【作者】 王小艳；

【导师】 喻树迅；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2014， 博士

【摘要】 棉花是我国重要的经济作物，在国民经济和社会发展中占有重要地位，但是我国的基本国情是人多地少，粮棉矛盾突出。所以短季棉的选育与推广是解决我国粮棉争地、保证粮食安全的重要途径。开花促进因子基因FLOWERING PROMOTING FACTOR1(FPF1)于1997年从拟南芥中克隆出来，经光周期诱导后，它在茎尖分生组织中的表达量迅速上调，对拟南芥开花时间起到正调控的作用，后来在其他作物如烟草中也证实其促进开花这一功能，但对于其调控开花时间的信号通路，目前还不清楚。本研究利用已经测序完成的雷蒙德氏棉和亚洲棉数据库为基础，在陆地棉中获得了FPF1基因的同源序列，对它们的表达模式进行分析筛选出候选基因，并对其功能进行了更加深入的研究，具体结果如下：1、以拟南芥中FPF1基因为参考序列在二倍体雷蒙德氏棉和亚洲棉数据库中分别比对得到六条蛋白质相似度比较高的序列。将这些序列于核酸和氨基酸水平在两个二倍体棉种间进行对比分析发现：六个基因的核酸序列均有不同程度的差异，相似度在84%-99%之间；有两个基因的氨基酸序列在两个物种中是完全相同的；其中五个基因含有较高的同义突变，这可能是由于不同物种分子水平上进化的差异性以及保守型决定的。2、从陆地棉中棉所36中克隆到FPF1的同源基因六条，分别命名为GhFPF1，GhFLP1，GhFLP2，GhFLP3，GhFLP4和GhFLP5，发现该基因家族基因片段均比较小，且不含内含子。研究各基因在陆地棉各组织器官中的表达模式发现，GhFPF1基因家族成员呈现出明显的表达特异性，主要在根和茎尖中表达。利用早熟棉中棉所36和遗传标准系TM-1筛选出候选基因GhFPF1基因，暗示其可能参与短季棉开花时间的调控。3、构建了融合表达载体pBI121-GFP-GhFPF1，采用基因枪轰击洋葱表皮的研究发现GhFPF1蛋白在细胞膜和核内均有表达。进一步通过5’-RACE及3’-RACE策略，扩增出了GhFPF1的全长转录本为701bp，包含56bp5’-UTR，315bp3’-UTR和330bp ORF区域。分析启动子区域发现其主要包含两大类顺式元件，光反应元件和植物胁迫相关的一些元件。外源激素处理试验表明GhFPF1能够响应SA和JA的处理，暗示GhFPF1可能参与植物的防御反应。4、将GhFPF1基因构建植物过表达载体分别转化拟南芥和棉花，分别获得了7个转基因拟南芥株系和9个转基因棉花株系。结果证实转基因拟南芥各株系的开花时间平均比野生型拟南芥提前5.4天，莲座叶和茎生叶的数目也减少。通过对比分析拟南芥内源开花时间相关基因在野生型和转基因中的表达量变化，推测GhFPF1在拟南芥中过表达促进开花很可能依赖于AtAP1和AtFLC。5、最新研究发现GhFPF1基因除了具有调节植物生育期、开花时间、植物叶数之外，转基因拟南芥与野生型相比，下胚轴伸长，叶柄加长，叶绿素的含量降低，AtPHYB基因的表达量在转基因拟南芥中也明显降低，我们推论GhFPF1基因很可能参与调控植物的“避荫”反应。6、将该基因采用农杆菌介导的方法过表达转化棉花，通过基因组和mRNA水平的检测共获得了9个T0代转基因株系，目前个别株系已收获T2种子，要想揭开GhFPF1在调控植物开花和“避荫”反应过程中所涉及的信号通路，还需进一步研究。更多还原

