单氰胺破除葡萄休眠生理生化响应及相关基因克隆与表达分析

Physiological and biochemical responses of grape dormancy breaking with cyanamide and expression analysis of related genes

关键词:  单氰胺，葡萄，休眠，转录因子，VvCBF，VvFTs

Key words:  cyanamide, grape, dormancy, transcription factors, VvCBF, VvFTs

摘要:

为探究单氰胺破除葡萄休眠的生理生化响应及分子调控机理，该研究以‘水晶’葡萄（Vitis vinifera×V. labrusca ‘Shuijing’）为试验材料，测定单氰胺处理 ‘水晶’葡萄后芽内SOD、POD和CAT活性、MDA和H2O2含量及氧自由基产生速率的变化，并通过RT-PCR技术克隆得到其葡萄芽的2个FT （Flowering locus T） 基因（VvFT1和VvFT2）和1个CBF（C-repeat binding factor）基因（VvCBF）的全长cDNA序列，分析其理化性质、系统进化、保守基序及结构域和单氰胺处理后其在葡萄芽中的表达水平差异等。结果表明：（1）生理生化指标测定分析表明，单氰胺处理后葡萄芽内SOD、POD和CAT活性、MDA和H2O2含量及氧自由基产生速率均明显增加。（2）‘水晶’葡萄VvFT1和VvFT2的cDNA全长为525 bp，编码174 aa；VvCBF的cDNA全长为714 bp，编码237 aa。（3）同源性分析表明，‘水晶’葡萄VvFT1与荔枝（Litchi chinensis，LcFT：AEU08960.1）和龙眼（Dimocarpus longan，DlFT2：ALA55998.1）的氨基酸同源性最高，VvFT2与LcFT（AEU08961.1）和DlFT2（AHF27444.1）的氨基酸同源性最高，系统进化分析表明VvFT1、VvFT2 、LcFT（AEU08960.1；AEU08961.1）和DlFT2（ALA55998.1；AHF27444.1）聚为一支，亲缘关系最近；VvCBF与野扁桃（Prunus ledebouriana，PlCBF：AEB69782.1）的氨基酸同源性最高，系统进化分析表明VvCBF和PlCBF聚为一支，亲缘关系最近。（4）qRT-PCR分析表明，单氰胺处理后葡萄芽中VvFT1、VvFT2基因的表达量显著增加，而VvCBF基因的表达量显著降低。综上所述，该研究较为全面地分析了VvFT1、VvFT2和VvCBF基因的系统进化和单氰胺处理后葡萄芽内各基因表达模式及生理生化指标变化，为单氰胺破除葡萄休眠的研究奠定了理论基础。

Abstract:

To explore the responses of physiological and biochemical and mechanism of molecular regulatory for grape dormancy breaking with cyanamide, Vitis vinifera×V. labrusca ‘Shuijing’ was used as experimental materials in this study to determine the changes in SOD, POD, CAT activity, MDA and H2O2 content, and oxygen free radical production rate, and RT-PCR technology was used to clone the full-length cDNA sequences of two FT (Flowering location T) genes (VvFT1 and VvFT2) and one CBF (C-repeat binding factor) gene (VvCBF) from its buds, then their physicochemical properties, phylogenetic evolution, conserved motifs and domains, and differences expression levels in grape buds after cyanamide treatment were analyzed. The results were as follows: (1) The analysis of physiological and biochemical indicators results showed that SOD, POD and CAT activities, MDA and H2O2 contents, and the rate of oxygen free radical production in grape buds were significantly increased after treated with cyanamide. (2) The full-lengths cDNA sequences of VvFT1 and VvFT2 genes were 525 bp from Vitis vinifera×V. labrusca ‘Shuijing’, encoding 174 aa, and the full-lengths cDNA sequences of VvCBF gene was 714 bp, encoding 237 aa. (3) The homology analysis results showed that VvFT1 of Vitis vinifera×V. labrusca ‘Shuijing’ had the highest amino acid homology with Litchi chinensis (LcFT: AEU08960.1) and Dimocarpus longan (DlFT2: ALA55998.1), VvFT2 had the highest amino acid homology with LcFT (AEU08961.1) and DlFT2 (AHF27444.1). The phylogenetic analysis result showed that VvFT1, VvFT2, LcFT (AEU08960.1; AEU08961.1) and DlFT2 (ALA55998.1; AHF27444.1) clustered into a branch, with the most closes genetic relationship; VvCBF had the highest amino acid homology with Prunus ledebouriana (PlCBF: AEB69782.1), and the phylogenetic analysis showed that VvCBF and PlCBF clustered into a branch, with the most closest genetic relationship. (4) qRT-PCR analysis showed that VvFT1 and VvFT2 expression levels were significantly increased in buds after treated with cyanamide, while VvCBF expression levels was significantly decreased. In summary, this study comprehensively analyzed the phylogenetic evolution of VvFT1, VvFT2, and VvCBF genes, as well as the expression patterns of these genes and physiological and biochemical indicators in grape buds after treated with cyanamide, laying a theoretical foundation for the molecular and physiological mechanisms of grape dormancy breaking with cyanamide.