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林业科学 ›› 2019, Vol. 55 ›› Issue (6): 176-183.doi: 10.11707/j.1001-7488.20190621

• 研究简报 • 上一篇    下一篇

蜡梅转录因子CpTAF10基因的克隆及功能分析

刘道凤, 王霞, 代银, 杨建峰, 马婧, 李名扬, 眭顺照   

  1. 西南大学园艺园林学院 南方山地园艺学教育部重点实验室 重庆市花卉工程技术研究中心 重庆 400715
  • 收稿日期:2018-06-28 修回日期:2018-08-15 出版日期:2019-06-25 发布日期:2019-07-11
  • 基金资助:
    重庆市基础与前沿研究一般项目(cstc2016jcyjA0153);重庆市社会民生重点研发项目(cstc2018jscx-mszdX0004);中央高校基本科研业务费重点项目(XDJK2015B013)。

Cloning and Function Analysis of CpTAF10 from Wintersweet (Chimonanthus praecox)

Liu Daofeng, Wang Xia, Dai Yin, Yang Jianfeng, Ma Jing, Li Mingyang, Sui Shunzhao   

  1. Chongqing Engineering Research Center for Floriculture Key Laboratory of Horticulture Science for Southern Mountainous Regions of Ministry of Education College of Horticulture and Landscape, Southwest University Chongqing 400715
  • Received:2018-06-28 Revised:2018-08-15 Online:2019-06-25 Published:2019-07-11

摘要: [目的]TATA框结合蛋白相关因子TAF10作为基本转录因子之一,在生长发育和胁迫响应过程中发挥着广泛的、重要的生物学作用。对蜡梅中TAF10同源基因CpTAF10的克隆与功能分析,有利于丰富对植物TAFs基因功能的认识,并为解析蜡梅抗逆形成的转录调节机理提供新的理论依据。[方法]以转录组数据库中获得的蜡梅TAFs家族基因序列,克隆得到CpTAF10基因的cDNA序列,并对其编码蛋白进行序列特征和进化树分析。采用实时荧光定量PCR技术分析CpTAF10基因在蜡梅不同组织及花期中的表达特性,以及高温、低温、盐胁迫及ABA处理后的表达变化。同时,构建CpTAF10基因的过表达载体,采用花序侵染法进行拟南芥遗传转化,对拟南芥转基因纯合株系进行表型观察和胁迫耐性分析。[结果]获得的CpTAF10基因cDNA序列为712 bp,包含405 bp的开放阅读框(ORF),编码134个氨基酸,蛋白理论分子量为15.21 kDa,预测的等电点pI值为5.19。CpTAF10蛋白序列与其他植物同源序列具有较高的同源性,蛋白多序列比对显示CpTAF10蛋白属于TAF10同源蛋白,并含有组蛋白折叠结构域。表达特性分析结果发现,CpTAF10基因在蜡梅的根、茎、子叶、幼叶、成熟叶和花6个不同组织中均有不同程度的表达,其中,在成熟叶中的表达量最高。CpTAF10在蜡梅花朵的不同花期中,呈现出波动的表达模式,在衰老期表达量最高。在低温、盐胁迫和ABA处理的蜡梅叶片中均能被诱导表达,但其表达变化各不相同。在拟南芥中过表达CpTAF10基因可提高盐胁迫下拟南芥种子的萌发率,相对于野生型植株,转基因植株的主根和侧根在盐胁迫下均表现出一定的生长优势。[结论]CpTAF10基因能在低温、盐胁迫和ABA处理后诱导表达,可能参与蜡梅逆境胁迫耐性的分子调控。在拟南芥中过表达CpTAF10基因显著提高了转基因拟南芥的萌芽率及主根和侧根的生长优势,在一定程度上可增强植物的盐胁迫耐性。

关键词: 蜡梅, TAFs, 基因表达, 盐胁迫

Abstract: [Objective] TATA box-binding protein associated factor TAF10, as one of the basic transcription factors, plays an extensive and important biological role in the regulation of growth, development and stress response. Cloning and functional analysis of TAF10 homologous gene CpTAF10 in wintersweet (Chimonanthus praecox) may help to enrich the understanding of plant TAFs gene, and provide theoretical basis for the analysis of the mechanism of transcriptional regulation of the resistant formation of wintersweet.[Method] Based on the cDNA sequence of TAF family gene in wintersweet transcription database, we obtained the CpTAF10 gene and analyzed the sequence characteristics and evolutionary tree. The expressions of CpTAF10 in different tissues and flowering stages, and the changes of CpTAF10 gene expression under heat, cold, NaCl stress and ABA treatments were analyzed using real-time quantitative PCR. Meanwhile, the overexpression vector of CpTAF10 was constructed, and transformed into Arabidopsis thaliana via floral dipping method, phenotypes and stress tolerance of the homozygous transgenic lines were analyzed.[Result] The cDNA sequence of CpTAF10 is 712 bp, including 405 bp of ORF (open reading frame). The open reading frame of CpTAF10 encoded a protein composed of 134 amino acids. The theoretical molecular weight of this protein was 15.21 kDa, and the predicted isoelectric point pI value was 5.19. The CpTAF10 protein has high homology with other plant homologous proteins. The multiple sequence alignment shows that CpTAF10 protein belongs to the TAF10 homologous protein and contains the histone folding domain. The results showed that the CpTAF10 gene expressed in the roots, stems, cotyledon, young leaves, mature leaves and flowers of wintersweet, and the mature leaves had the highest expression level. CpTAF10 showed a fluctuating expression pattern in different flowering stages, and showed the highest expression in the senescence stage. The expression of CpTAF10 was induced in cold, salt stress and ABA treatment in wintersweet. The overexpression of CpTAF10 gene increased the germination rate of transgenic Arabidopsis seeds under salt stress. Compared with the wild type, the main root and lateral roots of the transgenic plants showed growth advantage to some extent under salt stress.[Conclusion] The expression of CpTAF10 gene can be induced under cold, salt stress and ABA treatment, and CpTAF10 gene may be involved in molecular regulation of stress tolerance in wintersweet. The overexpression of CpTAF10 gene in Arabidopsis significantly enhanced the germination rate and the growth advantages of root under salt stress, enhanced the salt stress tolerance in transgenic Arabidopsis to some extent.

Key words: Chimonanthus praecox, TBP associated factors, gene expression, salt stress

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