棉花GhGT-2转录因子的克隆及其功能分析  

Cloning and Function Analysis of Cotton Transcription Factor GhGT-2

作　　者：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] 

机构地区：[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

出　　处：《中国农业科学》2015年第24期4872-4884,共13页Scientia Agricultura Sinica

基　　金：国家转基因生物新品种培育科技重大专项(2009ZX08009-090B)

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.emblheidelberg.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亲缘关系最近。实时荧光定量PCR表明,GhGT-2在棉花的根、茎�[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 gene in 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-2subfamily 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 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.[Result]The full-length c

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 

分 类 号：S562[农业科学—作物学]