陆地棉开花相关基因GhFLP5的表达及功能分析

The Expression Patterns and Function Analysis of Gh FLP5, a Gene Related to Flowering in Upland Cotton(Gossypium hirsutum L.)

【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] To provide more information for breeding, the gene Flowering Promoting Factorl-like Protein 5 （GhFLPS）, a member of the flowering promoting factor family in Gossypium hirsutum L., was cloned, and then its expression patterns were studied to characterize its functions in flowering process. [Method] According to the sequences on NCBI, specific primers were designed by Oligo7 and GhFLP5 was cloned from cDNA of cultivar CCRI50. The protein properties were predicted via ExPASy. FPFs of other species were retrieved from NCBI. ClustalX2 was used for multiple sequence alignment and phylogenetic tree was constructed by MEGA6. The samples of different stages and different tissues of early maturity variety CCRI50 and later maturity variety Lu28 were used to study the spatial and temporal expression profiles of GhFLPS. A 1 500 bp fragment upstream the start codon was taken from the genomic database, then the cis-acting elements were analysed with PlantCARE. Based on the predictions, several phytohormones were selected to explore the responses of GhFLP5. pB1121-GhFLP5, a recombinant expression plasmid was constructed and transformed into Arabidopsis. The homozygous overexpression lines were observed and expression profiles were performed with quantitative real-time PCR （qRT-PCR）. [ Result] With a 300 bp coding frame, GhFLP5 encoded a 11.4 kD protein. There were three pretty conserved domains, which reveals that GhFLP5 has a close relationship with those of Glycine max, Medicago truncatula and Polulus trichocarpa. Spatial expression patterns showed that it expressed predominantly in leaves. And GhFLP5 was transcribed at a higher level in early maturity variety CCRI50 than in later maturity variety Lu28. Temporal expression patterns showed that it hit a peak at three-true-leaf stage in CCRI50 but at four-true-leaf stage in Lu28. There were mainly two kinds of cis-acting elements in promoter region： one was light-response elements and circadian elements, and the other was stress-response elements. According to the elements＇ distributions and functions, salicylic acid （SA）, abscisic acid （ABA） and jasmonic acid （JA） were selected to treat cotton seedlings. As a result, GhFLP5 was activated by SA and ABA, while it was suppressed by JA. The overexpression plants bolted about 9 days and flowered about 7 days earlier than the wild type. Meanwhile, the rosette leaves decreased, and all the differences were extremely significant. Quantitative analysis showed that the genes promoting flowering such as LEAFY （AtLFY）, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS （AtSOC1）, FLOWERING LOCUS T （AtFT）, APETALA1 （AtAP1） and FRUITFULL （AtFUL） were up-regulated in 35S：：GhFLP5 lines, while the gene AtFLC, delaying flowering, was down-regulated. Moreover, the auxin-responsive genes SMALL AUXIN UPREGULATED 20 （AtSAUR20） and SMALL AUXIN UPREGULATED 22 （AtSAUR22） were induced in transgenic Arabidopsis lines. GIBBERELLIN 20-OXIDASE 1 （GA2OOX1）, a gene involved in gibberellin （GA） biosynthesis, was also up-regulated more than two folds. [Conclusion] The Arabidopsis thaliana lines over-expressing GhFLP5 performed an obvious early-flowering phenotype. Moreover, the gene expression profiles indicated that GhFLP5 may play dual roles in the transition to flowering via both GA and IAA signaling pathways.