基于转录组信息的黑果枸杞MYB转录因子家族分析

Analysis of MYB Transcription Factor Family Based on Transcriptome Sequencing in Lycium ruthenicum Murr.

【目的】MYB基因家族是植物中最大的一类转录因子家族,广泛参与植物的生长发育和代谢调控过程。目前仍没有针对枸杞等木本作物MYB转录因子家族的系统分析。基于转录组数据鉴定分析黑果枸杞MYB基因家族,可为该类基因生物学功能和代谢调控机制的研究提供参考。【方法】基于黑果枸杞转录组测序(RNA-Seq)数据,利用NR、NT、Swiss-Prot和PFAM 4个数据库和NCBI网站,对黑果枸杞MYB基因进行筛选注释;利用Web Logo3、Prot Comp 9.0、MEGA5.0软件进行保守结构域、亚细胞定位和系统进化等生物信息学分析。基于转录组数据分析MYB基因在黑果枸杞果实不同发育期的差异表达模式,并采用荧光定量PCR方法进行验证。【结果】基于转录组测序数据,注释得到83个黑果枸杞MYB类转录因子基因,根据结构特性将其分为4大类:R2R3-MYB、1R-MYB、3R-MYB和4R-MYB。结构域分析表明:R2R3-MYB类转录因子的R2 MYB基序中包含3个极度保守的色氨酸残基,R3 MYB基序中的第一个色氨酸残基被疏水氨基酸替代。比较分析黑果枸杞MYB家族和拟南芥MYB家族共同构建的进化树发现,黑果枸杞的MYB家族在进化上包括3个大类,6个亚类。亚细胞定位预测结果显示,44个MYB转录因子定位于细胞质中,37个定位于细胞核中。基于转录组数据的MYB差异表达模式分析表明,黑果枸杞MYB基因可能参与了果实不同发育时期花青素变化的调控;基于荧光定量PCR的差异表达数据进一步验证了部分MYB转录因子在果实不同发育时期的花青素合成中可能起到调控作用。【结论】黑果枸杞MYB基因家族注释得到83个黑果枸杞MYB类转录因子基因,为进一步研究MYB家族的基因结构和生物学功能奠定了基础。

[Objective] MYB is one of the most common transcription factor families in plants. It is widely involved in plant growth, and metabolic regulations. So far, there is no systematic analysis of the MYB transcription factor family of tree crops. Analysis of MYB family based on the transcriptome data in Lycium ruthenicum Murr. was conducted in this study, which laid a foundation for the research on biological function, and mechanism of metabolic regulations of MYB genes. [Method] Based on the transcriptome sequencing（RNA-Seq） data, the NR, NT, Swiss-Prot, PFAM and NCBI sites were used at the same time to screen and classify the MYB genes of L. ruthenicum Murr. The Web Logo3, Prot Comp 9.0, and MEGA5.0 were also applied to conservative structure prediction, subcellular localization, and phylogenetic analysis. The expression pattern of MYB genes related to fruit development was obtained and Real-time fluorescence quantitative PCR was used to detect the specific expression of those genes. [Result] Based on the transcriptome sequencing（RNA-Seq） data, 83 transcription factors of MYB family were annotated, selected, and divided into four categories（R2 R3-MYB, 1 R-MYB, 3 R-MYB and 4 R-MYB） according to their structural characteristics. The R2 MYB motif of the R2 R3-MYB transcription factor contains three highly conserved tryptophan residues, and the first tryptophan residue in the R3 MYB motif is replaced by some hydrophobic amino acids. The phylogenetic trees of MYB family of L. ruthenicum Murr. and Arabidopsis thaliana were constructed, which showed that the MYB family of L. ruthenicum Murr. contained three major branches, and six evolutionary branches. The result of the subcellular localization demonstrated that 44 MYB transcription factors were located in the cytoplasm, and 37 MYB transcription factors were located in the nucleus. The analysis of differential expression of MYB genes of L. ruthenicum Murr. based on transcriptome sequencing（RNA-Seq） showed that MYB genes might be involved in the regulation of anthocyanin in three fruit development periods. Additionally, differential expression data based on fluorescence quantitative PCR confirmed that some MYB transcription factors might play a role in the regulation of anthocyanin synthesis in different fruit development periods of L. ruthenicum Murr.. [Conclusion] 83 transcription factors of MYB family were annotated of L. ruthenicum Murr. The findings have laid a foundation for further studies of the structures and biological functions of MYB family.