| Rapeseed belongs to Cruciferae and is a important oil crop. The main purpose of growing rapeseed is to harvest its seed. The inflorescence growth of rapeseed that links its pod number directly affects its seed production. In addition, the rapeseed flowering time has a great relationship with maturity time. Early maturity of rapeseed is advantageous to the popularization of rice-rice-rapeseed planting system in the south of china. TFL1 is a main gene that controls inflorescence growth and flowering time of Arabidopsis thaliana, BnTFL1 of Brassica napus is a homologous gene of TFL1. In this experiment, we cloned BnTFL1 genes, analyses their expression, and studied their functions.The shoot regeneration culture of rapeseed usually takes two-step regeneration method. In the process of two-step regeneration, explants of cotyledon or hypocotyl cutted cotyledonary node were cultured in the pre-culture medium to induce callus, then were transferred in differentiation medium to regenerate shoot. In terms of two-step regeneration culture, the operation is complicated and the period is long. The rapeseed transformation based on two-step regeneration had complicated process and long period. Innovatively took cotyledon or hypocotyl with meristem of cotyledonary node as explants, we established one-step regeneration culture system that explants were cultured only in differentiation medium to induce meristem cells of cotyledonary node to regenerate shoot. Compared with two-step regeneration culture, the one-step regeneration culture has simple, rapid and high frequency characteristics. Based on the one-step regeneration culture, we established rapeseed transformation system with merits of simplified transformation process and shortened transformation period significantly.The main research results are mentioned as follow:1.We isolated five TFL1 homologous genes BnTFL1-1, BnTFL1-2, BnTFL1-3, BnTFL1-4, BnTFL1-5 and their corresponding cDNA genes BnTFL1-1c, BnTFL1-2c, BnTFL1-3c, BnTFL1-4c, BnTFL1-5c from rapeseed. Bioinformatics analysis reveals that proteins encoded by BnTFL1-1c, BnTFL1-2c, BnTFL1-3c, BnTFL1-4c, BnTFL1-5c are hydrophilic and non-transmembrane, have a conserved PEBP domain, and locates in cytoplasm. A phylogenetic analysis suggests that the five BnTFL1 genes are all close in homology to TFL1 genes from other plants. Among the genetic relationship of the five BnTFL1 genes with TFL1 from Arabidopsis, the closest are BnTFL1-4 and BnTFL1-5, then is BnTFL1-2, the farthest are BnTFL1-1 and BnTFL1-3. This result indicates that the evolutions of BnTFL1-1 and BnTFL1-3 are highest, the evolution of BnTFLl-2 is middle, the evolutions of BnTFL1-4 and BnTFL1-5 are least.2. We studied the expression of the five BnTFL1 genes in diverse issue/organ and developmental stage, and found that BnTFL1 genes mainly transcripted in stem tip, flower and bud. BnTFL1 genes had strong expression in the stem tip of flower bud differentiation stage, had medium expression in the stem tip of vegetative growth stage and young bud of primary bolting stage, and had weak expression in the flower and bud of bloom stage. In the same tissue or organ, BnTFL1-4 and BnTFL1-5 transcripted strongly, BnTFL1-1, BnTFL1-2 and BnTFL1-3 transcripted weakly.3. We isolated promoters of BnTFL1-1 and BnTFLl-3 that were named BnTFL1-1-promoter and BnTFL1-3-promoter respectively. Using the online promoter prediction tool of PlantCARE to analysis BnTFL1-1-promoter and BnTFL1-3-promoter, the results showed that the two promoters contained many light reaction elements, which indicated that BnTFL1-1-promoter and BnTFL1-3-promoter belonged to light inducible promoters and participated in flowering regulation induced by light signal. In addition, BnTFL1-1-promoter and BnTFL1-3-promoter also contained other specific expression elements, such as endosperm expression elements and stress-induced elements, it indicated that BnTFL1-1-promoter and BnTFL1-3-promoter probably also related to endosperm development, disease and stress resistance.4. The BnTFL1-1c transgenic plants of Arabidopsis thaliana were achieved. Compared with wild plant, the T1 transgenic plants bolted, flowered and matured later obviously, the height of the T1 transgenic plants were significantly higher, the branch and pod numbers of the T1 transgenic plants were significantly less, but the pod numbers of the main branch of the T1 transgeic plants had less difference. It indicated that the main functions of BnTFL1-1 are to delay flowering and mature, promote vegetative and inflorescence growth, inhibit producing branch. The main function of BnTFL1-1 and TFL1 are basically same, the difference is that BnTFLl-1 inhibits producing branch, and TFL1 promotes producing branch.5. Five BnTFL1 RNAi transgenic T0 rapeseed plants named TY1, TY2, TY3, TY4 and TY5 were achieved. Compared with wild plant, TY1, TY2, TY3 and TY4 bolted, flowered and matured earlier obviously, the plant height of TY1, TY2, TY3 and TY4 were significantly shorter, the branch numbers of TY1, TY2, TY3 and TY4 were significantly more. It indicated that the main function of BnTFL1-1 is to delay flowering and mature, promote vegetative and inflorescence growth, inhibit producing branch. The function analysis of BnTFL1 in rapeseed and BnTFL1-1c in Arabidopsis are same.6. Using BnTFL1 RNAi technology, we got transgenic rapeseed plants that flowered and matured earlier than wild plant. It opens up a new way for rapeseed genetic improvement and quickly getting early-maturing germplasm through biotechnology.7. We established a one-step shoot regeneration system using hypocotyl with meristem of cotyledonary node as explant. The content of the system is that hypocotyls with meristem of cotyledonary node from five days seedlings of Zhongshuang 11 were cultured in the medium MS+4.0mg/L 6-BA+30g/L sucrose+2.6g/L phytagel. The shoot regenerated from the explants five days later, one explant regenerated 8.0 shoots on average and the regeneration frequency is 100%. Based on the one-step shoot regeneration system, a transformation system was established and a BnTFL1 RNAi vector was transformed into Zhongshuang 11 successfully. In the whole transformation, the period from seeding to getting rooted resistant seedlings spanned about 70 days. Compared to 130 days of traditional transformation period, this new transformation method obviously shortened transformation period, simplified transformation process and improved transformation efficiency.8. We established a one-step shoot regeneration system using cotyledon with meristem of cotyledonary node as explant. The content of the system is that cotyledons with meristem of cotyledonary node from five days seedlings of Zhongshuang 11 were cultured in the medium MS+3.0mg/L 6-BA+30g/L sucrose+2.6g/L phytagel. The shoot regenerated from the explants five days later, one explant regenerated 5.5 shoots on average and the regeneration frequency is 100%. Based on the one-step shoot regeneration system, a transformation system was established and a BnTFL1 RNAi vector was transformed into Zhongshuang 11 successfully. In the whole transformation, the period from seeding to getting rooted resistant seedlings spanned about 70 days. This new transformation method obviously shortened transformation period, simplified transformation process and improved transformation efficiency. |
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