长筒石蒜花色变异的分子基础
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摘要
长筒石蒜(L. longituba)为我国所特有,长筒石蒜种内花色花型变异最为丰富,这为种质创新和新品种选育奠定了基础。
本研究以长筒石蒜花瓣为材料,对长筒石蒜花色变异的分子基础进行研究。主要结果如下:
1.对43个长筒石蒜变异类型和长筒石蒜模式种的花色进行评价。HPLC-DAD/ESI-MS确定长筒石蒜中的花青素组成,初步阐明了长筒石蒜花色形成化学机理。
2.通过RACE方法从长筒石蒜白色和紫红色花瓣的cDNA中克隆到了ANS,CHS,DFR,F3H,F3’5’H和FLS六个基因的全长cDNA序列。
3.用实时定量RT-PCR分析了长筒石蒜ANS,CHS,DFR,F3H,F3’5’H和FLS六个基因在不同花色长筒石蒜中的时空表达情况。
4.构建植物表达载体对矮牵牛进行遗传转化,转基因矮牵牛花朵的颜色有不同程度的变化。
5.预测了348个转录因子。其中58个转录因子在花瓣中较叶片而言有特异性高表达,同时对其中22个转录因子分析了它们在不同花发育阶段的表达模式。
6.从前ESTs序列中开发了809个EST-SSR,并进行了初步分析。
7.不同倍性石蒜(L.radiata)基因组MSAP分析表明DNA甲基化水平与倍性高低关系不明显,而三倍体石蒜表现出较为显著的低甲基化水平特征。
8.用流式细胞仪对石蒜属植物中9个种进行基因组大小的测定。石蒜属植物基因组大小与其花色、叶形有着密切的关系,并且石蒜属植物基因组大小与染色体数目有关。
The genus Lycoris have colorful flowers favorable for instance as Lycoris longituba. The flowers of L. longituba exhibit a great deal of diversity in both color and floral form, and its bulb has some medicinal potential.
In this research, the Molecular Biology of Color Variation in L.longituba was studied. The main results were showed as following:
1、The flower color of 43 L. longituba varieties and the Type species were divided into nine groups: red, orange, yellow and purple series.Anthocyanin composition of L. longituba determined using HPLC-DAD/ESI-MS. The result of multiple linear regression analysis (MLR) indicated that Cy derivatives were the major factors affected the value of color parameters greatly.
2、Six complete-length cDNAs encoding ANS, CHS, DFR, F3H, F3’5’H and FLS were isolated from the petals by RACE.
3、The spatial expression analysis of ANS, CHS, DFR, F3H, F3’5’H and FLS in L. longituba indicated that these genes were preferentially expressed in flowers stems and leaves. The temporal expression analysis showed that different expressed patterns in different color flowers.
4、The function of ANS, CHS, DFR and F3’5’H gene was confirmed by Agrobacterium-mediated transformation of petunia.These results indicated that these genes normally expressed in the transformant.
5、Based on more than thirty thousand ESTs sequenced in the flower tissue of Lycoris longituba, 349 putative TFs were predicted, and then validated by real time RT-PCR.
6、Totally 809 SSRs distributed in 738 ESTs were mined out. These results provide a base for the development and future application of EST-SSR markers in genus Lycoris.
7、Analysis of the level and patterns of gemomic DNA methylation in different ploidy Lycoris radiate by MSAP indicated that 3128 genetic loci were produced by 36 pairs of selective primers. However, the results implied that the DNA methylation levels were not closely associated with the autopolyploidy level in L.radiata.
8、The nuclear DNA content in Lycoris exhibited significant differences from that of the standard (L. longiflorum). Each species group was allied based on genome size, chromosome number, flower color and leaf morphology; larger genome size was associated with a lower the chromosome number.
本研究以长筒石蒜花瓣为材料,对长筒石蒜花色变异的分子基础进行研究。主要结果如下:
1.对43个长筒石蒜变异类型和长筒石蒜模式种的花色进行评价。HPLC-DAD/ESI-MS确定长筒石蒜中的花青素组成,初步阐明了长筒石蒜花色形成化学机理。
2.通过RACE方法从长筒石蒜白色和紫红色花瓣的cDNA中克隆到了ANS,CHS,DFR,F3H,F3’5’H和FLS六个基因的全长cDNA序列。
3.用实时定量RT-PCR分析了长筒石蒜ANS,CHS,DFR,F3H,F3’5’H和FLS六个基因在不同花色长筒石蒜中的时空表达情况。
4.构建植物表达载体对矮牵牛进行遗传转化,转基因矮牵牛花朵的颜色有不同程度的变化。
5.预测了348个转录因子。其中58个转录因子在花瓣中较叶片而言有特异性高表达,同时对其中22个转录因子分析了它们在不同花发育阶段的表达模式。
6.从前ESTs序列中开发了809个EST-SSR,并进行了初步分析。
7.不同倍性石蒜(L.radiata)基因组MSAP分析表明DNA甲基化水平与倍性高低关系不明显,而三倍体石蒜表现出较为显著的低甲基化水平特征。
8.用流式细胞仪对石蒜属植物中9个种进行基因组大小的测定。石蒜属植物基因组大小与其花色、叶形有着密切的关系,并且石蒜属植物基因组大小与染色体数目有关。
The genus Lycoris have colorful flowers favorable for instance as Lycoris longituba. The flowers of L. longituba exhibit a great deal of diversity in both color and floral form, and its bulb has some medicinal potential.
In this research, the Molecular Biology of Color Variation in L.longituba was studied. The main results were showed as following:
1、The flower color of 43 L. longituba varieties and the Type species were divided into nine groups: red, orange, yellow and purple series.Anthocyanin composition of L. longituba determined using HPLC-DAD/ESI-MS. The result of multiple linear regression analysis (MLR) indicated that Cy derivatives were the major factors affected the value of color parameters greatly.
2、Six complete-length cDNAs encoding ANS, CHS, DFR, F3H, F3’5’H and FLS were isolated from the petals by RACE.
3、The spatial expression analysis of ANS, CHS, DFR, F3H, F3’5’H and FLS in L. longituba indicated that these genes were preferentially expressed in flowers stems and leaves. The temporal expression analysis showed that different expressed patterns in different color flowers.
4、The function of ANS, CHS, DFR and F3’5’H gene was confirmed by Agrobacterium-mediated transformation of petunia.These results indicated that these genes normally expressed in the transformant.
5、Based on more than thirty thousand ESTs sequenced in the flower tissue of Lycoris longituba, 349 putative TFs were predicted, and then validated by real time RT-PCR.
6、Totally 809 SSRs distributed in 738 ESTs were mined out. These results provide a base for the development and future application of EST-SSR markers in genus Lycoris.
7、Analysis of the level and patterns of gemomic DNA methylation in different ploidy Lycoris radiate by MSAP indicated that 3128 genetic loci were produced by 36 pairs of selective primers. However, the results implied that the DNA methylation levels were not closely associated with the autopolyploidy level in L.radiata.
8、The nuclear DNA content in Lycoris exhibited significant differences from that of the standard (L. longiflorum). Each species group was allied based on genome size, chromosome number, flower color and leaf morphology; larger genome size was associated with a lower the chromosome number.
引文
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