蒺藜苜蓿生物钟基因MtTOC1a的克隆及功能验证

Cloning and functional verification of circadian clock gene MtTOC1a in Medicago truncatula

【目的】分析蒺藜苜蓿Medicago truncatula生物钟基因MtTOC1a的蛋白结构,探究MtTOC1a在生物钟系统中的生物学功能,比较其与拟南芥Arabidopsis thaliana的AtTOC1功能相似性和差异性。【方法】通过生物信息学分析,在全基因组范围内鉴定了TOC1在蒺藜苜蓿中的同源基因。构建MtTOC1a基因的表达载体,利用农杆菌介导法引入到拟南芥野生型Col、及相应的功能丧失突变体toc1-2中,进行遗传互补分析。【结果】MtTOC1a和MtTOC1b均具有保守的功能结构域和三维结构。遗传分析表明,在早期光形态建成中,外源转化的MtTOC1a完全恢复了toc1-2的下胚轴伸长表型,但对toc1-2的提前开花表型没有显著影响。在引入CAB::LUC报告基因的株系中,外源转化MtTOC1a在连续光照下使短周期突变体toc1-2的近日节律周期延长,但仍不能完全恢复至野生型水平。【结论】MtTOC1a和拟南芥AtTOC1的功能存在相似性,但在不同的下游调控途径中所扮演的角色存在差异。本研究结果为进一步探索MtTOC1a基因的功能,利用MtTOC1a基因改造苜蓿的重要性状提供了理论依据。

【Objective】The goal of this study is to analyze the protein structure of Medicago truncatula MtTOC1a,explore the biological function of MtTOC1a in the circadian clock system,and compare its similarities and differences in function with its ortholog AtTOC1 in Arabidopsis thaliana.【Method】The orthologous genes of TOC1 in Medicago were identified through bioinformatics analysis,the expression vector of MtTOC1a gene was constructed and introduced into Arabidopsis wild-type Col and the corresponding loss-of-function mutant toc1-2 by Agrobacterium mediated method for genetic complementation analysis.Both MtTOC1a and MtTOC1b have conserved functional domains and protein structures.The genetic analysis indicated that during early photomorphogenesis,exogenously transformed MtTOC1a fully restored the hypocotyl elongation phenotype of toc1-2,but had no significant effect on the premature flowering phenotype of toc1-2.In the CAB::LUC reporter lines,MtTOC1a lengthened the period of the short period mutant toc1-2 under continuous light conditions,yet the mutant could not fully recover to the wild-type level.【Conclusion】MtTOC1a and AtTOC1 have similar functions,but their roles in drownstream pathways are still different.The results provide a theoretical basis for further exploring the function of MtTOC1a gene and using MtTOC1a gene to modify the important traits in Medicago.