| Chromatin in the eukaryotic nucleus is the material basis of all genetics process, and histone is present in the eukaryotic chromosomes within a class of small molecules with DNA binding basic proteins. Histone is an important component basic structural unit of chromosomes. Histone octamer is formed by the core histones H2A, H2B, H3and H4.H3constitutes a class nucleosome lysine-rich in the histone. It’s more conservative in evolutionary. H3.1and H3.2-type are mainly divided into plants. H3.1is replication-dependent histone mainly present in the cell division stage expression.The H3.2type is non-replication-dependent histone. This protein is usually highly acetylated in meristem of plant. The main difference is that weatner have introns in them. Histone regulates gene expression level by acetylation, methylation, phosphorylation, ubiquitination, SUMO.Chimonanthus praecox (L.) Link, a shrub originated from China, blossos lordly in deep winter, possessing many advantages, such as resistance of coldness and drought. It is our main purpose that whether H3is involved in the defense mechanism.We have cloned a H3sequence from the cDNA library, with the Genebank accession number JQ217379. And then analyzes the transcriptional level of CpH3gene in different tissues and stress response. A plant expression vector is constructed and the vector is transformed into tobacco. The main results are as follows:1The cloning of CpH3gene and characterization of the encoded proteinBased on a cDNA library constructed from Chimonanthus praecox flowers and EST analysis, a new gene was cloned by randomly cloning and sequencing, named as CpH3. The CpH3gene has an open reading frame (ORF) of411bp, encoding a putative polypeptide of136amino acid residues. BLAST analysis showed that the gene sequence has a high homology with other plant. It cloned1200bp secquence used DNA template. This sequence contains three intron used sequence analysis, the intron belong to GT-AG type. Bioinformatics analysis showed that the large protein was presence of nucleus, no signal peptide, composed with50%α-helix and47.8%random coil, and there are3serine phosphorylation sites,5threonine phosphorylation.2Transcriptional expression of CpH3It is showed that the expression level of CpH3in climax leaves is higher than that in roots and tender leaves by Real-time fluorescence quantitative PCR. And there is a higher expression level in outer perianths and medial perianths. In the developmental stages of flower, the expression level of CpH3in bloom period is more abundant than that in other periods. CpH3gene expression was induced under various abiotic stresses such as high salinity. But the high temperature, hypothermia, drought and ABA can inhibit the expression level of CpH3. It’s showed that the gene may be involved in plant development and stress responses.3Eukaryotic expression of CpH3The target CpH3gene was linked with plant expression vector pCAMBIA2301g by restriction enzyme digestion, ligation, transformation, and then transformed into Agrobacterium tumefaciens LBA4404. The plant expression vector containing CpH3was transformed into wild tobacco plants through Agrobacterium-mediated leaf disc method, transgenic tobacco were obtained by means of resistance screening, GUS staining and PCR amplification test. Treated with salt stress in transgenic tobacco, the chlorophyll, malondialdehyde and proline content does not change compared with the control. It is shows that the CpH3gene has no significant function on resistence of salt stress. |
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