陆地棉株高相关基因GhGA20ox6的克隆及功能初探

Cloning and preliminary functional analysis of plant height related gene GhGA20ox6 in upland cotton

【目的】对棉花GA20ox基因GhGA20ox6进行克隆和初步的功能验证,分析其对棉花株高的影响。【方法】通过分子克隆的方法,在陆地棉标准系TM-1的茎中成功获得GhGA20ox6基因;利用进化树分析、氨基酸序列比对明确其与拟南芥、水稻中同源基因的进化关系;利用实时定量聚合酶链式反应分析GhGA20ox6在TM-1中的组织特异性表达;利用亚细胞定位分析其在细胞中的定位,并在拟南芥中进行了功能分析。【结果】GhGA20ox6的开放阅读框长度为1155 bp,编码384个氨基酸残基,编码产物的分子质量为43.32 ku,预测的等电点为6.37。进化树分析显示GhGA20ox6与水稻绿色革命基因sd1(OsGA20ox2)的相似性最高。组织特异性表达分析发现,GhGA20ox6在TM-1茎中的表达量高于其在其他器官中的表达量。在烟草表皮细胞的亚细胞定位分析发现,GhGA20ox6蛋白定位在细胞膜。在拟南芥中过表达GhGA20ox6能显著促使赤霉素不敏感矮化基因GID1(gibberellin insensitive dwarf1)表达量上升,转基因拟南芥的株高增加、抽薹提前。【结论】通过对GhGA20ox6的克隆和功能分析,推测它可能与棉花体内的赤霉素合成相关,进而影响棉花株高。这些结果为进一步在分子水平验证GhGA20ox6在棉花生长发育中的生物学功能奠定基础。

[Objective]Gibberellin 20 oxidase gene GhGA20ox6 was cloned and the preliminary functional analysis was performed for plant height.[Method]The GhGA20ox6 gene was successfully cloned from the stem of upland cotton standard line TM-1.The evolutionary relationships of GhGA20ox6 and its homologous genes in Arabidopsis thaliana and rice were confirmed by phylogenetic analysis and multiple alignment of amino acid sequences.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to analyse the expression pattern of GhGA20ox6 in multiple tissues of TM-1.In addition,subcellular localization was used to analyze its specific subcellular localization.And its function in Arabidopsis was analysed.[Result]The open reading frame of GhGA20ox6 is 1155 bp and encodes 384 amino acids.The molecular mass of GhGA20ox6 is 43.32 ku and the predicted isoelectric point is 6.37.Phylogenetic analysis showed that GhGA20ox6 was highly similar to the green revolution gene sd1(OsGA20ox2)in rice.The qRT-PCR results showed that GhGA20ox6 was higher expressed in the stem than in other tissues of TM-1.Furthermore,according to the results of subcellular localization analysis in tobacco epidermd cells,GhGA20ox6 was localized in the cell membrane.Overexpression of GhGA20ox6 in Arabidopsis significantly increased the expression of a downstream gene GID1(gibberellin insensitive dwarf1),resulting in the increase of plant height and earlier bolting.[Conclusion]Based on the cloning and functional analysis of GhGA20ox6,it is speculated that the gene may be related to gibberellin synthesis in cotton,and may affect the plant height of cotton.These results lay a foundation for further molecular verification of the biological function of GhGA20ox6 in cotton growth and development.