| Remorins form a superfamily of plant-specific filamentous proteins which anchored to plasma membrane/lipid rafts. They comprise a multigene family in angiosperms, gymnosperms and bryophytes. Remorin monomer can form filaments that may play roles in cytoskeleton and membrane skeleton. Another important function of remorin proteins is that they may participate in plant resistant and transduction pathways of growth signals. And the functions of remorin proteins may differ in different plants. While Chimonanthus praecox is a winter flowering plant, with strong adaptability to adversity. The study was conducted to clone the gene of remorin from Chimonanthus praecox, and transcriptional analysis of remorin family gene under stress, which laid the foundation for the study of the response mechanism on resistance to adversity and the molecular mechanisms of winter-blooming of Chimonanthus praecox.In this study, based on a cDNA library constructed from Chimonanthus praecox flowers and analysised EST0253, a new remorin gene was cloned with its cDNA of CH0676, named as CpREM (GenBank accession No. DW222919). And we analyzed the function of this gene by using the methods of bioinformatics analysis, quantitative PCR and the overexpression in model plantlet. The main results were as follows:1. Molecular characteristics of CpREMThe length of the gene is958bp, with an open reading frame (ORF) of612bp encoding a putative polypeptide of203amino acid residues. Its predicted protein molecular weight is22.23kD, an isoelectric point of8.38, with no transmembrane domain and no signal peptide. CpREM is comprised of a conserved C-terminal domain and a variable N-terminal region like other remorins. Sequence alignments and phylogenetic analysis revealed the homology between remorin in Chimonanthus praecox and that in Vitis vinifera is up to87%. Remorin genes from Chimonanthus praecox and Ricinus communis are closely related and directly evolved from a common ancestral gene.2. The expression analysis of CpREM at the transcriptional levelReal-time fluorescent quantitative PCR analysis showed that CpREM was expressed in various tissues of Chimonanthus praecox. In particular, the highest expression levels in the petals indicated that the gene expression had a strong tissue-specificity. The expression of CpREM gradually increased through Chimonanthus praecox flower blooming, up to highest when came to the wither period of flowering, indicated that the gene expression had a strong time-specific. Under different stress treatments, the expression of CpREM in Chimonanthus praecox leaves were as follows. The expression of CpREM was fluctuant under NaCl stress; Under PEG stress treatment, CpREM showed a rapid response and then decreased; Under ABA stress treatment, its expression showed no significant change when compared with the control. It showed that the expression decreased slightly in a short time and then rebounded gradually. It was presumed that CpREM would be related with the response mechanism on resistance to adversity in Chimonanthus praecox.3. Overexpression of CpREM gene in transgenic tobaccosWe had constructed plantlet expression vector of pC2301g-CpREM successfully. And then transformed pC2301g-CpREM into the tobacco plantlets by agrobacterium-mediated leaf-disk transformation. Transgenic seedlings were identified by antibiotics resistance gene screening, GUS staining, and qRT-PCR. Eight transgenic tobacco lines were obtained. Transgenic tobacco plantlets had an enhanced resistence when plantlets were treated with salt stress. According to the above, we can speculate preliminarily that CpREM gene has a certain function related with salt tolerance mechanism. |
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