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。
【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.
出处
《中国农业科学》
CAS
CSCD
北大核心
2010年第13期2735-2743,共9页
Scientia Agricultura Sinica
基金
国家自然科学基金项目(30871679)
国家"863"计划重点项目(2006AA100108)
山东省农业良种工程项目
