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| 青稞转录因子HvnANT1基因的克隆与表达分析 |
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陈林1,2,3,姚晓华1,2,3*,苏乐平1,2,3,4,姚有华1,2,3,魏婵1,2,3,吴昆仑1,2,3
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| (1.青海大学 农林科学院, 西宁 810016;2.国家麦类改良中心青海青稞分中心, 西宁 810016;3.青海省青稞遗传育种重点实验室, 西宁 810016;4. 延安市农业科学研究所, 陕西 延安 716000) |
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| 摘要: |
| 为探究HvnANT1基因在青稞粒色形成过程中的基因表达模式，以紫粒青稞‘涅如姆扎’和白粒青稞‘昆仑10号’为试材，利用简化基因组GBS(Genotyping-by-Sequencing)对青稞紫粒进行基因定位，克隆到HvnANT1基因，对其进行生物信息学分析。结果表明:1)在7H染色体的84.30—86.00 cM获得1个MYB类转录因子，命名为HvnANT1。2)该基因长1 033 bp，其完整开放阅读框为762 bp，编码253个氨基酸。HvnANT1蛋白分子量为27.14 kU，是亲水性的不稳定碱性蛋白且不存在跨膜结构，无信号肽。该蛋白的二级结构主要是由无规卷曲、α-螺旋、延伸链和β-转角组成，具有2个SANT结构域(分别位于第13—第63个；第66—第114个氨基酸)。3)同源比对与系统进化分析表明:青稞HvnANT1蛋白与大麦、乌拉尔图小麦、高梁、玉米、二型花、小米、水稻、土瓶草、车轴草和杨梅10种植物的ANT1蛋白序列相似性分别为100.00%、88.85%、54.64%、60.95%、60.82%、58.46%、57.61%、37.76%、36.05%和36.33%；这些序列都具有2个高度保守的2个SANT结构域；与大麦的亲缘关系最近，其次是与乌拉尔图小麦，与水稻最远。4)亚细胞定位结果表明，该基因定位在细胞核内。5)实时荧光定量PCR(qRT-PCR)结果显示:与籽粒颜色形成的乳熟早期和乳熟晚期相比，软面团期的HvnANT1基因在‘涅如姆扎’中表达量极显著上调，而在‘昆仑10号’中各时期的表达量较低且差异不显著；且软面团期的‘涅如姆扎’籽粒HvnANT1基因的表达量极显著高于‘昆仑10号’(P<0.01)。花青素合成相关的结构基因HvnCHI、HvnANS和HvnDFR与该基因表达量模式相似。综上，青稞HvnANT1蛋白结构中的SANT结构域在物种进化过程中比较保守；该基因定位在细胞核内，符合转录因子特性；HvnANT1基因表达模式与结构基因HvnCHI、HvnANS和HvnDFR相似，在籽粒颜色形成的软面团期表达量极显著升高。 |
| 关键词: 青稞 HvnANT1 HvnCHI HvnANS HvnDFR 基因表达 |
| DOI：10.11841/j.issn.1007-4333.2022.08.06 |
| 投稿时间：2021-08-31 |
| 基金项目:国家自然科学基金(31960427)；国家大麦产业技术体系(CARS-05)；国家重点研发项目(2020YFD1001403) |
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| Isolation and expression analysis of a transcription factor HvnANT1 gene in hulless barley |
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CHEN Lin1,2,3,YAO Xiaohua1,2,3*,SU Leping1,2,3,4,YAO Youhua1,2,3,WEI Chan1,2,3,WU Kunlun1,2,3
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| (1.College of Agriculture and Forestry Sciences, Qinghai University, Xining 810016 China;2.Qinghai Subcenter of National Hulless Barley Improvement, Xining 810016 China;3.Qinghai Key Laboratory of Hulless Barley Genetics and Breeding, Xining 810016 China;4.Yan'an Institute of Agricultural Sciences, Yanan 716000 China) |
| Abstract: |
| To explore the expression of HvnANT1 gene in the process of purple-grain color development in Hulless barley, purple grain coat hulless barley ‘Nierumuzha' and white grain coat hulless barley ‘Kunlun 10' were taken as test materials. The gene of hulless barley purple grain was located by simplified genome GBS(genetic-by-sequencing), and the HvnANT1 gene was cloned for bioinformatics analysis. The results showed that: 1)A MYB transcription factor was obtained at 84. 30-86. 00 cM of chromosome 7H and named as HvnANT1. 2)The gene was 1 033 bp in length, and its complete open reading frame was 762 bp, encoding 253 amino acids. The molecular weight of HvnANT1 protein was 27. 14 kU, and it was an unstable hydrophilic basic protein with no transmembrane structure and no signal peptide. The secondary structure of the protein is mainly composed of random coil, α-helix, extended chain and β-rotation, and has two SANT domains(located at the 13th to the 63th; the 66th to the 114th amino acids). 3)The results of homology alignment and phylogenetic analysis showed that the sequence similarity between the HvnANT1 protein of hulless barley and the ANT1 protein of Hordeum vulgare, Triticum turgidum, Sorghum bicolor, Zea mays L, Dichanthelium oligosanthes, Setaria italica, Oryza rufipogon, Cephalotus follicularis, Trifolium affine, Morella rubra were 100. 00%, 88. 85%, 54. 64%, 60. 95%, 60. 82%, 58. 46%, 57. 61%, 37. 76%, 36. 05%, 36. 33%; these sequences all have two highly conserved SANT domains. The HvnANT1 protein of hulless barley had the closest relationship with H. vulgare, followed by Triticum urartu and the farthest relationship with Oryza sativa. 4)The results of subcellular localization showed that the gene was localized in the nucleus. 5)The results of qRT-PCR showed that the expression of HvnANT1 gene in the soft dough stage was extremely significantly up-regulated in ‘Nierumuzha' compared with the early milk stage and late milk stage. The expression level of ‘Kunlun 10' was lower and the difference was not significant in each period; and the expression of HvnANT1 gene in the grains of ‘Nierumuzha' at soft dough stage was significantly higher than that of ‘Kunlun 10'(P<0. 01). The anthocyanin synthesis-related structural genes HvnCHI, HvnANS and HvnDFR were similar to the gene expression patterns. In conclusion, the SANT domain in the HvnANT1 protein structure of hulless barley is relatively conserved in the evolution of species. The gene is located in the nucleus, which is consistent with the characteristics of transcription factors, and the expression pattern of HvnANT1 gene is related to the structural genes HvnCHI, HvnANS and HvnDFR. Similarly, the expression level is significantly increased in the soft dough stage of grain color formation, which provids a theoretical basis for in-depth study of the regulation of hulless barley HvnANT1 gene on grain color formation. |
| Key words: tibetan hulless barle HvnANT1 HvnCHI HvnANS HvnDFR gene expression |
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