利用cDNA-AFLP技术鉴定菊花品种‘紫荷’的抗白锈病相关基因

doi:10.3864/j.issn.0578-1752.2012.05.013

摘要/Abstract

摘要： 【目的】筛选菊花中抗白锈病相关基因，研究菊花抗白锈病的分子机理。【方法】以菊花中对白锈病完全免疫的品种‘紫荷’为供试材料，对其叶片喷施106个白锈病冬孢子/mL蒸馏水，处理时间梯度为0 h、3 h、6 h、12 h、24 h、48 h、72 h、96 h，使用cDNA-AFLP技术分析这一接种过程中的差异表达基因，并采用RT-PCR方法对表达结果进行验证。利用RACE技术克隆其中重要蛋白基因的cDNA全长，采用MEGA 4.0程序进行序列分析。【结果】利用76对引物组合，共筛选出了约4 950条差异表达的cDNA片段，对其中80个表达差异显著的片段进行测序，最终得到51个差异片段的核苷酸序列。Blastx同源性比对发现，其中18个序列与已报道的抗病基因具较高同源性，其功能涉及到抗病、信号转导、光合作用和光呼吸、反转录转座子以及植物基础代谢。RT-PCR验证结果表明，利用cDNA-AFLP技术获得了稳定表达的序列；使用RACE技术获得了其中两个编码植物14-3-3蛋白的基因：CmGFR01、CmGFR02的cDNA全长序列分别为1 062 bp和1 098 bp，分别编码含260和271个氨基酸残基的开放阅读框。对序列进行分析发现，CmGFR01、CmGFR02属于高等植物中一类新的14-3-3基因亚家族。【结论】构建了菊花品种‘紫荷’在白锈病胁迫下的基因表达谱，从基因组水平上识别了一批与抗白锈病相关的基因，这些基因有助于解析植物抗白锈病的分子机理。分离得到的CmGFR01、CmGFR02可用于栽培菊花抗白锈病的分子育种。

关键词: 菊花, 白锈病, cDNA-AFLP, 14-3-3基因

Abstract: 【Objective】An experiment was conducted to study the molecular mechanism of Chrysanthemum morifolium ‘Zihe’ response to the white rust, and to screen the white rust resistance genes. 【Method】 Infections were conducted by spraying the C. morifolium ‘Zihe’ leaves with suspension of 106 white rust sporangia per mL pure water for 3, 6, 12, 24, 48, 72, 96 h. cDNA-AFLP was used to moniter the gene expression under the white rust infections, and RT-PCR was used to validate the gene expression patterns. RACE method was used to get two fully cDNA sequences encoding 14-3-3 proteins, and phylogenetic analysis was also performed. 【Result】 Seventy-six primer combinations were used to investigate 4 950 cDNA fragments. After sequencing of 80 ESTs, the nucleic acid sequences of 51 ESTs were obtained, and among them, 18 ESTs were obtained, and showed homologous to the resistance-related genes of other species. These 18 ESTs were sorted into six functional categories: disease resistance protein, signal transduction, photosynthesis and photorespiration, retroelement polyprotein-like, senescence and metabolism pathways. RT-PCR showed that the cDNA-AFLP results was accurate and believable. Furthermore, two fully cDNA sequences of genes encoding 14-3-3 proteins were obtained: CmGFR01, CmGFR02. These two genes were 1 062 and 1 098 bp, encoded ORFs of 260 and 271 amino-acid residues. Phylogenetic analysis showed that the CmGFR01 and CmGFR02 might belong to a new subfamily of the higher plants 14-3-3 genes family. 【Conclusion】Gene expression profiling in response to white rust and differentially expressed genes of C. morifolium ‘Zihe’ were identified via cDNA-AFLP analysis. These genes could help us to understand the mechanism of response of plants to white rust. Furthermore, the isolated two genes, CmGFR01 and CmGFR02, could be used for the gene enigeering of the cultivated chrysanthemum resistance to the biotic stresses.

Key words: Chrysanthemum morifolium, white rust, cDNA-AFLP, 14-3-3 gene

黄河, 王顺利, 戴思兰. 利用cDNA-AFLP技术鉴定菊花品种‘紫荷’的抗白锈病相关基因[J]. 中国农业科学, 2012, 45(5): 926-935.

HUANG He, WANG Shun-Li, DAI Si-Lan. cDNA-AFLP Analysis of White Rust Response Genes in Chrysanthemum morifolium ‘Zihe’[J]. Scientia Agricultura Sinica, 2012, 45(5): 926-935.

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