【摘 要】
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龙眼(Dimocarpus longan Lour.)是重要的亚热带果树。本试验以‘红核子’和‘四季蜜’龙眼为材料,克隆龙眼GIGANTEA (GI)和FLAVIN-BINDING, KELCH REPEAT, F-BOX1(FKFl)基因,
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龙眼(Dimocarpus longan Lour.)是重要的亚热带果树。本试验以‘红核子’和‘四季蜜’龙眼为材料,克隆龙眼GIGANTEA (GI)和FLAVIN-BINDING, KELCH REPEAT, F-BOX1(FKFl)基因,初步研究龙眼两个基因的功能,研究结果有助于了解龙眼GI和FKFl的功能以及开花调控的分子机制。本试验主要获得了以下研究结果:1.以‘红核子’龙眼叶芽为试验材料,克隆了龙眼GI和FKFl同源基因cDNA编码区全长序列,命名为DIGI和DlFKFl。荧光定量PCR分析DIGI和DIFKF1在‘红核子’龙眼花芽分化过程中的表达。结果显示,在11月份(龙眼花芽分化生理分化期),DIGI和DIFKF1基因表达量均达到顶峰,在2月份(龙眼花芽分化进入形态分化期),DIGI和DIFKF1的表达量最低。2.将DIGI和DIFKF1构建植物重组表达载体,农杆菌介导转入拟南芥中,对转基因植株表型进行分析发现。在长日照条件下,DIGI和DIFKF1过量表达导致促进转基因拟南芥不定根的形成和早花现象,荧光定量PCR结果表明DIGI和DIFKF1促进拟南芥体内生长素的合成。在短日照条件下,DIGI过量表达拟南芥表现出早花现象,而DIFKF1过量表达使拟南芥表现出晚花现象,同时两种转基因植株都出现下胚轴伸长的现象;同时通过荧光定量PCR对赤霉素合成途径中关键基因在拟南芥根和叶片的表达结合液相色谱数据表明,DIGI拟南芥和DIFKF1拟南芥GA3的激素水平均显著低于野生型,说明短日照下,DIGI和DIFKF1抑制拟南芥体内GA3的合成,同时DlGI有促进开花的功能。结合龙眼花芽分化过程DIGI和DIFKF1在生理分化期表达量上升的现象,推测DIGI和DIFKF1在龙眼中可能通过调节内源激素水平促进花芽分化。3.将构建好的DIGI和DIFKF1基因表达载体转化普通烟草,DIGI和DIFKF1转基因烟草都表现出不定根增加的现象,DIGI烟草的根长显著比对照短,与长日照条件下转基因拟南芥的表型相似,进一步验证了DIGI和DIFKF1能调控激素合成的功能。同时在两个品种的转基因烟草中,都出现了不同时期的生理落花现象。4.以‘红核子’和‘四季蜜’龙眼DNA为材料,分别克隆出DIGI基因1338 bp和1587 bp启动子,序列分析显示,‘红核子’和‘四季蜜’启动子中有22个相同的顺式元件,有10个不同的顺式元件,‘四季蜜’中存在着TCA-element、TGA-element Unnamed-2特有的顺式元件,红核子仅有一个特有的顺式元件MBSI,DIGI在两个龙眼品种中的表达差异可能与启动子的顺式元件差异有关。
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