| Chinese cabbage (Brassica rapa subsp. pekinensis) has different cold sensitivities in different varieties. Early bolting of weaker winterness varieties becomes a limiting factor affecting income. On the other hand, producing seeds is difficulty for strong winterness varieties. Therefore, research on the flowering mechanisms in Chinese cabbage will have important scientific and practical value. Although some studies on flowering molecular regulation in Chinese cabbage have been conducted, the information is still lacking compared to the flowering molecular mechanism of Arabidopsis. Completion of the whole genome sequencing and continuous improvement of gene annotation in Chinese cabbage, and the extensive application of high-throughput sequencing technology have provided a new method for the study of flowering mechanism in Chinese cabbage.In this study, the experiment established a optimum flowering system for Chinese cabbage. The high-throughput sequencing technology was used to sequence the shoot apex that was without low temperature treatment (CK1), immediately flower bud differentiation (V1) and flower bud differentiation stage 1 (V2) after low temperature treatment to screen the genes related to flowering. LFY homologue was cloned and its alternative splicing and expression pattern were analyzed. In addition, the content change of auxin (IAA), jasmonic acid (JA), gibberellin (GA1+3), cytokinin (Z+ZR) and abscisic acid (ABA) during the various developmental stages of plant were analyzed, and the roles of these endogenous hormones in the flowering process were clarified, so as to further improve the molecular mechanism of flowering regulation in Chinese cabbage. The main results were as follows:1. The experiment compared the state of floral bud emergence and blossom in 4 Chinese cabbage lines'3#','7#','8#'and'9#'after low temperature treatment for 10 d,20 d and 30 d, the results showed that the time needed for initiation period of flower bud differentiation and appearing, blossom were rduced with the extension of treatment time, and the number of leaves during the stage of flower bud differentiation and appearing were reduced too. The winterness difference of these lines was significant, the winterness of'9#'was the strongest, followed by'8#','3#'and'7#'were the weakest. Comprehensive analyzed the results,'3#'and '7#'could be used for the research of mechanisms underlying the floral development, and the treatment time under low temperature for promoting flowering was 20 d, while'9#'could be used for a comparative research, however the treatment time was 30 d at least.2. The high-throughput sequencing technology was used to sequence the shoot apex of CK1, VI and V2. The results showed that there were 1680 differentially expressed genes (DEGs) in CK1_vs_V1, including 780 up-regulated genes and 900 down-regulated genes. The function of DEGs in CK1_vs_V1 were analyzed using GO functional classification annotation, the results showed that the enriched terms were mainly focused on chloroplast fractions in the annotation of cellular component, on some oxidoreductase activity in the annotation of molecular function, and on some factors related to flowering in the annotation of biological process, such as response to blue light, far red light and sucrose, salicylic acid and jasmonic acid mediated signaling pathway, MAPK cascade etc. The KEGG pathway enrichment analysis results showed that flavonoid and glucosinolate were important to flowering in Chinese cabbage.3. The flower genes published in BRAD were compared with gene expression profiles, the results showed that there were 10 DEGs. The expression of most genes were consistent with the prediction, which suggested that the sequencing results were reliable. There were 4 high expression change DEGs whose variation trend were consistent with the function, they were promoting flowering gene Bra000180, Bra009813 and inhibiting flowering gene Bra024350, Bra026653. These results suggested that these 4 genes may participate in the process of flowering in Chinese cabbage, so they should be subjected to further analysis.4. Two homologues of LFY were cloned and the ORF was 1,248 bp and 1260 bp respectively. Named the two genes as BrpLFYl and BrpLFY2, and uploaded sequence to NCBI, GenBank accession number were KR190435 and KR190436. Alternative splicing analysis revealed that these 2 genes were different alternative splicing variants of BrpLFY. The reason for the difference of 12 bp was because of alternative 5'exon ends, and one of which retained the first intron sequence of 12 bp.PCR analysis revealed that there almost only the expression of BrpLFY2 in all tissues during different development stages of Chinese cabbage in different lines. This result was consistent with the expression in alternative splicing analysis and suggested that BrpLFY2 may play a major role in the process of flowering in Chinese cabbage.5. Real-time PCR analysis results showed that BrpLFY mRNA was detected in all tissues. BrpLFY expression in the shoot apex increased gradually during vegetative growth and increased dramatically at stage 1 of flower bud differentiation. The relative expression peaked at stage 5 and then decreased in later stages. Moreover, the trend in BrpLFY expression level change in the shoot apex was similar regardless in spring or autumn plants with winter storage. But the peak of expression was higher in the strong winterness varieties, it suggested that the strong winterness varieties may need higher BrpLFY expression to induce flowering.The experiment compared BrpLFY expression in different tissues, the results showed that at stage 5, the BrpZFY expression was highest in the young leaves, somewhat less in the mature leaves and shoot apex, and lowest in the roots. In addition, the expression in flower buds and flowers were less. BrpLFY expression were higher in young and mature leaves than other tissues during flower bud differentiation, it suggested that BrpLFY may function in the growth and maturation of leaves.6. The endogenous hormones IAA, JA, GA1+3, Z+ZR and ABA in shoot apex during various developmental stages of plant in low temperature treatment group and control group of Chinese cabbage lines '7#' were determined by ELISA. The results in low temperature treatment group indicated that the higher content of IAA, JA and GA1+3 were beneficial to flowering transition within a certain range, while bolting and floral bud emergence required a higher content of IAA, JA and Z+ZR. The hormone levels were relatively low in stage 3, which suggested that there may be a short pause during the differentiation process. In addition, the main genes which resulted in the content change of IAA, JA, Z+ZR and ABA in the process of flowering were screened by KEGG pathway analysis.Compared to group CK, the higher GA1+3/IAA, Z+ZR/IAA, ABA/IAA, GA1+3/Z+ZR, ABA/GA1+3 and ABA/Z+ZR were required for the whole process of flowering in Chinese cabbage. In low temperature treatment group, the higher ABA/IAA, GA1+3/IAA, Z+ZR/IAA, JA/LAA, JA/GA1+3, JA/Z+ZR and JA/ABA were beneficial to flowering transition within a certain range, while the differentiation process needed lower JA/IAA, JA/GA1+3, JA/Z+ZR and JA/ABA. In addition, the lower GA1+3/IAA, ABA/IAA, GA1+3/Z+ZR, ABA/Z+ZR and higher ABA/GA1+3, JA/GA1+3, JA/ABA were required for bolting and floral bud emergence. Moreover, it was inferred that the ratio of ABA/IAA and GA1+3/Z+ZR was 1 may be the key value of flower bud differentiation in Chinese cabbage, and higher than this value was beneficial to flowering. |
PDF Full Text Request