小麦烯醇化酶基因 ENO2 的可变翻译分析和原核表达.

[Objective]The objective of this study is to identify the genes(TaENO2s)encoding the enolase family member ENO2 in the important crop plant of wheat(Triticum aestivum L.)and to determine the alternative translation feature and protein enolase activity of TaENO2s.[Method]Wheat TaENO2s were identified by bioinformatics. One of the identified TaENO2s was selected as a representative for protoplast-based exogenous expression and characterization of alternative translation products. This selected TaENO2 was also subjected to prokaryotic expression for purification of recombinant protein. The enolase activity of the purified recombinant protein was determined with in vitro assays.[Result]The hexaploid wheat genome had three homeologous TaENO2s which sequences encoded a conserved enolase catalytic center and had a putative alternative translation start site at the internal ATG codon encoding the residue M94(ATGM94) of the enolase sequence. When exogenously expressed in protoplasts, the representative TaENO2 encoded two proteins, one full-length form of cytoplasmic and nuclear enolase protein and one N-terminal truncated form of nuclear transcriptional repressor TaMBP-1, while the variant of the same gene containing mutated ATGM94 only encoded the full-length form of enolase protein. Soluble recombinant GST-TaENO2 protein expressed in Escherichia coli was successfully purified and verified to have an in vitro enzymatic activity to catalyze the conversion from 2-phosphoglycerate (2-PGA)to phosphoenolpyruvate(PEP)[Conclusion]The wheat genome has three TaENO2 genes encoding the enolase protein ENO2. Under the condition of exogenous expression, TaENO2 encodes two protein products via mRNA alternative translation, one enolase protein translated from the first start codon and one TaMBP-1 protein translated from the internal start codon(2-PGA)to phosphoenolpyruvate(PEP).[Conclusion[The wheat genome has three TaENO2 genes encoding the enolase protein ENO2. Under the condition of exogenous expression, TaENO2 encodes two protein products via mRNA alternative translation, one enolase protein translated from the first start codon and one TaMBP-1 protein translated from the internal start codon ATGM94. The prokaryotically expressed recombinant protein GST-TaENO2 possesses in vitro enolase activity.. The prokaryotically expressed recombinant protein GST-TaENO2 possesses in vitro enolase activity. [ABSTRACT FROM AUTHOR]