摘要
山茶花是我国传统十大名花之一,也是世界名贵花卉。重瓣型山茶花由于丰富的造型而深受喜爱,但野生状态下的山茶花为单瓣花型,在漫长的生物进化中单瓣花是如何变化为重瓣花类型的,是本研究之目的。从花发育的“ABC模型”理论可知,B类功能基因是同时参与了真核植物花瓣和雄蕊的发育从而影响到花器官的第二轮和第三轮结构确定性的关键因子,暗示了B功能基因在山茶重瓣花形成中起到关键作用。尽管B类基因的功能在模式植物中研究的较为清楚,但在山茶中B功能基因扮演什么角色及是否引起山茶重瓣花形成等均不为人知。
采用RT-PCR (Reverse transcription polymerase chain reaction)和RACE(Rapid amplification of cDNA end)法从中国传统重瓣花型茶花名种‘红十八学士’(Cameliia japonica‘Hongshibaxueshi’)中分离出B功能基因4个成员的cDNA全长,分别命名为CjHGLO1、CjHGLO2、CjHDEF和CjHTM6,探讨了这4个成员的功能及其在部分山茶属植物不同花类型山茶花中的表达模式。研究发现,分离的‘红十八学士’B功能基因在大肠杆菌E. Coli BL21中经IPTG诱导能表达出相应的蛋白,表明分离的四个成员结构完整,具有完整编码蛋白质的能力。将四个成员的正义基因导入拟南芥发现,过表达CjHGLO1基因引起萼片和花瓣的融合,这种融合的趋势是从萼片和花瓣的基部开始,逐渐向上;过表达正义CjHGLO2没有引起转基因拟南芥表型改变;而过表达CjHDEF基因引起拟南芥第一轮萼片转变为花瓣,使得原来有4片花瓣的拟南芥转变为具有8片花瓣的花;过表达CjHTM6基因造成拟南芥的雌蕊在花苞尚未展开时即显著伸长,引起花瓣和雌蕊的不一致性生长,同时6枚雄蕊消失。
采用Real-time PCR技术研究了B功能基因在山茶单瓣、半重瓣、托桂型、牡丹重瓣型和完全重瓣型等5个不同类型茶花中的表达模式发现,GLO1(GLOBOSA1)和GLO2(GLOBOSA2)的同源基因是在具有花瓣属性的部位高表达,而在具有或靠近萼片属性的部位表达量低或不表达,表明其在花瓣属性的决定中扮演重要角色;DEF(DEFICIENS)和TM6(TOMATO MADS BOX GENE6)同源异型基因是在雄蕊的融合处高表达,这种现象表明它们可能在雄蕊瓣化为花瓣的过程中扮演重要角色。在山茶单瓣花转变为重瓣花的过程中,是伴随着山茶花瓣数目的增加和花器官轮数的逐渐减少,其中B功能基因的表达域也逐渐缩小并稳定的在花瓣器官中高表达,这种现象表明B功能基因的功能域是逐渐的在向着中心部位的花瓣迁移,说明B功能基因在决定山茶花瓣属性中具有高度的保守性。
通过本研究发现,B功能基因拓展了表达域是引起山茶萼片瓣化现象的原因之一,这种情况会造成山茶花瓣数目的增加是山茶重瓣花形成的原因之一。山茶重瓣花涉及积累起源、苞(萼)片起源和雌雄蕊等多种起源方式。
Camellia is one of the top ten traditional flowers in China, and also is one of the precious flowers in the world. In the natural states the camellia flowers is signal form and how to changes into double flowers in biology evolution is our research purpose. From“ABC model”theory of floral development we knew that B-function was key role in the second and third whorl in eukaryote plants, so, this phenomenon was implied that B-function may be play key factor for double flowers formation in camellia. Although the function of B-function gene was clearly in model plant, but it’s role in double flowers formation were unknown in camellia.
To investigate the mechanism of B-function gene for double flower development in Camellia, B-function gene cDNA was cloned by RT-PCR and RACE methods and named CjHGLO1, CjHGLO2, CjHDEF and CjHTM6 respective, and its function and expression pattern were analyzed. The results showed that the coding protein of B-function gene was completely expressed in Escherichia coli BL21 by IPTG induced, this phenomenon indicated that the isolated genes had completed structure. The sense gene of B-function was transforred into Arabidopsis and the results showed that the sepals and petals were fused by CjHGLO1 gene, and the fused tendency started from based part. The phenotype was not changed by sense CjHGLO2 gene. The first-whorl sepals were completely transforrmed into petals by sense CjHDEF gene, so that all of pepals was eight. The stigma was extended when the flower buds was not opening in Arabidopsis by sense CjHTM6 gene, and the petals growth was not cohered with stigma, meanwhile, the stamens has disappeared.
The expression pattern of B-function gene among signal、semi-double、anemone form、peony form and formal double was studied by Real-time PCR method. The results showed that B-function gene GLOBOSA1 (GLO1) and GLOBOSA2 (GLO2) had high expression at the part with petal identity, and had low or no expression at or near the part with sepal identity, and these kinds of expression showed that gene played an important role in determining petal identity. B-function gene DEFICIENS (DEF) and TOMATO MADS BOX GENE6 (TM6) had high expression at the fused part of stamens, and this implied the importance of gene when stamen transforrmed into petal. The petals were increased and the flower whorls were decrease when the signal transformed into double flowers in camellia, and expression domain of B-function was also toward to the central petals, the simplest explanation for this was that the B-function was removal toward the flower center, this phenomenon explained that the B-function gene was high conservative.
Our analyses of the paper, the phenomenon of the sepals petaloid in camellia is extented of expression domain by B-function gene, and it is key factor in double flower formation. It has multiple origins including accumulation origin,sepal origin and stamens-carpels origin in double flowers formation in camellia. Thus, B-function gene is very important when signal evolved into double flowers in camellia.
引文
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