青素苷的积累量降低,使花色变浅。同时干扰内源DFR的表达,会使花青素苷合成相关的其他结构基因的表达量下调,结果证明了CmDFR与菊花花色形成直接相关,而且作用十分关键。以叶柄tTCLs为外植体,通过共转化的方式同时导入菊花F3′H的RNA干扰载体和SCFH过表达载体。通过对1250个叶柄tTCLs进行共转化,共获得31个PCR阳性的转化苗,转化率为2.48%。PCR结果表明有10个为SCFH过表达株系,5个为F3′H干扰株系,而16个为双基因转化株系,双基因苗占51.6%。RNA干扰株系表现出F3′H的表达量的下调,但花色变浅幅度有限。在F3′5′H异源表达,同时F3′H表达受到干扰的株系中,转化苗花色较对照红移和蓝移。本研究的主要结论如下：利用菊花横切薄层(tTCLs)能建立高效的遗传转化体系,可以有效解决菊花遗传转化中存在的品种特异性和转化率低的问题。CHS、CHI、F3H、F3’H、DFR和ANS是菊花花青素苷生物合成途径中的关键结构基因,这些基因的表达量下调或表达受到抑制,会影响最终花青素苷的合成。F3′5′H未参与菊花花青素苷的生物合成。DFR、F3’5’H和F3′H这三个结构基因是影响菊花蓝色花形成的关键基因。异源表达F3′5′H能增加花青素苷的含量,使花色红移和蓝移。过表达DFR能增加花青素苷的含量,使花色加深。而干扰DFR的表达,会使花色变浅,花青素苷含量下降。干扰内源DFR的表达后,会下调体内多个参与花青素昔生物合成的关键结构基因的表达,说明菊花DFR直接参与花色的形成,而且作用十分关键。F3′H表达受到抑制,对花色的影响较小,说明菊花基因组中可能存在F3′H基因的其他拷贝。异源表达F3′5′H,同时干扰体内F3′H的表达,会使菊花花色红移和蓝移。更多还原

【Abstract】 Chrysanthemum is a traditional famous flower in China, and is also an important cutted and potted flower, with the capacity to earn foreign exchange through exports. All color serials except blue have been bred through traditional breeding. Because of the limitation of gene resources, the novel blue serial can’t be bred through traditional breeding. The gene transformation technique can break the species restrictions, and it is likely to attain blue flower color varieties through induction new gene(s) into the gene pool of chrysanthemum. However, the mechanism of chrysanthemum flower coloration, chemical analysis of pigment and molecular breeding are very preliminary. Variety specificity and low genetic transformation efficiency have not been effectively resolved in chrysanthemum transgenic event. In this paper, we analysed the genes expression involved in anthocyanin synthesis, and established high efficiency shoots regeneration and gene transformation system. Then we analyzed the influence of key structural genes which affected the formation of blue flower chrysanthemum on flower color and gene expression of chrysanthemum.In this paper, we established adventitious buds regeneration system of chrysanthemum internode transverse thin cell layers (tTCLs). Adventitious buds regeneration did not demonstrate variety specificity using tTCLs as explants. Buds regeneration efficiency was influenced by explants’position, cultural temperature and concentration of agar. The latter two could influence the efficiency of buds vitrification. Evaluation of different explants in buds regeneration and transformation indicate that petiole tTCLs are the best explants for chrysanthemum transformation, with the best medium, MS+6-BA3.0+NAA0.5mg/L, for buds regeneration and MS+6-BA1.0+2,4-D0.1+Kan15+Carb400mg/L for resistant cell regeneration after Agrobacterium infection.The results of anthocyanin accumulation and RT-PCR analysis of key structural gens involved in anthocyanin synthesis of ray florets indicated that ray florets only accumulate cyanidin with the expression of F3’H in 9 different developmental capitulums of’Riqietaohong’(’DF-3’). The pattern of anthocyanin accumulation was similar to that of structural genes expression, but the latter was slightly earlier than the former. CmDFR and CmANS only expressed in ray florets. Besides, In addition, inactivation of transcriptional factors would suppress the expression of several genes involved in anthocyanin synthesis, and thus can’t accumulate anthocyanin in ray florets.After that, we optimized the concentration of selection pressure for buds regeneration and rooting. Then, we induced F3’5’H homologous gene, SCFH into chrysanthemum by Agrobacterium mediated. 865 explants were carried out, and 8 individual resistant lines were attained, with the efficiency of 0.92%.6 of 8 were SCFH positive lines after PCR amplification. SCFH transcript was detected in 5 of 6 lines. The color of transgenic lines was darker than control. The content of anthocyanin significantly increased in transgenic lines, but new type of anthocyanin was not detected by HPLC. To verify the activity of DFR to reduce DHM, full length cDNA of DFR homologus CmDFR was isolated from ray florets of’DF-3’through RT-PCR and RACE. Overexpression and RNAi of CmDFR in chrysanthemum indicated that CmDFR directly related to coloration of chrysanthemum flower. Overexpression could accumulate more anthocyanin, while suppression expression by RNAi could down regulate other key structural genes involved in anthocyanin synthesis.36 resistant lines were attained through 1250 chrysanthemum petiole tTCLs Agrobacterium mediated cotransformation of SCFH and F3’H RNAi vector, with the efficiency of 2.48%.10 lines were F3’5’H overexpression lines,5 lines were F3’H RNAi lines, and 16 lines were cotransgenic lines, with the ratio of 51.6%. The expression of F3’Hi lines was down regulated, but the extend of flower color alteration was limited. The flower color of F3’5’H heterologous expression and F3’H RNAi chrysanthemums became redder and bluer.In this paper, we established the chrysanthemum tTCLs transgenic system, and resolved the problems of variety specificity and low transformation efficiency. CHS, CHI, F3H, F3’H, DFR and ANS are key structural genes involved in anthocyanin synthesis. Down regulation or suppression the expression of these genes would influence the synthesis of anthocyanin. F3’5’H did not involve in the synthesis of anthocyanin. DFR, F3’5’H and F3’H were key structural genes which affected the formation of blue color chrysanthemum. Heterologous expression of F3’5’H could enhance the content of anthocyanin, and make the flower color redder and bluer. Overexpression of DFR in chrysanthemum could enhance the content of anthocyanin, and make the flower color redder. However, interference the expression of DFR in chrysanthemum through RNAi would decrease the content of anthocyanin, and make the flower color paler. Interference the DFR expression in chrysanthemum would down regulate the expression of other key structural genes involved in anthocyanin synthesis, which indicated that DFR involved in coloration of chrysanthemum directly, and the role of DFR was very important. The influence of expression suppression of F3’H on chrysanthemum coloration was limited, which indicate that may exist other F3’H copy(ies) in chrysanthemum genome. Heterologous expression and suppression the expression of F3’H in chrysanthemum could make the flower color redder and bluer.更多还原