甘蔗近缘野生种蔗茅EfGRAS基因克隆及表达分析

Cloning and Expression Analysis of EfGRAS Gene of Relative Sugarcane Wild Species Erianthus fluvus

为深入挖掘甘蔗品种的抗旱性能,本研究挑选甘蔗品种中参与调控抗旱机制的主要转录因子GRAS的基因进行表达分析。从转录组数据库中调取GRAS转录因子基因序列信息,采用RACE-PCR和qRT-PCR技术从‘蔗茅99-2’中克隆得到1个GRAS基因,将其命名为EfGRAS(GenBank登录号MT499789),并对其进行生物信息学分析与干旱胁迫表达分析。结果表明,EfGRAS基因编码547个氨基酸,CDS全长1644 bp,该蛋白的分子式为C2645H4149N747O816S21,相对分子质量为60.14 kDa,不稳定系数为54.85,脂肪系数为84.37,GRAVY值为‒0.293,是一类不稳定蛋白和亲水蛋白;亚细胞定位表明其位于细胞核内,EfGRAS蛋白主要的二级结构是α-螺旋和无规则卷曲,与高粱亲缘关系最近;qRT-PCR分析显示,受到干旱胁迫后,‘蔗茅99-2’中基因表达量相比于对照组上调约57.6倍,‘滇蔗01-58’中基因表达量相比于对照上调约27.4倍,‘崖城89-9’中基因表达量相比于对照组上调倍数较低。本研究结果为进一步研究EfGRAS基因在甘蔗中的功能提供理论基础。

In order to make deeper research into the drought resistance of sugarcane varieties,the genes of GRAS which playing an important role in the regulation of drought resistance mechanisms in sugarcane varieties for expression analysis were studied.In this experiment,we firstly retrieved the GRAS transcription factor gene sequence information from the transcriptome database,and used RACE-PCR and qRT-PCR techniques to clone a GRAS gene from‘Sucrose 99-2’and namde it EfGRAS(GenBank registration No.MT499789),then the bioinformatics analysis and expression analysis under drought stress were conducted on the EfGRAS gene.The results showed that the EfGRAS gene could encode 547 amino acids with a CDS 1644 bp in length,the molecular formula of the protein was C2645H4149N747O816S21,the relative molecular mass was 60.14 kDa,the instability coefficient was 54.85,the fat coefficient was 84.37,and the GRAVY value was‒0.293,which was a class unstable and hydrophilic protein.The subcellular location indicated that it was located in the nucleus.The main secondary structure of EfGRAS protein wasα-helix and random coils,which were closely related to sorghum;qRT-PCR analysis showed that after drought stress,the gene expression in‘Sugarcane 99-2’was up-regulated by about 57.6 times compared to the control group,and the gene expression in‘DZ 01-58’was up-regulated by about 27.4 times compared to the control group.Compared with the control group,the gene expression level in‘YC 89-9’had a lower fold of up-regulation.The results would provide a theoretical basis for the further study on the function of EfGRAS in sugarcane.