【摘要】 【目的】克隆新疆红肉苹果[Malus sieversii f.neidzwetzkyana(Dieck)Langenf]MYB10转录因子基因,进行序列分析和原核表达研究,为进一步探索红色发育机理及选育新的栽培红肉苹果奠定理论基础。【方法】根据MdMYB10基因编码区设计1对特异引物,以新疆红肉苹果叶片总RNA为模板,通过RT-PCR获得1个约700bp的cDNA片段,T/A克隆后进行序列测定,并对该序列进行分析。随后将该基因片段连接到原核表达载体pET-30a(+)中,构建融合表达质粒,转化到E.coli BL21(DE3)中进行表达。【结果】测序结果显示,RT-PCR获得的cDNA全长包含完整的cDNA开放读码框732bp,编码244个氨基酸,命名为MsMYB10。MsMYB10分子量为28.56kD,等电点为8.41,GenBank登录号为GQ500894。该蛋白具有R2R3MYB结构域,结构域中有保守的色氨酸残基,在C端有1个富含酸性氨基酸的转录激活区。与已知MdMYB10的氨基酸序列的同源性为98%。另外,MsMYB10没有信号肽,具有核定位信号。进化树分析表明,MsMYB10与调控花青苷合成的转录因子MdMYB10亲缘关系最近,处在同一进化枝。SDS-PAGE电泳检测结果表明,表达蛋白与预期蛋白大小一致。【结论】克隆了新疆红肉苹果转录因子MsMYB10基因,并可在大肠杆菌中转化表达。为进一步纯化和鉴定目的蛋白及研究其功能奠定了试验基础。更多还原

【Abstract】 【Objective】 Cloning,sequence analysis and expression in E. coli of MsMYB10 gene from Malus sieversii f. neidzwetzkyana was conducted to further explore the red development mechanism and breed cultivars of red-flesh apple. 【Method】 A cDNA fragment about 700 bp was amplified from the total RNA of leaves of Malus sieversii f. neidzwetzkyana by reverse transcription PCR(RT-PCR) with a pair of specific primers based on the sequences of MdMYB10. The recombinant prokaryotic expression vector pET30a-MsMYB10 was constructed by inserting the cDNA fragment into the prokaryotic expression vector pET-30a,and then transformed into E. coli BL21(DE3). 【Results】 Sequence analysis showed that the fragment contains a full coding region of 732 bp encoding 244 amino acid residues with a molecular mass of 28.56 kD. Its GenBank accession number is GQ500894. This deduced protein has a pI of 8.41. Results of the study showed that MsMYB10 exhibited typical features of the R2R3-MYB domain. Tryptophan residues are conserved in R2R3 domain. There is one transcriptional activation domain rich in acidic amino acids in the C-terminal. MsMYB10 exhibits a homology of 99% and 98% with MdMYB10 in nucleotide and amino acid levels,respectively. MsMYB10 has no signal peptide,but a nuclear localization signal. The homology tree showed that MsMYB10 is at the same evolutionary branch with MdMYB10. The SDS-PAGE displays that the expressed proteins consistent with the size of expected protein. 【Conclusion】 MsMYB10 was cloned from Malus and expressed in E. coli. These results have provided a foundation for further purifying and identifying target protein and function study of MsMYB10.更多还原