中国农业科学 ›› 2015, Vol. 48 ›› Issue (24): 4872-4884.doi: 10.3864/j.issn.0578-1752.2015.24.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

棉花GhGT-2转录因子的克隆及其功能分析

李月1,2,3,刘晓东3,谢宗铭1,董永梅1,武冬梅1,陈受宜2   

  1. 1新疆农垦科学院生物技术研究所/作物种质创新与基因资源利用兵团重点实验室,新疆石河子 832000
    2中国科学院遗传与发育生物学研究所/   国家植物基因组重点实验室,北京 100101
    3新疆农业大学农学院/农业生物技术重点实验室,乌鲁木齐 830052
  • 收稿日期:2015-05-19 出版日期:2015-12-16 发布日期:2015-12-16
  • 通讯作者: 谢宗铭,E-mail:xiezmchy@163.com。陈受宜,E-mail:sychen@genetics.ac.cn
  • 作者简介:李月,E-mail:liyue6905@126.com
  • 基金资助:
    国家转基因生物新品种培育科技重大专项(2009ZX08009-090B)

Cloning and Function Analysis of Cotton Transcription Factor GhGT-2

LI Yue1,2,3, LIU Xiao-dong3, XIE Zong-ming1, DONG Yong-mei1, WU Dong-mei1, CHEN Shou-yi2   

  1. 1Biotechnology Research Institute, Xinjiang Academy of Agricultural and Reclamation Science/Xinjiang Production and Construction Group Key Laboratory of Crop Germplasm Enhancement and Gene Resources Utilization, Shihezi 832000, Xinjiang
    2Institute of Genetics and Developmental Biology, Chinese Academy of Sciences/National Key Laboratory of Plant Genomics, Beijing 100101
    3College of Agronomy, Xinjiang Agricultural University/Key Lab of Agro-Biotechnology, Urumqi 830052
  • Received:2015-05-19 Online:2015-12-16 Published:2015-12-16

摘要: 【目的】棉花是重要的纤维作物,其生长常遭受非生物逆境危害,严重影响棉花的生长和产量。Trihelix转录因子在植物抵御各种逆境胁迫中扮演重要作用。克隆棉花Trihelix转录因子基因并分析其表达特性和功能,为最终利用转基因手段改良棉花抗逆性奠定基础。【方法】通过BLAST分析比对,从棉花EST数据库中获得1个高度同源基因,通过基因序列分析,发现其属于Trihelix转录因子GT-2亚家族,命名为GhGT-2。以棉花叶片总RNA为模板,根据EST序列设计引物,利用RT-PCR结合RACE技术,获得GhGT-2的编码序列。使用MEGA5对蛋白序列及其同源序列进行多序列比对分析,并构建同源物种间系统进化树,通过SMART网站(http://smart.embl- heidelberg.de/)进行蛋白结构预测。以陆地棉品种新陆早26号为研究材料,在棉花15 d苗龄时(一对真叶期),分别对其植株进行非生物胁迫和ABA处理0、1、3、6和12 h,然后采集相应时段棉苗叶片。另外采集同一品种棉花的不同发育时期的根、茎、叶、花、开花后当天胚珠以及开花后12 d(12 days post anthesis,DPA)纤维等不同组织样品,利用实时荧光定量PCR方法分析GhGT-2在棉花不同组织间的表达差异及其在低温、干旱、高盐和ABA处理下的表达模式。将GhGT-2克隆至GFP表达载体pBI221,和GAL4 DNA结合结构域载体,在拟南芥原生质体中验证GhGT-2在细胞内的定位情况和转录激活活性。利用凝胶迁移试验(EMSA)检测DNA结合元件。【结果】克隆了棉花GhGT-2的cDNA全长序列。该基因cDNA全长1 579 bp,开放阅读框为1 428 bp,编码475个氨基酸的蛋白,推导编码蛋白质的分子量为54.07 kD,等电点为8.96。SMART蛋白结构预测发现,该蛋白含有2个Trihelix家族典型的SANT蛋白结合域。系统进化树分析表明,GhGT-2属于Trihelix转录因子GT-2亚家族,与拟南芥AtGTL1、白杨PtaGTL1 GT2-Box、GT3-Box、GT-1b(BoxⅡ)和MYB元件MBS1、MRE1、MRE3、MRE4亲缘关系最近。实时荧光定量PCR表明,GhGT-2在棉花的根、茎、叶、花、开花后当天胚珠以及开花后12 d(12 DPA)纤维中均有表达,其中,在叶中表达量最高,在根中表达量最低。在冷胁迫下,GhGT-2除在3 h时表达量接近0 h外,在1、6和12 h的表达量均低于0 h,呈现抑制表达特征。在高盐、干旱和ABA处理3种胁迫下,GhGT-2在1 h的表达量均低于0 h,但3、6和12 h的表达量均高于0 h,表现为先抑制后上调表达特征。推测该基因可能参与棉花ABA信号通路中对逆境胁迫的抗性反应。利用拟南芥原生质体分析,GhGT-2主要定位于细胞核中,转录激活活性不明显。凝胶阻滞(EMSA)分析发现,GhGT-2可以结合GT元件。【结论】获得棉花GhGT-2的全长cDNA序列,其编码蛋白含有2个SANT蛋白结合域,属于棉花Trihelix转录因子GT-2亚家族。在干旱、高盐和ABA逆境胁迫下,GhGT-2属于依赖于ABA胁迫响应基因调控网络,推测GhGT-2在陆地棉的非生物胁迫适应过程中可能具有重要的作用。

