棉花DELLA蛋白GhGAI4b基因的克隆及功能初步分析

[目的]DELLA蛋白已在不同的物种中被广泛鉴定,并且功能区高度保守,是GA信号通路中的负调控因子,在棉花纤维细胞起始和发育中起重要的作用.研究棉花中DELLA基因的功能.[方法]从棉花中克隆DELLA蛋白的同源基因GhGAI4b,构建35S:GhGAI4b植物过量表达载体,通过农杆菌侵染花序的方法转入野生型拟南芥中,观察转基因植株的表型.[结果]序列分析表明GhGAI4b全长开放阅读框1 617 bp,编码538个氨基酸,具有DELLA蛋白的典型结构域.采用Clustal X将GhGAI4b的氨基酸序列与其他已知的DELLA蛋白氨基酸序列进行相似性比对,发现GhGAI4b氨基酸序列与陆地棉、拟南芥的相似性较高,达到47;以上.转基因株系表现出叶片颜色深绿,叶片缩小且肥厚,叶片细长呈锯齿状;有些株系会出现多重莲座叶,雄蕊发育不全,晚花,株高变矮等赤霉素不敏感突变体的表型.[结论]过表达GhGAI4b能够抑制拟南芥莲座叶的生长和发育,影响转基因拟南芥花器官的发育,严重时引起转基因拟南芥的不育.

Cloning and Preliminary Functional Analysis of GhGAI4bGene Encoding DELLA Protein from Gossypium hirsutum

【Objective】DELLA protein has been identified widely in different species and its function areas are highly conserved.It is a negative regulator of GA signaling pathway,and DELLA plays an important role in the initiation and development of the cotton fiber cells.【Method】The gene GhGAI4b encoding the DELLA protein in Gossypium hirsutum is isolated and cloned.We have constructed an over-expression vector 35S:GhGAI4b and transformed the vector into Arabidopsis thaliana L.with the methodologies of Agrobacterium-mediated transformation.Over-expression lines of GhGAI4b are got.【Result】Sequence analysis indicates that GhGAI4b contains a putative ORF which is 1 617 bp in length and encodes 538 amino acids.The typical domains of DELLA proteins are also found in the GhGAI4b proteins.CLUSTAL X analysis shows that the similarity of GhGAI4b’s is greater than 47% between Gossypium hirsutum.L and Arabidopsis.The leaves of transgenic plants are dark green,narrow and thick,thin blade indented;Several transgenic lines have multiple rosette leaves,some of which display stamens infertility,late flower,and severely dwarfs.【Conclusion】Over-expression GhGAI4b can inhibit the growth and development of Arabidopsis’s rosette leaves,influence the development of floral organ,and even more cause the infertility of transgenic Arabidopsis.