成花素FT基因在柑橘维管组织中移动受限机制和解除策略

Flowering is the foundation of fruit breeding and production. Recent study has revealed that woody plants goes similar mechanism in development transition and flowering relying on FT expression. In model herbaceous plants, it has been confirmed that the FLOWERING LOCUS T (FT) proteins can be transported from the leaves to the apical meristem through vascular bundles. However, it is not effective in shortening the juvenile period in adult fruit trees by juvenile material top-grafted on flowering root-stock. In a few recent research reported that juvenile apple and poplar grafted on FT transgenic material did not display early flowering as expected, indicating that the movement of FT in woody plants may be limited. in woody plants. This project intends to compare the similarities and differences of phloem structure, FT protein size and amino acids in key position between citrus and Arabidopsis as well as tomato. By making exchange of the key element of FT protein between citrus and model plants, modified FT protein will be transformed into tomato and citrus respectively to compare the mobility of FT in transgenic plants. In sieve tube and companion cell of citrus, interaction ability of FT-INTERACTING PROTEIN 1 (FTIP1) and phloem-specific Myb-related protein (FE) to FT protein and their dynamic expression will be examined to reveal the refine mechanism of FT protein restriction in citrus. The FT movement within the scion will be also investigated when the infant shoots are grafted by seedling-grafting method on transgenic FT-GFP material. Meanwhile, to further explore the possibility of increasing the mobility of FT proteins, a binary vector of FT and phosphatidylcholine synthesis-related genes will be constructed, transformed into citrus, and supplemented with external treatments to observe its effect on the movement of FT proteins. Thus, the project is expected to improve our knowledge about the basis of flowering in citrus plants and provide a practical strategy to shorten juvenile period and enhance breeding efficiency in citrus.

开花是果树育种和生产的基础。近期研究已揭示木本果树的成花转变与模式植物一样，也依赖成花素FT基因表达。在草本植物中已证实FT蛋白可以通过维管束从叶片转运到顶端分生组织，但在已开花的成年果树上高接童期材料，却并不能有效缩短童期；转化FT早花材料上嫁接童期苹果和杨树，也未获得早花表型转移的预想结果，表明FT在木本植物中的移动受到限制。本项目拟在前期基础上，对比研究柑橘与拟南芥、番茄维管结构与成分、FT蛋白关键位置氨基酸异同，并互换FT相应区域氨基酸分别转化番茄和柑橘，比较FT移动性；检测柑橘FTIP及FE与FT蛋白互作能力和表达动态变化，以揭示柑橘中FT蛋白移动受限分子机制；采用芽苗嫁接法检验FT在细嫩接穗内移动情况；构建FT与磷脂酰胆碱合成相关基因的双元载体，转化柑橘，并辅加外施处理，观察其对FT蛋白移动的影响，探索提高FT蛋白移动能力的可能性，为提高柑橘育种效率和早结丰产提供新思路。

开花是果树育种和生产的基础。在草本植物中已证明FT蛋白可以通过嫁接从砧木转运到接穗并促进其开花，但在已开花的成年果树上高接童期材料，却并不能有效缩短童期。为探究木本果树中FT蛋白是否能够随嫁接从砧木传递到接穗，本研究分别克隆了拟南芥AtFT (草本来源)和枳ToFT（木本来源）基因，转化番茄（草本）和枳（木本），观察成花表型和信号运转。研究发现，不管是草本来源还是木本来源的FT都能促进转基因番茄和枳早花，在同物种内的作用无明显差异；番茄中超表达AtFT和ToFT砧木上嫁接野生型都能提早开花，但在柑橘中具早花能力的转ToFT或AtFT枳砧木上嫁接实生材料并无早花效果；通过实体荧光成像和Western检测发现，在番茄和枳中，带GFP标签的AtFT和ToFT蛋白都能在嫁接体系中双向迁移，且在同物种内嫁接体系之间的迁移能力无明显差异，而其mRNA并未检测到迁移信号；定量分析发现，在枳和番茄嫁接口上同等高度接穗叶片中，番茄中运输的AtFT和ToFT蛋白的积累量高于枳，表明番茄中FT蛋白的迁移能力强于枳。对内源FT和外源FT蛋白定量分析发现，在枳中外源FT蛋白迁移的同时，枳内源的FT蛋白水平显著下降，可能是木本植物嫁接后FT蛋白虽然有迁移却不能引起早花的原因。克隆了枳FTIP， 在体内和体外条件下检测到与ToFT之间存在互作，且表达动态和表达部位一致；在番茄中超表达ToFTIP能够提早开花，与ToFT共同超表达有协同早花效应；超表达ToFTIP接穗嫁接到超表达ToFT砧木上也能比同样砧木上嫁接的野生型更早开化，表明ToFTIP在番茄体内可能促进了外源ToFT的移动。从柑橘中克隆的脂类相关基因LIP2A，在拟南芥中超表达具有早花功能，但与FT双超表达没有复合效应；转LIP2A基因拟南芥茎薄壁细胞增大，可能有利于FT蛋白的运输，而LIP2A蛋白本身与FT基因没有直接互作的能力。研究过程中，通过授粉杂交和胚抢救，获得转FT基因枳与葡萄柚、克里曼丁橘和四季橘等柑橘品种的杂交后代，部分材料在试管苗阶段就提早开花，为开展柑橘种间高世代杂交育种和遗传规律研究提供了宝贵材料。