Gene cloning and functional identification of AfFT3 gene in Aechmea fasciata（Lindl.） Baker
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摘要: 【目的】克隆蜻蜓凤梨成花素基因AfFT3,并鉴定其生物学功能,为深入解析凤梨科植物开花分子机制提供理论依据。【方法】克隆AfFT3基因,利用生物信息学软件进行序列分析,并通过实时荧光定量PCR探究其在低温(18℃)、常温(25℃)和高温(35℃)及外源乙烯处理下的表达情况,采用农杆菌浸花法将AfFT3基因转入拟南芥,观察转基因植株表型,通过异源表达预测其生物学功能。通过酵母单杂交试验初步鉴定其启动子与AfEIN3蛋白的互作关系。【结果】从蜻蜓凤梨中克隆获得AfFT3基因全长570 bp,编码区(CDS)序列为465 bp,编码154个氨基酸残基,蛋白分子量为17.3 kD,为稳定的亲水性蛋白,无跨膜螺旋区,含有PKC、PKA、cdc2、INSR和GSK3等多个蛋白激酶磷酸化位点。同源序列比对发现,AfFT3蛋白中有2个氨基酸残基138Trp和140Gln突变为138Met和140Glu。系统发育分析结果显示,该蛋白属于PEBP家族的FT-like蛋白亚家族,与TFL-like亚家族的亲缘关系较近。与对照(大棚正常条件下栽培植株)相比,高温和低温处理下AfFT3基因在蜻蜓凤梨中的相对表达量显著(P<0.05,下同)或极显著(P<0.01,下同)升高。在高温、常温和低温条件下,使用外源乙烯处理后,AfFT3基因在蜻蜓凤梨中的相对表达量均较对照显著或极显著升高,在常温处理下其相对表达量最高。共获得6个转基因拟南芥株系(L1~L6),其中转基因株系L3和L6较转空载体拟南芥和野生型拟南芥延迟抽薹8 d。通过分段扩增获得AfFT3基因启动子3段序列(AfFT3-P1、AfFT3-P2和AfFT3-P3),长度分别为390、1077和552 bp,通过酵母单杂试验推测启动子序列AfFT3-P1和AfFT3-P3可与AfEIN3蛋白发生互作。【结论】高温、低温胁迫和乙烯均不同程度诱导AfFT3基因的高效表达,但在高温和低温条件下乙烯对AfFT3基因的表达诱导效果较常温有所降低,其转基因株系延迟抽薹,说明AfFT3基因参与调控蜻蜓凤梨开花过程,且响应温度和乙烯信号。Abstract: 【Objective】The flowering-related gene AfFT3 in Aechmea fasciata (Lindl.) Baker was cloned and its biological function was identified,which provided a theoretical basis for further analysis of the molecular mechanism of flowering of Bromeliaceae plants.【Method】AfFT3 gene was cloned and its sequence was analyzed by bioinformatics software.The expression levels of AfFT3 at low temperature(18℃),normal temperature(25℃)and high temperature(35℃)and exogenous ethylene treatment were detected by realtime fluorescence quantitative PCR(qRT-PCR).AfFT3gene was transferred into Arabidopsis thaliana (L.) Heynh by Agrobacterium flower dipping.The phenotype of transgenic plants was observed,and its biological function was predicted by heterologous expression.The interaction between the promoter and AfEIN3 protein was preliminarily identified by yeast one-hybrid assay.【Result】The full length of AfFT3gene cloned from A.fasciata was 570 bp,and the coding sequence (CDS) was 465 bp,encoding 154 amino acid residues.AfFT3 protein had a molecular weight of 17.3 kD,was a stable hydrophilic protein without a transmembrane helical region and contained several phosphorylation sites of protein kinases including PKC,PKA,cdc2,INSR and GSK3.The homologous sequence alignment revealed that two amino acid residues of AfFT3 protein 138Trp and 140Gln mutated to138Met and 140Glu.Phylogenetic analysis indicated that AfFT3 protein belonged to the FT-like protein subfamily of the PEBP family,and was close to the TFL-like subfamily.Compared with the control (plants cultivated in normal conditions in greenhouse),the relative expression of AfFT3 gene in A.fasciata was increased significantly (P<0.05,the same below) or extremely significantly (P<0.01,the same below) under the treatment of high temperature and low temperature.Under the conditions of high temperature,normal temperature and low temperature,after exogenous ethylene treatment,the relative expression of AfFT3 gene in A.fasciata was increased significantly or extremely significantly compared with the control,with the highest relative expression at normal temperature.A total of six transgenic A.thaliana plants (L1 to L6) were obtained,in which the transgenic plants L(3 )and L6 were about 8 d delayed than the empty vector and wild-type A.thaliana.The AfFT3 gene promoter sequences (AfFT3-P1,AfFT3-P2 and AfFT3-P3) were obtained by segmented amplification with the lengths of 390,1077 and 552 bp respectively.The results of yeast one-hybrid experiment preliminarily suggested that promoter sequence AfFT3-P1 and AfFT3-P3 might interact with AfEIN3 protein.【Conclusion】The effective expression of AfFT3 gene is induced by high temperature,low temperature stress and ethylene in different degrees,but the induction effect of ethylene on the expression of AfFT3 gene under high temperature and low temperature conditions is lower than that of normal temperature.The genetically modified plants delay bolting,indicating that AfFT3 gene is involved in the regulation of flowering process of A.fasciata,and respond to temperature and ethylene signals.
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Keywords:
- Aechmea fasciata(Lindl.) Baker /
- AfFT3 gene /
- flowering /
- temperature /
- ethylene /
- functional identification /
- promoter
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