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Screening and Identification of Differentially Expressed Endodormant Genes from Latent Buds and Prompt Buds by Suppression Subtractive Hybridization
- MIN Zhuo-1, GENG Wan-Gang-1, FANG Yu-Lin-1, 2 , ZHANG Zhen-Wen-1, 2 , LI Hua-1, 2
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Scientia Agricultura Sinica. 2014, 47(5):
1029-1040.
doi:10.3864/j.issn.0578-1752.2014.05.019
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【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.
