Comparision Of Gene Expression Profiles During Floral Transition And Cloning And Expression Analysis Of BrcLFY In Pak Choi (Brassica Rapa Ssp. Chinensis)

The transition from vegetative growth to reproductive growth in plants is not only relevant to the perpetuation of species, but also related closely to human life. Pak choi (Brassica rapa ssp. chinensis; Brassica campestris ssp. chinensis Makino) originated from China and belongs to the Brassicaceae family, it is one of the more cultivated and consumed vegetables in China and other Asian countries. Pak choi was recently introduced to North America and Europe and became a world wide vegetable. Whole genome sequencing recently completed and the constant enrichment of information on gene annotation in Chinese cabbage will facilitate the research on flowering mechanism in pak choi and other B. rapa plants. Although some studies on flower molecular regulation in Brassica species have been conducted, information is still lacking compared with the flowering molecular mechanism of the model plant A. thaliana. Therefore, further studies on the flowering mechanism of pak choi are very important in breeding B. rapa, as well as other plants within Brassicaceae.Using pak choi as the materials, flower bud differentiation process was identified by using paraffin sectioning and the scanning electron microscopy based on the optimized flowering system. Digital gene expression profiling technology was performed to examine gene expression profile changes in the shoot apex among flower bud differentiation stage 1 and two controls so as to screen the differentially expressed genes (DEGs) and identify the pathways involved in floral transition in pak choi. The contents of protein and soluble sugar in different developmental stages were detected and DEGs of related nitrogen metabolism, sucrose and starch metabolism pathway were analysed during floral transition. BrcLFY gene, controlling transition from inflorescence meristem to floral meristem and flowering time, was isolated from the shoot apex of pak choi variety ’75#, by reverse transcription-polymerase chain reaction (RT-PCR), the sequence and temporal and spatial expression were analysed, which might further improve the understanding of the plant flowering molecular mechanism.The main results were as follows:1. The flowering of pak choi varieties (lines)’75#,and’78#,were compared after the germinated seeds were treated at 4℃ for 10 d,20 d and 30 d respectively. The results showed that leaf number is less and the budding time is earlier with prolonged cold treatment. The treatment time under low temperature for promoting flowering of ’75#,was 20 d, while the’78#,need 30 d or more. Comparing the flowering of ’75#,at different planting density, the results showed that the plants in 50 hole plug flowered earlier than in 72 hole plug. The budding rate with 36.11% of plants cultivated in 50 hole plug at designated time is higher than in the 72 hole plug. So for the flowering research in pak choi, it is more appropriate that’75#,is treated at low temperature for 20 d and planted in 50 hole plug.2. Flower bud differentiation is the morphological mark of the transition from the vegetative growth to reproductive growth. The paraffin section of apex from pak choi were stained only with safranine for 2 h and the structure was clear and the dyeing effect was uniform and stable; while the tissue cells could not be observed clearly after staining with only fast green for any definited time; stability and clarity by staining with both fast green and safraine were less than with only safraine. Therefore, staining for 2 h with safranine was the best method for apex of pak choi. Based on the scanning electron microscopy and paraffin section, flower development process in pak choi was divided into 6 stages:vegetative meristem, vegetative meristem transit to reproductive meristem, early flower primordia, floral differentiation, pedicel elongation, individual flower differentiation.3. Gene expression profile in shoot apex of the three developmental stages were examined using HiSeqTM Illumina 2500 platform in pak choi. A total of 1,486 DEGs during floral transition (between Stagel and CK) were identified, of which 505 were upregulated and 981 were downregulated. Gene ontology enrichment analysis showed that the proteins encoded by DEGs were mainly located in 8 cell regions including the apoplast, plant-type cell wall, and chloroplast etc. They had 8 kinds of molecular function such as transcription factor and oxidoreductase, and involved in 72 biological processes containing jasmonic acid and salicylic acid metabolism, glucose metabolism, and the MAPK cascade so on. Further pathway enrichment analysis showed that 4 metabolic pathways were significantly enriched by DEGs. DEGs that exhibited more than 10-fold difference were analyzed in detail. Results showed that flavonoid metabolism, nitrogen metabolism and phenylpropanoid biosynthesis (or pathways) were significantly enriched and the corresponding gene number was the most. Moreover,15 genes among the highly expressed genes were involved in reproductive development, of which 5 homologues in A. thaliana were directly related to floral transition, which suggested that these genes play some roles in floral transition from vegetative to reprocductive growth.4. 962 DEGs were identified in "Combination of Stage 1 and UVCK", of which 258 were upregulated and 704 were downregulated when the flower bud was differentiated, fold changes were mainly two-five fold. Gene ontology enrichment analysis showed that the proteins encoded by DEGs were mainly located in 7 cell regions including the vacuole, extracellular region, apoplast, plant-type cell wall, cytoplasm and endoplasmic reticulum lumen. They had 5 kinds of molecular function such as iron ion binding and heme binding, and involved in 56 biological processes containing response to sucrose, response to cold, response to high light intensity and heat, oxidation-reduction process so on. Further pathway enrichment analysis showed that 4 metabolic pathways were significantly enriched by DEGs, namely, protein processing in endoplasmic reticulum, nitrogen metabolism, linoleic acid metabolism and flavonoid metabolism (or pathways). In addition,69 DEGs that exhibited more than 10-fold difference were analyzed in detail.7 genes among the highly expressed genes were involved in reproductive development, among which the homologues of Bra034357 and Bra000506 in A. thaliana were directly related to flowering transition. It means that the genes are directly related to flowering in pak choi.5. The contents of protein and soluble sugar in different developmental stages were detected and DEGs of related nitrogen metabolism, sucrose and starch metabolism pathway were analysed during floral transition in pak chio, the results showed that protein, soluble sugar and starch content increased slowly from germinated seeds to the front of the flower bud differentiation, and were significantly increased at stage 1, furthermore constantly increased until stage 3. After stage 3, protein and sugar content began to decline, until bud visible stage then rose again. However, the starch continued to decline from stage 3. The expression of the DEGs in sucrose and starch metabolism pathway was beneficial to the accumulation of sucrose and starch during floral transition, and the result was consistent with the content of soluble starch and sugar.6. BrcLEAFY(BrcLFY), a FLO/LF7 homologue, was cloned from pak choi by RT-PCR, the full cDNA of BrcLFY was 1,353 bp in length with an open reading frame of 1,260 bp encoding 419 amino acids. Sequence analysis showed the BrcLFY was more closely related to the LFY-like proteins from cruciferous vegetables, especially Brassica vegetables. Quantitative real-time PCR results showed that BrcLFT expression gradually increased during the vegetative growth stage and increased sharply at stage 1, with the highest expression level at flower differentiation stage 5. When comparing BrcLFY expression in different organs, we concluded that the level in young and mature leaves was significantly higher than in the apex and other tissues at flower differentiation stage 3 and visible flower bud stage and fully-bloom stage. The BrcLFY expression in germinated seeds and seedlings with five leaves was analyzed during cold treatment for 0 d,10 d and 20 d at 4℃, respectively, and the results showed that BrcLFY gene expression rose gradually with prolonged low-temperature. These result indicated that high level expression of BrcLFY promote flower differentiation in pak choi.