摘要
【目的】葡萄新梢上的芽有冬芽和夏芽两种,根据这两种芽休眠特性的差异,筛选与芽生理休眠相关基因,进而从分子水平研究葡萄芽生理休眠的相关机制。【方法】以‘赤霞珠’葡萄新梢上冬芽和夏芽为材料,夏芽作为驱动方(driver),冬芽作为实验方(tester),采用抑制消减杂交技术(suppression subtractive hybridization,SSH)构建冬芽和夏芽的SSH-cDNA文库,筛选冬芽和夏芽差异表达的基因片段。通过NCBI同源比对,寻找与葡萄芽生理休眠相关的基因,并对差异表达基因进行功能注释和分类。最后,运用Real-time PCR技术对部分候选基因进行检测,以验证其与葡萄芽生理休眠的相关性。【结果】共获得359条有效、高质量的差异基因片段,经CAP3软件聚类拼接,得到106个unique EST。在GenBank中进行BLASTN同源比对,有98条EST与已知蛋白或基因具有不同程度的同源性,占全部EST的92.4%。进行BLASTX同源比对,有54条EST与功能已知蛋白或基因具有不同程度的同源性,占全部EST的51%。已知功能的EST涉及休眠、逆境胁迫、细胞代谢、胞内物质运输等方面,且绝大多数基因信息来源于葡萄、苜蓿、橄榄、油菜、高粱、棉花、大蒜、拟南芥等植物。对差异基因片段进一步的功能分析发现,调控开花的基因占生物学过程的20%;与环境胁迫相关的基因占生物学过程的6%;与细胞代谢相关的基因占生物学过程的20%;与胞内物质运输相关的基因占总生物学过程的6%;其他包括DNA转录、减数分裂、细胞死亡及细胞壁松弛作用等。这些基因的分子功能主要有半胱氨酸型肽链内切酶、甲基转移酶、蛋白激酶、催化活性、结构分子活性、蛋白结合功能、DNA结合功能、ATP结合功能、蛋白质二聚体催化活性及丙酮酸氨基转移酶活性等。经Real-time PCR检测得知,在夏芽萌发过程中,候选基因在冬芽和夏芽中表达量呈不规律变化,且同一基因在相同节位的冬芽和夏芽中表达量均有较大差异。【结论】发现了一些在冬芽中表达频率较高的基因,如编码MADS FLC-like蛋白、钙依赖蛋白激酶、NADP依赖型的苹果酸酶、细胞壁水解酶、细胞壁松弛蛋白、外被体蛋白β亚基、热激蛋白、衰老相关蛋白、细胞色素P450、细胞壁关联蛋白等基因,认为线粒体蛋白基因、未知蛋白基因、开花相关基因、ATP合成酶β亚基基因、MADs FLC-like蛋白基因等与葡萄芽生理休眠具有相关性。
【Objective】There are two kinds of buds, latent buds and prompt buds, along shoots of grapevine. The aim of this study is to screen the genes associated with the bud endodormancy of grapevine depending on the different characteristics of grape latent buds and prompt buds in dormancy, on the basis of which the mechanism of grapevine bud endodormancy was explored.【Method】The SSH-cDNA library of latent buds and prompt buds of Cabernet Sauvignon was constructed using the technology of suppression subtractive hybridization (SSH) to screen the differential expressed gene fragments between them, during which latent buds were used as driver and prompt buds as tester. These fragment sequences were analyzed based on the NCBI and the gene functions were annotated. Furthermore, real-time PCR was employed to detect the expression profile of part of candidate genes with bud development to confirm the relationship between candidate genes and bud endodormancy. 【Result】Totally 359 effective differentially expressed gene fragments with high quality were obtained and assembled by CAP3 Assembly Program, and 106 unique expressed sequence tags (ESTs) were obtained. After comparing the sequences with BLASTN in the Genbank, it was found that 98 unigenes, accounting for 92.4% of all the unigenes, showed homology with the known genes or proteins. And after analysis with BLASTX, it was found that 54 unigenes, accounting for 51% of all, showed homology with the known proteins or genes, which were involved in the dormancy, cell metabolism, abiotic and biotic stress and intracellular transportation etc. Moreover, most of the gene information came from plants such as grape, alfalfa, olive, rape, sorghum, cotton, garlic, arabidopsis etc. Gene ontology (GO) analysis revealed that these ESTs were involved in the regulation of flower development, response to environmental stress, cell metabolism, intracellular material transport, and they accounted for 20%, 6%, 20%, and 6% of all the biological processes, respectively. The rest part included DNA transcription, male meiosis, cell death and cell wall loosening etc. As for the molecular function, these genes were found to have cysteine-type endopeptidase activity, methyltransferase activity, protein kinase activity, catalytic activity, structural molecule activity, protein binding, DNA binding, ATP binding, protein dimerization activity and pyruvate aminotransferase activity, etc. Real-time PCR detection showed that the expression of candidate genes varied greatly with the bud break of prompt buds and showed a big difference between these two kinds of buds even in the same node. 【Conclusion】 Some genes which had high expression in latent buds were indentified such as MADS flowering FLC-like protein, calcium-dependent protein kinase, NADP-dependent malic enzyme, cell wall-associated hydrolase, cell wall loosening protein, subunit beta of coatomer protein complex, heat shock protein, senescence-associated protein, cytochrome P450, cell wall-associated protein etc, and it showed that mitochondrial protein gene, unnamed protein gene, flower bloom associated gene, ATP synthase subunit beta gene, and MADs FLC-like protein gene played important roles in bud endodormancy of grapevine.
出处
《中国农业科学》
CAS
CSCD
北大核心
2014年第5期1029-1040,共12页
Scientia Agricultura Sinica
基金
陕西省科技攻关项目(2012K01-25)
国家"十二五"科技支撑计划(2012BAD31B07)