用嫁接和标签示踪研究柑橘成花信号传导与相互作用

Flowering is the foundation of fruit breeding and production. Perenial woody plant shares similar molecular mechanism in flowering formation with annual model plants but differed greatly in flowering habit. FT has been considered to be the hypothesized florigen which was capable of graft-transmission from leaves to shoot tip through phloem and promoting flower transition in shoot apical meristem. However, the juvenile scion from woody plants could not be induced early flowering by using rootstock of both adult tree and FT-overexpressed transgenic plants, which was supposed to produce abundant FT protein. Thus we hypothesize that the movement of FT protein may possibly restrained by signals imported from the scion. In this project, Clementina citrus will be grafted up/down to transgenic Poncirus trifoliata carried fusion gene of FT:GFP or FLC:GUS respectively driven by CaMV 35S promoter. Transduction ability of FT or FLC will be evaluated by tracing of the GFP fluorescence and GUS activity respectively in the phloem between the scion and rootstock, as well as the transduction route and the rule for distribution. The signals in the form of RNAs and protein exchanging between rootstock and scion will be analyzed by use of the great mass of SNP variations between Clementina and Poncirus trifoliata and proteinomics methods of iTRAQ (isobaric tag for relative and absolute quantitation) respectively. Real-time PCR will be performed to analysis the relationship between imported signals of RNAs or protein and gene expression changes in flowering related genes. Based on above analysis, the results will reveal whether flowering signals are transmissible between shoot apical meristem and leaves, as well as their forms, transmission route and distribution rule, so as to enhance our knowledge about plant phase development and flowering transition. Also, the fulfill of this project will do helpful as reference for study of the interaction between rootstock and scion, and provide theoretical foundation for accelerating fruit breeding and utilization of transgenic plants to produce un-transgenic fruit.

花形成是果树育种和产量的基础。木本果树与草本植物在成花转变过程具相似的分子基础和显著的习性差别。FT被多数研究者认同是假想的成花素，已用模式植物嫁接证明可通过韧皮部向顶端分生组织转运并促进成花。成年木本果树及超表达FT转化苗作砧木却不能使童期接穗提早成花，表明FT移动受上部接穗的信号限制。本项目拟以克里曼丁橘分别与超表达FT:GFP或FLC:GUS转基因枳正、反嫁接，以GFP荧光和GUS染色分别示踪研究FT及FLC在柑橘中的传导能力、方向、途径和分配规律;利用克里曼丁橘与枳属间SNP差异、蛋白组分析和实时定量，比较超表达FT或FLC时砧穗间RNAs和蛋白信号交流及与成花基因表达关系，以探明顶端分生组织与基端叶片之间成花信号类型、传导方式及相互作用，进一步丰富植物阶段转变和生殖发育调控理论，并为砧穗互作研究、采用嫁接方式加速育种和性状遗传规律研究，以及利用转基因材料生产非转基因果实探索新路。

嫁接是园艺作物中广泛应用的一项繁殖技术，并用以增强栽培品种的抗逆性、提高产量和品质。本实验将克里曼丁橘嫁接于枳上，用接穗的母树克里曼丁橘和未嫁接的枳作为对照。利用接穗和砧木间特异的SNP作为标签对mRNA进行标记，从而研究嫁接后接穗和砧木间mRNA的移动和嫁接后砧穗间基因表达变化。此外，也检测了成花素基因FT在柑橘中运输情况。主要结果如下：（1）比对分析发现，在接穗中检测到砧木特异的SNP有563个，砧木中检测到接穗特异的SNP有1268个；接穗中检测到砧木的InDel有3个，砧木中检测到接穗的InDel有33个。结果表明嫁接植株上的砧木和接穗组织在嫁接前后发生了SNP和InDel变化，表明植物在嫁接复合体中接穗与砧木间存在mRNA的移动,检测到的向上移动和向下移动的mRNA种类数分别为348和710，双向移动的基因有15个。可移动的mRNA中有较高比例的基因数含有类似tRNA的茎环结构。在可移动的基因中，单生物体细胞化过程、原初代谢过程调控、防御反应和含氮化合物代谢等相关途径的基因比例最高。（2）嫁接不但存在基因mRNA传递，而且也对基因的表达产生显著的影响。嫁接前后砧木有598个基因表达量显著上调，993个基因表达量显著下调；而接穗嫁接前后只有53个基因表达量显著上调，69个基因表达量显著下调。因此从嫁接移动的基因数目和基因表达变化看，上部接穗材料对下部的砧木影响比下部对上部的影响更大；（3）采用GFP标记FT基因转化枳和番茄，获得了早花的转基因植株，转基因番茄上嫁接野生型目前尚未能观察到早花表现，有可能是嫁接时期和方法对表型有影响；试管内嫁接在转FT-GFP枳上的普通枳，在嫁接口上部观察到GFP荧光，表明FT蛋白在试管嫁接的情况下可以通过嫁接部上移进入接穗，但移动过程中GFP荧光迅速衰减，表明FT移动受到限制；（4）改造FT基因结构，增加发夹结构，在柑橘上胚轴上的再生芽直接成为花芽，表明其诱导成花的能力更强。上述研究结果为进一步利用可移动基因、调控柑橘成花提供了很好的思路。