Functional Analysis Of Flowering Regulation Through FT-likes In Chrysanthemum

Chrysanthemum morifolium is one of the world’s top four cut flowers and one of Chinese ten traditional flowers, it occupies high ornamental value and owns a very important position in the international flower trade. According to the flowering season, chrysanthemum is divided into summer chrysanthemum, autumn chrysanthemum and winter chrysanthemum. According to the response to different photoperiod, chrysanthemums can be divided into short-day chrysanthemum and day neutral chrysanthemum. Chrysanthemum owns variety spieces, however, their flowering time are relatively concentrated at some timepoints. Day neutral chrysanthemum was flowering under mostly long-day summer; short day flowering chrysanthemums are concentrated in autumn or winter. Therefore, research of blossom mechanism from the perspective of molecular level of chrysanthemum will be conducive to find out the differences which exist between day-neutral and short-day chrysanthemum. Elaborate chrysanthemum flowering mechanism from different aspects, will provide a strong theoretical basis to improve flowering regulation and production. The main contents and conclusions of this study are as follows:1. The FT gene which is called ’Florigen’ plays an important role in the process of plant flowering.In order to study chrysanthemum flowering mechanism between different varieties and their different mechnism, we used ’Jinba’ (SD chrysanthemum) and ’Yuuka’ (day-neutral chrysanthemum) as the materials, CmFT-like1(CmFTL1), CmFT-like 2 (CmFTL2) and CmFT-like 3 (CmFTL3) were cloned. Through the analysis, we found that the three genes were coexist in’Jinba’and’Yuuka’. The length of CmFTL1, CmFTL2 and CmFTL3 gene were all 525 bp, encoding 174 amino acids. Through FT sequence homology comparison with that from Arabidopsis thaliana, sugar beet, rice and lettuce, we found CmFTL1, CmFTL2 and CmFTL3 were a kind of relatively conservative sequence. Through the system evolution analysis indicated that all of CmFTLl,2,3 fall in FT subfamily of PEBP family.Through qRT-PCR, a time and space expression model of CmFTLl, CmFTL2 and CmFTL3 in chrysanthemum’Jinba’and’Yuuca’was built. We found there was a significant difference of CmFTL1 expression between the day-neutral and the typical short day chrysanthemum cultivars. CmFTL1 was induced under night break condition, but was almost wholely suppressed under short day condition. Expression peak of CmFTL2 appeared both in’Jinba’and’Yuuka’flower bud differentiation stage. Under long day condition, the expression of CmFTL3 in flower bud differentiation stage is far lower than that of CmFTL1 and CmFTL2, but significant higher under short day condition.2. The functional analysis of CmFTL1 was carried on. The subcellular localization results of CmFTLl indicated that its protein products are widely distributed in the whole cells. Through function complementary of Arabidopsis mutant ft-10, CmFTL1 has the function of recovering late flower phenotype of the plant. The OX-CmFTL1 transgenic chrysanthemum flowered earlier, and CmFTL1-amiRNA transgenic chrysanthemum flowered later comparing with wild type plants, which suggested CmFTLl was an very important florigen in day-neutral chrysanthemum’Yuuka’. Four kinds of FT alternative splicing trancripts, CmFTL1astE134 (without a second exons), CmFTLl1astIn1-1, CmFTL1astIn1-2 and CmFTL1astIn1-3(retained first introns), were found during the process of CmFTL1 cloning pMDC43-CmFTL1astE134, pMDC43-CmFTL1astIn1-1, pMDC43-CmFTLlastIn1-2 and pMDC43-CmFTLlastIn1-3 four expression vectors were constructed, respectively. Through genetic transformation of Arabidopsis ft-10, it was found that CmFTLlastIn1-1 and CmFTLlastIn1-3 could recover the late flower phenotype. Through transgenic confirmation at cDNA level, CmFTLl and CmFTL1ast were coexist, indicating a posttranscriptional modification of chrysanthemum CmFTLl gene.3. The functional analysis of CmFTL2 was carried on. CmFTL2 was involved in floral bud differentiation process under both LD and SD condition. CmFTL2 also could completely recover the phenotype of Arabidopsis ft-10 mutation. After spraying 100 mM sucrose to’Jinba’, the expression of CmFTL2 was found highly induced, the results showed that CmFTL2 was a bridge between sucrose, photoperiod and flowering.4. The functional analysis of CmFTL3 was carried on. Through the subcellular localization and function complementary through Arabidopsis ft-10, CmFTL3 wad found to be a flowering hormone. In the cloning process of CmFTL3, CmFTLpsl, CmFTLps2, CmFTLps3, CmFTLps4 were found. Their sequences are highly similar, differences only exist among 18 amino acid sites. By construction of expression vector, we used AtFT promoter to drive the expression of CmFTLps1,2,3,4. Through function complementary of Arabidopsis ft-10 mutants, the transgenic plants of CmFTLpsl, CmFTLps2, CmFTLps3 were found to be late flowering recoverable, however, CmFTLps4 cannot.4 amino acid sites were replaced in CmFTLps4 comparing with CmFTL3, they might contribute crucial roles to FT function. Through application of Clontech kit, one cDNA yeast library of SAM from ’Yuuka’ was built. Use pGBKT7-CmFTL3 as bait plasmid, five protein potentially interact with CmFTL3 were found by yeast two hybrid experiments, function prediction shows that the five protein may be interact with CmFTL3 during transportation and transfer in cell.5. The RNA-Seq platform was used to characterize the high-temperature stress response of Chrysanthemum nankingense. After a threshold of ratio change>2 and a q-value of<0.05 were applied, the number of differentially transcribed genes was reduced to 3,955 response to the heat stress. Among the most differentially expressed genes between the two libraries were well-recognized high-temperature responsive protein families, such as heat shock factors and heat shock proteins, various transcription factor families, and a number of RNA metabolism-related genes. Seven sucrose metabolism-related genes were found associated with the impact of flowering under high temperature stress and were all suppressed. The promoter of heat shock protein gene CnHsp 17.4, which might be heat specific response, was cloned, may be used in efforts to applications of chrysanthemum flowering regulation.