甘蔗赤霉素氧化酶基因ScGA20ox1的克隆及功能分析

Cloning and functional analysis of ScGA20ox1 gibberellin oxidase gene in sugarcane

GA20-氧化酶(GA20-oxidase,GA20ox)是赤霉素(gibberellic acid,GA)合成过程中的关键限速酶,但甘蔗GA20ox1基因(ScGA20ox1)的功能及其表达模式尚未明确。本研究利用RT-PCR和RACE技术克隆了甘蔗GA20-氧化酶基因(ScGA20ox1),ScGA20ox1基因全长1574 bp,含有一个1125 bp的完整开放阅读框(open reading frame,ORF),编码375个氨基酸。ScGA20ox1蛋白分子量为42.3 kD,理论等电点为5.95,不含信号肽,不含跨膜结构域,为可溶性蛋白。实时荧光定量PCR结果表明该基因在甘蔗幼苗中茎秆的表达量最高,叶片次之,根的表达量最低;干旱、低温和赤霉素处理均会改变该基因在不同组织的表达模式。利用农杆菌介导法转化拟南芥,获得过表达ScGA20ox1的转基因拟南芥植株,转基因拟南芥植株存在株高增高,节间长度增长的表型变异。本研究结果表明,ScGA20ox1参与甘蔗的生长发育并在响应非生物逆境中发挥重要调控作用,过表达转基因拟南芥形态发生变化,这为深入探究ScGA20ox1生物学功能,解析甘蔗株型调控分子机制提供理论依据。

GA20-oxidase(GA20ox)is a key rate-limiting enzyme in the synthesis of gibberellic acid(GA),but the biological function and expression pattern of gene GA20ox1 in sugarcane remain unknown.In this study,we cloned GA20-oxidase gene(ScGA20ox1)from the sugarcane variety ROC22 sequence by RT-PCR and RACE,which had a total length of 1574 bp and a complete open reading frame(ORF)of 1125 bp that encoded 375 amino acids.The ScGA20ox1 protein is a hydrophilic protein with a molecular weight of 42.3 kD,an iscelectric point(pI)of 5.95,and no transmembrane structure or signal peptide.Real-time fluorescence quantitative analysis revealed that ScGA20ox1 had the highest expression levels in stems,medium in the leaves,and the lowest in roots of sugarcane seedlings.Drought stress,low temperature stress,and GA3 treatment change the expression patterns of ScGA20ox1 in different tissues.ScGA20ox1 was transformed into Arabidopsis via Agrobacterium-mediated transformation.Transgenic Arabidopsis thaliana plants with overexpressed ScGA20ox1 were obtained from the transformed Arabidopsis thaliana via Agrobacterium-mediated transformation,which showed a phenotypic variation of plant height and internode length increase.In conclusion,ScGA20ox1 was involved in sugarcane’s growth as a crucial regulator in response to abiotic stress and the phenotypic variation of overexpressed transgenic Arabidopsis thaliana changed,which would lay a theoretical basis for future in-depth studies on ScGA20ox1’s biological function and analysis of the molecular mechanism of sugarcane plant shape regulation.