烟叶苣苔RAD类基因的招募及进化意义研究

In the model organism Antirrhinum majus, two TCP genes CYCLOIDEA (CYC) and DICHOTOMA (DICH) determine dorsal floral identity mainly through directly activating a MYB gene RADIALIS (RAD) in the dorsal floral domains. Overexpression of CYC gene in heterogenous Arabidopsis thaliana can activate RAD gene of A. majus but cannot activate endogenous RAD-like genes of A.thaliana, hinting that the co-option of RAD gene into the regulatory network of CYC gene may play an important role in the generation of zygomorphic flowers in A.majus. A central question is that whether RAD-like genes have been recruited into the regulatory network of CYC-like genes in Gesneriaceae, one of the basal-most groups in Lamiales from which A. majus also belongs to. Primulina heterotricha represents an ideal candidate to address this question due to its typical zygomorphic flowers, previously related studies on two CYC-like genes (PhCYC1C and PhCYC1D), and the development of a promising plant regeneration and genetic transformation system.In preliminary experiments of this project, we isolated one RAD-like gene, namely PhRAD, from P. heterotricha. RT-PCR and RNA in situ hybridization results showed that PhRAD is specifically expressed in the dorsal floral regions during early stages. Promoter analysis showed that its promoter contains a putative CYC-binding site which could be weakly bound by recombinant PhCYC1 proteins in electrophoresis mobility shift assays. The above results hint that PhRAD gene may have been recruited into the regulatory network of PhCYC1 genes. Here, we will further investigate the regulatory relationship between PhCYC1 and PhRAD genes and its evolutionary significance by using a series of transgene methods. Combining previous studies in A. majus and A. thaliana, the results of this project will provide great insights into the origin and evolution of the regulatory network of CYC-like and RAD-like floral symmetry genes and the origin of zygomorphic flowers.

CYC基因对RAD基因的招募在金鱼草两侧对称花的形成中起着重要作用。在被子植物两侧对称花的起源与进化方面，一个亟待解决的问题是，在金鱼草所属广义唇形目的基部类群中，RAD类基因是否已经被招募到CYC类基因的调控网络中。具有典型两侧对称花的烟叶苣苔（苦苣苔科）具有深厚的研究基础，并且可以进行转基因，是研究以上问题的首选材料。在本项目的前期研究中，我们发现烟叶苣苔PhRAD基因早期具有背特性表达模式，且其启动子含有一个能够与重组PhCYC1蛋白发生弱相互作用的DNA结合位点，暗示PhRAD基因可能已经被招募到PhCYC1基因的调控网络中。在本项目接下来的研究中，我们将采用各种转基因方法深入探讨烟叶苣苔CYC类与RAD类基因的调控关系。结合金鱼草和拟南芥中的研究以及烟叶苣苔所处的系统位置，该项目的研究结果将对理解CYC类与RAD类基因调控网络的起源与演化以及两侧对称花的起源具有重要意义。

在模式植物金鱼草中，两个TCP基因CYCLOIDEA（CYC）和DICHOTOMA（DICH）在花分生组织背部区域的特异性表达激活了一个MYB基因RADIALIS（RAD）在相同区域的表达。RAD基因通过转录后抑制作用而拮抗另一个MYB基因DIVARICATA（DIV，腹部特征基因），使其仅在腹部花瓣上起作用。研究表明，在金鱼草这一支系中，CYC/DICH对RAD基因的招募在两侧对称花的形成过程中起着重要作用。那么，一个亟待解决的问题是，在金鱼草所属唇形目的基部类群中，RAD类基因是否已经被招募到CYC类基因的调控网络中，CYC类基因对RAD类基因的招募是否是两侧对称花形成的必要条件。.本项目以具有典型两侧对称花的苦苣苔科植物烟叶苣苔和斑叶草为研究对象，开展CYC类和RAD类基因的功能以及调控关系研究，从而深刻阐述苦苣苔科植物两侧对称花产生的分子调控及进化机制。.在烟叶苣苔中的研究表明：PhRAD1特异表达于腹部可育雄蕊上，而PhRAD2早期短暂表达于花分生组织的背部区域；PhRAD1受到PhCYC1的负调控，而PhRAD2受到PhCYC1的正调控。在斑叶草中的研究表明：CpCYC1、CpCYC2是控制斑叶草两侧对称花形成的关键基因，它们在特定区域的表达导致背部花瓣特征的形成以及背部和侧部雄蕊的败育，它们的沉默导致辐射对称花的形成；CpRAD具有促进雄蕊生长和发育的作用；CpCYC1、CpCYC2负调控CpRAD。综合以上研究，我们认为：在苦苣苔科中，CYC类基因对RAD类基因的招募可能刚刚发生，它们之间的调控关系还没有达到金鱼草中的那种紧密程度（PhRAD2仅在花发育的早期短暂表达于花分生组织的背部区域）；在这个招募过程中，RAD类基因的复制和分化事件（产生两个具有不同表达模式的旁系同源基因，如PhRAD1和PhRAD2）可能起着重要作用。