棉花转录因子GhGT30基因的克隆及转录功能分析

Cloning and Transcription Function Analysis of Cotton Transcription Factor GhGT30 Gene

Trihelix转录因子广泛参与植物生长发育、非生物胁迫应答等一系列生理活动。本研究根据表达序列标签(EST)电子拼接,结合cDNA末端快速扩增技术(RACE)和RT-PCR技术,从棉花中克隆了一个cDNA全长为2210bp的新基因,其开放读码框为2025bp,编码一个675氨基酸的蛋白,分子量约76.26kD,等电点为6.21。SMART蛋白结构预测发现,该基因在N端和C端各有一个trihelix结构域,属于GT-2型trihelix转录因子,被命名为GhGT30(GenBank登录号为JQ013098)。氨基酸序列比对显示,该蛋白和其他高等植物的GT蛋白有较高的同源性。进化树分析表明,GhGT30基因和大豆GmGT-2B基因处在同一进化树分支。拟南芥原生质体中瞬时表达分析表明,GhGT30主要定位于细胞核中。实时荧光定量PCR分析表明,该基因在棉花的花、纤维(12DPA)中的表达量明显高于根、茎、叶及胚珠(0DPA),同时,该基因对干旱、高盐、低温及脱落酸(ABA)等处理都有一定程度的响应,推测该基因对棉花非生物胁迫的调控起重要作用。

Trihelix transcription factors play an important role in the regulation of plant growth and development, as well as in their response to many kinds of abiotic stress. According to expressed sequence tag （EST）, full-length cDNA sequence of trihelix transcription factor was cloned from upland cotton （Gossypium hirsutum L.） using the methods of rapid-amplification of cDNA ends （RACE） and RT-PCR. Sequence analysis showed that the full-length of GhGT30 （GenBank accession No. JQ013098） was 2210 bp, containing a 2025 bp open reading frame which encoded a protein of 675 amino acids with predicted molecular weight of 76.26 kD and a isoelectric point of 6.21. SMART analysis showed GhGT30 with each trihelix domain at N-terminal and C-terminal, belonging to GT-2-type factors. Amino acid sequence alignment revealed that N-terminal of GhGT30 shared high degree of identity with other higher plant GT proteins. The phylogenetic tree showed that GhGT30 was located at the same branch with GmGT-2B. Transient expression of recombinant plasmid GhGT30/PBI221-GFP in Arabidopsis protoplasts showed that GhGT30 was located in cell nuclei. Real-time quantitative PCR （qPCR） showed that GhGT30 was expressed in a higher level in flower, fiber （12 DPA） than in root, stem leaf, and ovule （0 DPA）. The gene was differentially respondes to various abiotic stresses （dehydration, high salinity, and low temperature） and ABA, indicating that it may play important roles in response of cotton plant to abiotic stresses.