摘要
果树花芽形成是产量形成的基础,而大多数果树在第一次开花结果之前常常需要经历一个漫长的营养生长期——童期。童期的存在严重阻碍了杂交育种的进程,延长了育种周期。基因工程技术的发展为解决这一难题提供了新途径。最近,FT蛋白己被证明是植物中的“成花素”,苹果MdFT基因是拟南芥AtFT基因的同源基因,本研究开展了该基因的克隆和功能鉴定工作,取得的结果有:
1、通过RT-PCR扩增,从苹果栽培品种‘嘎拉'叶片cDNA中克隆了MdFT基因,同时,从拟南芥生态型Columbia中克隆了AtFT基因作为对照。序列分析表明,在氨基酸水平上,MdFT与AtFT的同源性为72.6%。
2、聚类分析表明,推导的MdFT蛋白与来自拟南芥、水稻、柑桔、杨树等的FT同源基因推导的蛋白同归于FT类。
3、构建了植物表达载体35S::MdFT和35S::AtFT。利用根癌农杆菌介导法,将它们导入拟南芥生态型Columbia和番茄栽培品种‘中蔬一号'中,得到了抗卡那霉素的阳性转化植株。经PCR扩增检测,证明它们已经导入转化的拟南芥基因组;经PCR扩增和半定量RT-PCR检测,证明它们已经导入转化的番茄基因组,并在转录水平得到了表达。
4、转基因拟南芥的Quantitative RT-PCR检测表明,MdFT基因通过调节下游成花相关基因的表达诱导拟南芥产生早花现象。
5、亚细胞定位结果显示,MdFT-GFP蛋白主要定位在细胞膜上。
6、形态鉴定结果表明,无论是转基因拟南芥植株还是番茄植株,均表现出早花性状。在短日照条件下,转35::MdFT拟南芥植株在8.9片莲座叶期即开始抽薹开花,而野生型植株通常要在莲座叶长到60片以后才开始抽薹开花。在温室栽培条件下,5株35S::MdFT转基因番茄植株的花芽分别为第4、8、11、7和9节位形成,而非转基因再生对照植株均在第1l节位以上形成花芽。
It is of great importance both for developmental phase transition and field yield for fruittrees to generate flower buds. Generally, most fruit trees belong to perennial woody plantwhich characterized by a long phase for vegetative growth before the first onset of flowering,called juvenility, a time in which any mathods are failed to induce flowering and fruiting. Theexistence of juvenility also badly hampers and prolongs the hybrid breeding process becausemany evaluation tasks about characterizations cann't be conducted during this period. Manypractices have proved that conventional ways are limited for juvenility reduction, while thedevelopment of genetic engineering sheds strong light for this target. Recently, FT protein hasbeen proved to be a florigen in higher plants. In apple, the FT homologus is MdFT, which wasisolated and characterized in this study, and the results are summaried as follows:
1、Full-length cDNA of MdFT gene was amplified from apple (cv. GALA) leaf cDNAs withRT-PCR, in parallel, AtFT gene was also amplified from Arabidopsis (Columbia) leafcDNAs as positive control. Sequence analysis showed that MdFT shared 72.6% similarityto that of AtFT.
2、phylogenetic analysis indicated that MdFT protein, together with those come from arabidopsis, Oryza sativa, Citrus, Oryza sativa and so on, was grouped into FT-like proteins.
3、Plant expression construct containing MdFT or AtFT gene driven by cauliflower mosaic virus 35S promoter was obtained and separately introduced into Arabidopsis(Columbia ecotype) and tomato cultivar ZHONGSHU NO.1 with Agrobacterium-mediated transformation. Finally, transgenic Arabidopsis and tomato lines overexpressing MdFT and AtFT, respectively, were regenerated from selection medium containing kanamycin. PCR amplification verified the integration of exogenous genes into the host genome of Arabidopsis; PCR amplification also, together with semiquantitative RT-PCR assay, displayed the successful transformation of MdFT and AtFT and their ectopic overexpression in transgenic tomatos.
4、Quantitative RT-PCR assay suggested that MdFT gene induced early flowering by regulated downstream flowering-related genes in transgenic Aradopsis
5、Subcellular location assay demonstrated that MdFT-GFP fusion protein primarily distributed on membrane.
6、Morphological observation found that transgenic lines, either Arabidopsis or tomato, flowered earlier than non-transgenic controls. Under shoot day condition, 35::MdFT lines of Arabidopsis flowered at the 8.9 rosettes, which were much earlier than that of non-transgenic lines that usually harbored more than 60 rosettes. Similarly, when tomato plants were grew in greenhouse, 5 transgenic regeneration plants set flower at the node of fourth, eighth, eleventh, seventh and ninth, respectively, which showed earlier than non-transgenic regeneration plants that often formed flower above the eleventh node.
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