关键词: 棉花, Trihelix转录因子, 非生物胁迫, 亚细胞定位, 转录激活, 凝胶迁移试验

Abstract: 【Objective】Cotton (Gossypium hirsutum L.), the most important textile crop worldwide, often encounters abiotic stresses during its growth season and its productivity is significantly limited by adverse factors. Trihelix transcription factors are important proteins involved in response to abiotic stresses in plants. Understanding the molecular mechanisms of the Trihelix transcription factor genein cotton will lay the foundation for improving the stress tolerance of cotton by gene manipulation.【Method】We searched the cotton ESTs database using BLAST (the Basic Local Alignment Search Tool). One GT unigene, named GhGT-2, was obtained. Sequence analysis of GhGT-2 was performed to confirm that this gene is a member of the Trihelix GT-2 subfamily gene. The total RNA from the leaves of cotton was used as the template to design the degenerate primers based on expressed sequence tags. The full-length cDNA of GhGT-2 were cloned using the method of Rapid amplification of cDNA Ends (RACE) combined with reverse transcription-PCR (RT-PCR). Homologous analysis and multiple alignments were performed with MEGA 5. SMART online tools were used for protein sequence analysis. Seedlings were treated with conditions simulating drought, salinity, cold and abscisic acid (ABA) at the 15-day-old seedling stage in Xin luzao 26. After treatment for 0 , 1 , 3 , 6 , and 12 h, the seedlings were harvested respectively. In the meantime, cotton roots, stems, leaves, flowers, ovules (0 DPA) and fibers (12 DPA) were harvested at different growth periods. The expression profiles of the GhGT-2 in various tissues and under cold, drought, salt and ABA treatments were investigated by quantitative real-time PCR (qRT-PCR) analysis. The GhGT-2【Result】The full-length cDNA sequence was obtained successfully from upland cotton, which was 1 579 bp in lenght, containing a 1 428 bp open reading frame that encoded a protein of 476 amino acids with a predicted molecular weight of 54.07 kD and an isoelectric point of 8.96. SMART analysis showed GhGT-2 contained two typical SANT motifs of GT-2 subfamily. Phylogenetic analysis showed that GhGT-2 belonged to the GT-2 subfamily of the Trihelix family and was most closely related to AtGTL1 and PtaGTL1. Real-time quantitative PCR (qRT-PCR) analysis revealed that GhGT-2 constitutively expresses in all tested tissues, such as roots, stems, leaves, flowers, ovules (0 DPA) and fibers (12 DPA). The expression level of GhGT-2 was highestin leaves, and was very low in roots. The expression profiling revealed that GhGT-2 was induced by ABA, high salt and drought at 3, 6, and 12 h points, but was weakly repressed at 1 h point. In contrast, expression of GhGT-2 was weakly repressed at 3, 6, and 12 h points under cold treatment apart from the 3 h point. These results suggested that GhGT-2 might be involved in stress resistance-related response of ABA signaling pathways in cotton plant. Using the Arabidopsis protoplasts assay system, we found that GhGT-2 protein was located in cell nuclei but its trans-activation activity could not be detected clearly. Electrophoretic Mobility Shift Assay (EMSA) showed that GhGT-2 protein could bind to GT2-Box, GT3-Box, GT-1b (BoxⅡ), MBS1, MRE1, MRE3 and MRE4. 【Conclusion】A full-length cDNA of GhGT-2 was isolated and the typical SNAT binding domain were found in the deduced protein, belonging to the Trihelix GT-2 subfamily. The expression of GhGT-2 was up-regulated under drought, high salt and ABA treatments, showing that GhGT-2 was involved in the ABA dependent stress-responding gene network. It was deduced that GhGT-2 may play important roles in upland cotton plant abiotic stress adaption. coding sequence was cloned onto GFP expressing vector pBI221, and GAL4 DNA-binding domain vector, transformed into Arabidopsis protoplasts to verify the localization in the cell and the transcriptional activation ability of the GhGT-2 protein. The gel-shift assay was performed to detect DNA binding elements.

Key words: cotton (Gossypium hirsutum (EMSA)L.), Trihelix transcription factor, abiotic stress, subcellular localization, trans- activation activity, electrophoretic mobility shift assay