Expression Analyses Of BCHSP81-4Gene In Polcms Of Non-Heading Chinese Cabbage And Functional Verification Of BCFLC

Cytoplasmic male sterile (CMS) lines do not only play a major role in cross breeding of crops, but also they are taken as ideal materials for studying pollen development, cytoplasmic inheritance, nucleo-cytoplasmic interaction and time-dependent expression. Accordingly, studies on the molecular mechanism of cytoplamic male sterile are very significant. Non-heading Chinese cabbage(Brassica campestris ssp. chinensis Makino) which originated from China, is one of the most widely cultivated vegetables in China, especially in the south of China. In recent years, with the development of molecular biotechnology, studies about cytoplasmic male sterility increased gradually. However, there is little kownledge about gene recombination and its expression. The buds of newly bred Pol CMS of non-heading Chinese cabbage and its maintainer line as plant materials, genes differentially expressed transcripts were analyzed by molecular biology. Therefore, this study laid a good foundation on revealing the molecular mechanism of CMS line. Meanwhile, earlier bolting reduces the yield and quality of the products in non-heading chinese cabbage. The introduction of late bolting gene FLC could avoid this phenomenon. but FLC has a passive effect on plant fertility. Therefore, studying the relations of bolting and plant fertility has a great significance.The main results are as follows:1-Suppression subtractive library was constructed using flowers of Pol cytoplasmic male sterile lines of Non-heading Chinese cabbageUsing suppression subtractive hybridization, the suppression subtractive cDNA library was builded, Pol cytoplasmic male sterile lines of Non-heading Chinese cabbage as the tester, and maintain as the driver. Colony were detected by PCR, the effective recombination rate is91%, and the size of inserts mainly between100bp and1000bp. Through dot blot screening,32positive clones were sequenced. Homology comparison analysis showed that15clones were unknown function, and the rest related to multiple aspects of the hormonal signal transduction, such as second messenger, photosynthesis, fatty acid metabolism and aging.2. Cloning and expression analysis of BcHSP81-4gene in the PolCMS of Non-heading Chinese CabbageBcHSP81-4gene, a member of heat shock proteins, was identified from a suppression subtractive hybridization cDNA library in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino). The deduced amino acid sequence of the BcHSP81-4cDNA revealed that it has high homology to other plant organelle isoforms and similar homology to both cytoplasmic and prokaryotic HSP90s. To study the regulation of gene expression, BcHSP81-4genes in maintainer and sterility lines were monitored at different development stages and at different stress treatments. Real-time PCR was used for quantification of BcHSP81-4mRNA. These results indicate that BcHSP81-4is not responsive to heat shock at least at35℃, while it is very responsive to salt and cold stress. And high expression of BcHSP81-4in the bud of sterile line suggests that it may play prominent roles in sterility of pol CMS in non-heading Chinese cabbage. Then the ORF had been cloned into PGEX-4T-3vector and transformed into the host BL21(DE3). Results of SDS-PAGE showed that the specific fusion protein was successfully induced to express by IPTG, and further proved that the gene belongs to heat shock protein90family。The heat shock protein BcHSP81-4gene of Brassica campestris ssp. Chinensis was analyzed by using a series of online software in the following aspects:physical and chemical characters, hydrophobicity, transmembrane topologieal structure, subcellular localization, and structure of the protein. The results indicated that the protein contains699amino acid residues. Its molecular weight is80.05KD, and the theoretical pI is4.95. TMpred analysis shows that it contained one distinguished transmembrane helice which correspond with the hydrophobic region that predicted by ProtScale. It is located in cytoplasm by Target P server. Structure analysis shows that a-helix and random coil are the main secondary structures of this protein.3. Fertility and Cold-resistance were affected in Arabidopsis by the overexpression of BcFLC isolated from Non-heading Chinese cabbageFLOWERING LOCUS C (FLC) in Arabidopsis encodes a dosage dependent repressor of flowering. In this paper, we isolated a FLC-related sequence from Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino). Constitutive expression of the BcFLC genes in Arabidopsis significantly delayed flowering by10days, confirming the requirement of this gene for floral repression. In35S::BcFLC overexpression line, the level of SOC1mRNA expression has decreased, but LEAFY (LFY) and FLOWERING LOCUS T (FT) expression in35S::BcFLC were significantly higher than that in wild-type when flowering. The analysis revealed that the35S::BcFLC overexpression line in Arabidopsis requires a greater dose of LFY and FT to flower than the wild type. Here, we reported that the reduced fertility in Arabidopsis by the overexpression of BcFLC may be related with the enhanced expression of anther filaments suppressor, RGA and RGL gene, and slightly declined expression of SEPALLATA3(SEP3) gene, encoding a MADS-box transcription factor involved flower and ovule development. We also found that, CBF1,2,3and COR15a expression were gradually increased in35S::BcFLC overexpression line, suggesting that increased BcFLC expression could activate the expression of CBF1,2,3and COR15a, and then improving the cold resistance.4. Exogenous Nitrate or NaCl regulates floral induction in Arabidopsis thalianaFlowering, the transition from the vegetative to reproductive phase in plants, is regulated by both endogenous and environmental signals. Exposure to an extended period of stress (such as low nitrate or NaCl) can also promote flowering in many species, but little is known about how these forms of stress regulate floral induction. We found that stress induced by low concentrations of nitrate or NaCl activated the biosynthesis of gibberellin (GA) as evidenced by increased expression of the GA biosynthetic enzyme GA1. Expression of CO and SOC1were also enhanced, leading to an acceleration of flowering. The effects of nitrate and NaCl on the photoperiod pathway were distinct, however. Two genes related to the photoperiod pathway, CCA1and LHY, were repressed only under low NaCl treatment, while expression was unaltered by nitrate. Previous studies have shown that SOC1integrated all endogenous pathways:vernalization, autonomous, long-day, and GA pathways. We suggest that the SOC1MADS-box gene may play an important role in integrating signals induced by exogenous stress with endogenous signals to regulate flowering in Arabidopsis.