花椰菜BobACT基因的克隆及其作为内参基因的研究
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福建省农业科学院作物研究所/福建省农业科学院蔬菜研究中心/福建省蔬菜工程技术研究中心,福州350013

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福建省属公益类科研院所基本科研专项(2018R1026-10);国家大宗蔬菜产业体系(CARS-23-G-53)


Molecular Cloning of Actin Gene and Study on This Gene as Reference Gene in Cauliflower(Brassica oleracea L . var. botrytis L.)
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Crops Research Institute,Fujian Academy of Agricultural Sciences/Vegetable Research Center,Fujian Academy of Agricultural Sciences/Fujian Engineering Research Center for Vegetables,Fuzhou 350013

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Foundation project :Fujian Provincial Public Welfare Research Institute basic Scientific Research Project (2018R1026-10);National bulk vegetable Industry system (CARS-23-G-53)

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    摘要:

    实时荧光定量PCR 技术是探索植物基因功能和调节机理的有效手段。选择合适的内参基因是获得实时荧光定量PCR 准确性数据的必备条件。ACT基因高度保守且表达稳定,常作为内参基因被广泛应用。为了获得花椰菜ACT基因,以转录组测 序和RT-PCR方法为手段克隆得到花椰菜肌动蛋白基因Actin。该基因等电点为5.395,理论分子量为41.77 kD;其cDNA 开 放阅读框长1134 bp,编码氨基酸377 个,GenBank 登录号为MG598643。Wolf Psort分析发现, BobActin蛋白亚细胞定位于 细胞质基质中。Motif Scan分析显示, BobActin蛋白质的氨基酸序列4-377位为Actin保守结构域。进化分析表明,同源序列 基因编码的蛋白质与同为十字花科的甘蓝、芜菁和油菜同源蛋白的相似性达到90%以上, 具有高度的保守性。在此基础上, 设 计了一对的荧光定量PCR引物, 分析显示, 该引物具有较高的特异性和扩增效率, 在花椰菜根、茎、花、花球、叶片等不同组 织和低温、高温、盐处理、干旱处理、ABA处理等胁迫处理下均能稳定表达, 适合在花椰菜基因表达研究中作为内参基因,为 开展花椰菜重要功能基因的挖掘、表达模式以及调控机理的研究提供参考。花椰菜在内参基因方面的研究还处于初步阶段, 今后可继续克隆其他内参基因,丰富花椰菜的内参基因库,从而进一步提高花椰菜基因表达分析研究的稳定性、重复性和准 确性。

    Abstract:

    Real-time fluorescence quantitative PCR is an effective method to quantify the transcriptional profile of target genes. Use of proximal gene as internal reference is essential when performing qRT-PCR experiments. The Actin (ACT) gene is highly conserved cross species and expressed stably and is often used as an internal control. In order to obtain the ACT gene of cauliflower, the ACT gene of cauliflower was cloned by RT-PCR method (GenBank ID: MG598643)1. The open reading frame (ORF) is 1,134 bp, encoding 377 amino acids with a predicted molecular weight of 41.77 kD and a hypothetical isoelectric point of 5.395. Wolf Psort analysis indicated that BobActin protein was located in the cytoplasmic matrix, and Motif Scan analysis showed that BobActin protein had the conserved actin at position of 4-377 sites. BobActin shared 90% identity with the homologous proteins from genus Brassicacea, such as Brassica oleracea var. oleracea, Brassica rapa and Brassica napus. A pair of qRT-PCR primers was designed from the BobActin gene sequence, and this combination showed high specificity and amplification efficiency. qRT-PCR analysis indicated that the BobActin gene was stably expressed in tissues of cauliflower, including root, stem, flower ball, leaf, even under various stress treatments (low temperature, high temperature, salt, drought and ABA). Thus, this gene might serve as an internal reference being suitable for gene expression in cauliflower.

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林珲,朱海生,黄丽芳,等.花椰菜BobACT基因的克隆及其作为内参基因的研究[J].植物遗传资源学报,2019,20(3):781-789.

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  • 收稿日期:2018-09-18
  • 最后修改日期:2018-09-18
  • 录用日期:2018-11-20
  • 在线发布日期: 2019-05-13
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