【Abstract】 Cotton (Gossypium spp.), one of the most important natural fiber crops occupies animportant position in national economy and social development in China. Because of thecontradiction between grain and cotton, more people and less land, the earliness of cotton hasbecome a vital subject for plant breeders to solve the problem and ensure the food security.FLOWERING PROMOTING FACTOR1(FPF1) gene was originally cloned fromArabidopsis and understood on account of its role in flowering. FPF1was expressed in apicalmeristems immediately after the photoperiodic induction of flowering in long-day plants thatcould flower in response to long days. Up till now, homologous genes of FPF1have beencharacterized to regulate flowering positively tobacco (Nicotiana tabacum) but the molecularmechanism was still unclear.Databases of G. raimondii L. genome and G. arboreum L. genome were screened toidentify and compare homologs of AtFPF1from the two diploid cotton species. The geneswere cloned and characterized from G. hirsutum L. as one of the major cultivated species. Toselect excellent candidate gene, their expression pattern in cotton were compared. In addition,more deep research work was conducted on its function. The specific results were as follows:1. Twelve FPF1homologs were identified from the diploid cotton genomic databases ofG. raimondii L. and G. arboretum L with the coding region of Arabidopsis FPF1gene as thereference sequence. Orthologous sequences from the two cotton species were compared witheach other, suggesting that nucleic acid sequences of the six pairs of orthologs were distinct(similarities,84%-99%), though two pairs of orthologous genes possessed the same deducedprotein sequence as a result of codon degeneracy. High constraints of genetic divergencemight occur during speciation for five genes had higher synonymous changes between thetwo species.2. Six genes were identified from CCRI36and named as GhFPF1, GhFLP-1, GhFLP-2,GhFLP-3, GhFLP-4, and GhFLP-5. Until now, as were characterized in several species,members of this gene family were short in length as well as lacking in intron of their genomicsequences. GhFPF1gene family displayed tissue-specific expression because abundant transcripts of the six genes were found in roots and floral apices, but were barely detectable inleaves or fibers. More importantly, we focused on the contrastive analysis of gene expressionin floral apices of CCRI36(a short-season cotton variety) and TM-1(a genetic standard line).Results uncovered that GhFPF1had more than four-fold transcript levels in CCRI36than inTM-1. Higher expression of GhFPF1in the short-season cotton suggested that it was the mostpossible FPF1orthologous gene as AtFPF1involved in the promotion of flowering.3. The fusion expression vector pBI121-GFP-GhFPF1was introduced into onionepidermal cells by the particle bombardment method. The onion epidermal cells carryingrecombinant plasmid emitted fluorescence throughout the entire cytoplasm and the nucleus. A5’-and3’-RACE strategy was performed to gain transcription initiation and termination sitesof GhFPF1. A full-length cDNA of701bp composed of56bp5’-UTR,315bp3’-UTR and330bp ORF was isolated. Cis-acting regulatory elements involved in light, defense and stressresponsiveness were found in the promoter of GhFPF1. The hormone treatment assayindicated that GhFPF1could respond to SA and JA, suggesting that GhFPF1might beinvolved in the defense responses of plants.4. After transforming GhFPF1into Arabidopsis and cotton, seven and nine transgeniclines were obtained, respectively. Results indicated that transgenic Arabidopsis flowered5.4days earlier with fewer rosette and cauline leaves than the wild-type. Contrast analysis ofArabidopsis thaliana endogenous genes related to flowering time in wild-type and transgeniclines suggested that the early flowering conferred by GhFPF1over-expression in Arabidopsiswas possibly mediated through AtAP1and AtFLC.5. Except accelerated flowering and fewer leaves, compared with wild-type, transgenicArabidopsis generated longer hypocotyl and petioles, lower content of chlorophyll. Moreover,for the first time we found that expression of PHYB in transgenic plants was decreased bymore than fifty percent. Taken together, transgenic plants were recognized as so-calledshadow-avoidance syndrome (SAS) which was also a new discovery for GhFPF1involved inthe regulation of plant growth and development.6. In addition, GhFPF1was introduced into cotton through Agrobacterium tumefaciensmediated transformation, nine transgenic lines were obtained after checking the DNA andmRNA. T2generation of genetically modified cotton was in the stage of seedling. To uncoverthe signaling pathways of GhFPF1involved in regulation of plant growth and developmentprocess, further work still needs to be done.更多还原