利用抑制消减杂交法筛选低夜温诱导的蝴蝶兰生殖发育相关基因

摘要

花芽的发育是花品质形成与调控的生物学基础,温度是影响蝴蝶兰(Phalaenopsis hybrida)花芽分化与发育乃至花朵开放的重要因素.为鉴定低夜温诱导蝴蝶兰花梗芽分化和发育相关基因,以蝴蝶兰兄弟女孩品种为材料,利用抑制削减杂交技术,构建低夜温诱导的花梗芽与营养生长期顶叶的正向差减文库.PCR验证后,挑取300个阳性单克隆进行测序和分析,共获得207条非冗余序列(GenBank登录号:JK720764～JK720970).这些基因覆盖了宽广功能范围的开花相关基因,提供了从营养生长期向生殖生长期转变相关分子机理方面的有益信息.Real-time PCR检测了具不同功能的23个基因在营养生长期顶叶和不同大小花梗芽中的表达水平.这些基因的表达量变化趋势不同,但在一定大小花梗芽中的表达量较营养生长期顶叶均有上调.与在营养生长期顶叶中的表达量相比,flowering locus T(FT)、APETALA1(AP1)、茉莉酮酸酯-O-甲基转移酶(JMT)基因和NADH泛醌氧化还原酶基因在各阶段花梗芽中的表达量均显著上调,表明这4个基因可能在低夜温诱导蝴蝶兰花梗芽分化和发育中起重要作用.本研究为深入探究低夜温诱导蝴蝶兰开花的分子机理提供了重要的基础资料.%The development of floral buds is a biologic foundation for flower traits formation and regulation.Moreover,temperature is an important factor that affects the floral buds' differentiation and development of Phalaenopsis plants,even flowering.To gain genes related to cool-night temperature induced differentiation and development of spikes in Phalaenopsis hybrida cv.Brother Sister,we performed suppression subtractive hybridization,which allowed identification of flowering-related genes in spikes induced by cool-night temperature and the apical leaves in the vegetative growth phase.After PCR detection for each clone,300 positive single clones were selected for sequencing.Totally,207 non-redundant expressed sequence tags(ESTs)(GenBank accession Numbers:JK720764～JK720970) were obtained in the forward-subtracted library.These cDNAs covered a broad repertoire of flowering-related genes,provided helpful information for understanding molecular mechanism in transition from the vegetative to reproductive growth phase.Expression analysis by Real-time PCR of 23 genes from different functional categories validated the expression levels in vegetative apical leaves and spikes with different sizes induced by cool-night temperature.Expression patterns of these 23 genes showed diverse trends.However,they accumulated higher transcripts of these genes in spikes of some stage relative to apical leaves in vegetative phase.Compared with the expression levels in vegetative apical leaves,the transcripts of genes encoding flowering locus T(FT),APETALA1(AP1),jasmonate-Omethyltransferase(JMT) and NADH-ubiquinone oxidoreductase in different sizes spikes significantly increased.These findings indicated that the four genes might presumably play an important role in the process of coolnight temperature induced differentiation and development of spikes.This study supplies an important foundation for further exploration about the molecular mechanisms of cool-night temperature induction of flowering in Phalaenopsis orchids.