| 黄瓜生长素反应因子(ARF)家族鉴定及表达特异性分析 |
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| 引用本文： | 盛慧,秦智伟,李文滨,周秀艳,武涛,辛明.黄瓜生长素反应因子(ARF)家族鉴定及表达特异性分析[J].中国农业科学,2014,47(10):1985-1994. |
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| 作者姓名： | 盛慧 秦智伟 李文滨 周秀艳 武涛 辛明 |
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| 作者单位： | 东北农业大学园艺学院/农业部东北地区园艺作物生物学与种质创制重点实验室;哈尔滨市农业科学院;东北农业大学农学院; |
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| 基金项目： | 国家“863”计划项目(2012AA100105) |
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| 摘 要： | 【目的】鉴定黄瓜生长素反应因子（ARF），预测small RNAs并验证ARF与small RNAs和生长素的关系；分析ARF在种子萌发过程中的表达模式，推断ARF在黄瓜单性结实和种子萌发过程中是否起到关键作用。【方法】利用拟南芥和水稻ARF蛋白质序列检索黄瓜基因组数据库，对检索到的黄瓜ARF家族进行结构分析和small RNAs预测；将预测到的gma-MIR160o precursur构建到pCAMBIA2301植物表达载体上，利用农杆菌介导法将其导入到单性结实黄瓜品种中，并对PCR检测为阳性的转基因植株进行RT-PCR验证；利用real-time RT-PCR方法分析ARF家族成员在生长素诱导后、开花时期及种子萌发阶段的表达模式。【结果】通过与拟南芥和水稻ARF蛋白序列的比对，共得到18个黄瓜ARF蛋白质序列。将其连同拟南芥和水稻的ARF蛋白质序列共分为4大类。从结构上看，外显子数目2-18不等，但同一类之间基因结构较为相似。进化树分析显示，18个基因之间的相似性不高；18个黄瓜ARF基因均有相对应的small RNA。其中， Csa010564、Csa011935、Csa015176、Csa020560和Csa022361可能都是miR160的靶基因。转基因试验进一步说明Csa010564、Csa011935和Csa015176在表达量上出现下降趋势，而Csa020560和Csa022361的表达量与对照相比略有上升，说明Csa010564、Csa011935和Csa015176是miR160的靶基因；在生长素诱导表达的试验中，Csa007296、Csa011935和Csa015176在根、茎和叶中的表达量均高于对照，说明ARF的表达受IAA的正调控；同时，以上3个基因在叶片和雌花花冠中的表达量均是开花第2 d低于开花当天，而在子房中的表达量确是第2天高于开花当天，尤其是Csa011935和Csa015176上调显著，说明ARF在子房发育过程中起到至关重要的作用；ARF在种子萌发过程中的表达分析显示：大部分基因在吸涨后12 h和48 h处于表达最高峰。【结论】ARF受生长素和相应的small RNAs调控。在黄瓜单性结实和种子萌发过程中，ARF可能起到了关键性作用。
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| 关 键 词： | 黄瓜 ARF small RNAs 生长素 表达模式 |
| 收稿时间： | 2013-10-16 |
Genome-wide Identification and Expression Analysis of Auxin Response Factor (ARF) Family in Cucumber |
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| SHENG Hui-,QIN Zhi-Wei-,LI Wen-Bin-,ZHOU Xiu-Yan-,WU Tao-,XIN Ming-.Genome-wide Identification and Expression Analysis of Auxin Response Factor (ARF) Family in Cucumber[J].Scientia Agricultura Sinica,2014,47(10):1985-1994. |
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| Authors: | SHENG Hui- QIN Zhi-Wei- LI Wen-Bin- ZHOU Xiu-Yan- WU Tao- XIN Ming- |
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| Affiliation: | 1、College of Horticulture, Northeast Agricultural University/Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in Northeast China, Ministry of Agriculture, Harbin 150030;
2、Harbin Academy of Agricultural Sciences, Harbin 150029;
3、College of Agriculture, Northeast Agricultural University, Harbin 150030 |
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| Abstract: | 【Objective】 The objective of this study is to identify the cucumber auxin response factor (ARF), forecast the small RNAs and verify the relationship between ARF with small RNAs and auxin. The expression pattern of ARF during seed germination was analyzed and the effect of ARF on the process of parthenocarpy and seed germination was deduced. 【Method】 The Arabidopsis and rice ARF proteins were used to search the cucumber genome database. Then, the retrieved cucumber ARF family structure was analyzed and the small RNAs were predicted. The predicted gma-MIR160o precursur was built into the pCAMBIA2301. By Agrobacterium-mediated method, it was imported into parthenocarpy cucumber varieties. Transgenic plants were validated by RT-PCR. Using real-time RT-PCR method, the expression patterns of ARF were analyzed in auxin treatment, flowing and seed germination. 【Result】 By comparison with the Arabidopsis and rice ARF protein sequences, there are 18 cucumber ARF protein sequences. ARF proteins were divided into four classes. The number of exons varied from 2 to 18, and the structure was similar among the same class. Phylogenetic tree showed that the similarity of 18 genes was not high. Small RNAs corresponds to ARF genes in cucumber were found. Csa010564, Csa011935, Csa015176, Csa020560 and Csa022361 were miR160 targets. Transgenic test further exhibited mRNA abundance of Csa010564, Csa011935 and Csa015176 decreased, while Csa020560 and Csa022361 rose slightly, indicating that Csa010564, Csa011935 and Csa015176 were miR160 targets. The results of real-time RT-PCR exhibited that mRNA abundance of Csa007296, Csa011935 and Csa015176 in roots, stems and leaves were higher than wild type, indicating the expressions of these genes were positively regulated by auxin. Meanwhile, the expressions in leaves and female flowers were decreased on the second flowering day, while the expression of the ovary was the opposite, especially Csa011935 and Csa015176, indicating that ARF genes play a crucial role in the development of the ovary. In the experiment of the auxin treatment, the expressions in different tissues were up-regulated. The expression of ARF genes in the seed germination was analyzed. The results showed that the expression peak of most genes were 12 h and 48 h in imbibition. 【Conclusion】 The ARF genes were regulated by auxin and the corresponding small RNAs. ARF genes may play a key role in the parthenocarpy and seed germination of cucumber . |
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| Keywords: | cucumber ARF small RNAs auxin expression patterns |
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