The Functional Analysis And Regulatory Mechanism Of Flowering Related Genes In Upland Cotton

Cotton is an important economical crop. Upland cotton, owning wide plant areas, is the most cultivated species in China. Because of conflicts in land use involving grain vs. cotton cultivation, early maturation has become a key trait in short-season cotton breeding programs. Previous research showed that flowering had closely related with early maturity. Meanwhile, FT, SOC1 and SPL genes intergrate the signal of flower induction and promote the expression of floral organ identity genes. Thus, these genes played important roles in flowering regulation. FT and SOC1/SPL acts as florigen and flowering integrating factors, respectively. In this study, two works were carried out. One was the expression and functional analysis of flowering related genes in upland cotton, the other was the regulation of these flowering genes. The mainly results were as follows.1. In view of recent evidence that genes in the phosphatidylethanolamine-binding protein(PEBP) family play an important role in generating mobile flowering signals, we isolated the PEBP gene family in cotton and examined the regulation and function of parts of its members. In this study, nine PEBP genes were cloned from upland cotton and found to be clustered with four subclades(FT, MFT, TFL1 and PEBP)Tissue expression showed that function of GhFT, GhPEBPs and GhTFL1 s may focus on the leaf development and signal transduncion in the vegetative stages. While MFT could play function in the root development and seed germination. Cotton PEBP genes show distinctly different expression patterns with respect to different upland cotton materials, photoperiod responsive and different maturing cotton varieties. We also showed that GhFT but not GhPEBP2 could promote flowering under both long day and short day conditions.2. 30 SPL genes were identified in the reference genome of Gossypium raimondii and 24 GhSPLs were cloned from Gossypium hirsutum. G. raimondiiis regarded as the putative contributor of the D-subgenome of G. hirsutum. Comparative analysis demonstrated sequence conservation between GhSPLs and other plant species. GhSPL genes could be classified into eight subclades based on phylogenetic analysis, diverse intron-exon structure, and motif prediction. Within each subclade, genes shared a similar structure. Sequence and experimental analysis predicted that 18 GhSPL genes are putative targets of Ghmi R156. Additionally, tissue-specific expression analysis of GhSPL genesshowed that their spatiotemporal expression patterns during development progressed differently, with most genes having hightranscript levels in leaves, stems, and flowers. Finally, overexpression of GhSPL3 and GhSPL18 in Arabidopsis plantsdemonstrated that these two genes are involved in the development of leaves and second shoots and play an integral role in promoting flowering.3. Two MADS-box genes GhSOC1 and GhMADS42(Gossypium hirsutum L.) were cloned from upland cotton CCRI36 and transformed into Arabidopsis. GhSOC1 was additionally transformed into uplandcotton. Comparative analysis demonstrated sequence conservation between GhSOC1 and GhMADS42 and genes of other plant species. Tissue-specific expression analysis of GhSOC1 and GhMADS42 revealed spatiotemporal expression patterns involving high transcript levels in leaves, shoot apicals, and flowers. In addition, overexpression of both GhSOC1 and GhMADS42 in Arabidopsis accelerated flowering, with GhMADS42 transgenic plants showing abnormal floral organ phenotypes. Overexpression of GhSOC1 in upland cotton also produced variations in floral organs.4. The transcriptional regulation study of GhSOC1. We identified and cloned the promoter sequence of GhFT, Gh MADS41/42, GhSPL3 and GhSPL18 according to the genome information. Sequence analysis revealed that the five genes contain the typical CArG box motif, which is the binding site of MADS-box GhSOC1. We verified the result using chromatin immunoprecipitation assay, and the monoantibody of GhSOC1 prepared in our lab. The result demonstrated that the flowering integrator GhSOC1 may bind to the promoter of GhSPL3 and GhMADS41 and GhMADS42 but not GhSPL18 and GhFT to regulate flowering.5. We cloned MADS-box factors AP1/FUL/SVP-like proteins and also GhFD.Yeast two-hybrid and bimolecular fluorescence complementation approach were undertaken to better understand it. These experiments showed that GhSOC1 can interact with AP1/FUL-like proteinsGhMADS41, GhMADS42, GhMADS43 and GhMADS44 in cotton.