【目的】克隆陆地棉开花促进因子家族的一个基因Flowering Promoting Factor1-like Protein 5(Gh FLP5),并对其时空表达模式进行研究,解析其在开花调控过程中的作用,为创制早熟陆地棉材料奠定基础。【方法】根据NCBI数据库中的序列,用Oligo7软件设计引物,以中棉所50(CCRI50)的c DNA为模板,克隆获得Gh FLP5。在Ex PASy网站上预测其蛋白的理化性质,同时在NCBI中检索其他物种中的FPF蛋白,使用Clustal X2进行多重序列比对,并用MEGA6构建系统进化树;取陆地棉早熟品种CCRI50和中晚熟品种鲁棉研28(Lu28)不同生长时期、不同组织的样品,对Gh FLP5的时间和空间表达模式进行分析;从基因组数据库中调取Gh FLP5起始密码子上游1 500 bp的片段,用Plant CARE在线工具预测其顺式作用元件,选取相关激素对二叶期棉花幼苗进行叶面喷施处理,研究Gh FLP5的应答反应;构建植物表达载体p BI121-Gh FLP5,转化拟南芥,观察转基因株系的表型,并用q RT-PCR对其内源基因表达的变化进行测定。【结果】Gh FLP5开放阅读框长300 bp,编码的蛋白质分子量为11.4 k D,共包含3个比较保守的区域,与大豆、苜蓿和毛果杨的开花促进因子亲缘关系较近。空间表达模式分析表明,Gh FLP5在叶片中优势表达,且在早熟品种CCRI50中的表达量显著高于晚熟品种Lu28;时间表达模式分析表明,在CCRI50中其表达量高峰出现在三叶期,而在Lu28中四叶期表达量最高。Gh FLP5的启动子上主要存在两大类顺式作用元件,一类是光响应元件和生物钟元件,一类是胁迫响应元件。根据顺式作用元件的分布和功能选取水杨酸(SA)、脱落酸(ABA)和茉莉酸(JA)喷施处理棉花幼苗,结果表明,Gh FLP5能响应外源SA和ABA而上调,也会被外施JA抑制。组成型表达Gh FLP5的拟南芥株系抽薹时间提前约9 d,开花时间提前约7 d,莲座叶数目减少,差异达到极显著水平。荧光定量的结果表明转基因拟南芥中促进开花的基因LEAFY(At LFY)、SUPPRESSOR OF OVEREXPRESSION OF CONSTANS(At SOC1)、FLOWERING LOCUS T(At FT)、APETALA1(At AP1)和FRUITFULL(At FUL)表达量显著升高,抑制开花的基因Flowering Locus C(At FLC)表达量显著降低。在转基因拟南芥中生长素应答基因SMALL AUXIN UPREGULATED 20(At SAUR20)和SMALL AUXIN UPREGULATED22(At SAUR22)显著上调,具有生物活性的赤霉素(GA)合成的相关基因GIBBERELLIN 20-OXIDASE 1(GA20OX1)的表达量提高2倍。【结论】转基因拟南芥早花表型明显,Gh FLP5可能在调控中发挥了双重作用,通过IAA和GA途径促进拟南芥的开花转型。 【Objective】 Cloning a Flowering Promoting Factor 1-like Protein 5 (Gh FLP5) gene, which is a flowering promoter of Upland cotton, and studying its temporal and spatial expression patterns, and analyzing its role in flowering regulation, Lay the foundation. 【Method】 According to the sequence in NCBI database, primers were designed with Oligo7 software and cloned using CC DNA of CCRI50 as a template to obtain Gh FLP5. The physical and chemical properties of the proteins were predicted on the Ex PASy website. At the same time, the FPF proteins of other species were searched in NCBI. Multiple sequence alignment was performed using Clustal X2. The phylogenetic tree was constructed by using MEGA6. CCRI50 and CCRI50 The temporal and spatial expression patterns of Gh FLP5 were analyzed in samples of different growth stages and tissues from Lu Mian 28 (Lu28). A fragment of 1 500 bp upstream of the start codon of Gh FLP5 was retrieved from the genome database and analyzed with Plant CARE Online tools to predict its cis-acting elements, select the relevant hormones on the two-leaf stage cotton seedlings foliar spray to study Gh FLP5 response; construct plant expression vector pBI121-Gh FLP5, transformed Arabidopsis, observed transgenic lines Line phenotype, and q RT-PCR changes in their endogenous gene expression were determined. 【Result】 The open reading frame of Gh FLP5 was 300 bp in length and encoded a molecular weight of 11.4 kD. Three conserved regions of Gh FLP5 were found, closely related to the flowering promoters of soybean, alfalfa and Populus trichocarpa. The spatial expression pattern analysis showed that Gh FLP5 was predominantly expressed in leaves and its expression level in CCRI50 was significantly higher than that in late-maturing variety Lu28. The temporal expression pattern showed that the expression peak appeared in the three-leaf stage in CCRI50 The highest expression of four leaves in Lu28. There are two main cis-acting elements on the promoter of Gh FLP5, one is the light response element and the circadian clock element, the other is the stress response element. According to the distribution and function of cis-acting elements, cotton seedlings were sprayed with SA, ABA and JA. The results showed that Gh FLP5 was up-regulated in response to exogenous SA and ABA, Will be applied JA inhibition. The Arabidopsis thaliana constitutively expressed Arabidopsis thaliana about 9 d ahead of flowering time and about 7 d early flowering time. The number of rosette leaves decreased to a very significant level. Fluorescence quantification results showed that the genes for promoting flowering in transgenic Arabidopsis, LEAFY (At LFY), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (At SOC1), FLOWERING LOCUS T (At FT), APETALA1 (At AP1) and FRUITFULL Significantly increased flowering Locus C (At FLC), a flowering-restricted gene, was significantly reduced. The gene GIBBERELLIN 20-OXIDASE 1 (GA20OX1), which has a significant up-regulation of gibberellin (GA) synthesis in transgenic Arabidopsis, auxin response genes SMALL AUXIN UPREGULATED 20 (At SAUR20) and SMALL AUXIN UPREGULATED22 (At SAUR22) ) Increased the expression of 2 times. 【Conclusion】 The early flowering phenotype of transgenic Arabidopsis thaliana is obvious. Gh FLP5 may play a dual role in the regulation, and promote the flowering transformation of Arabidopsis by IAA and GA.