Bioinformatics and Construction of Expression Vector for Longan MYB14
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摘要:
目的 克隆龙眼MYB-related基因家族中MYB14基因，并对其进行生物信息学分析和基因表达载体的构建，为进一步研究该基因的生物学功能提供基础数据。 方法 通过转录组测序及生物信息学分析得到龙眼MYB14基因全长序列，进一步设计特异引物对其开放阅读框（ORF）全长序列进行克隆和生物信息学分析，同时构建超表达载体。 结果 MYB14基因属于MYB-related家族的TBP-like亚家族，ORF长度831 bp，编码276个氨基酸，有一个MYB结合位点。该蛋白属于非跨膜亲水蛋白，亚细胞定位预测定位于细胞质，存在57个氨基酸磷酸化位点。其二级结构主要由无规则卷曲以及α-螺旋构成，二级结构与三级结构预测结果高度一致，同源基因进化树分析表明该基因与大麻亲缘关系最近，同时成功构建了植物超表达载体pBI121-MYB14。 结论 本研究克隆得到了龙眼MYB14基因，成功构建表达载体pBI121-MYB14，为该基因功能的研究奠定了坚实的基础。 -
关键词:
- 龙眼 /
- MYB-related /
- 基因克隆 /
- 表达载体构建
Abstract:Objective MYB14 in Longan was cloned, its bioinformatics studied, and expression vector constructed. Method Full-length sequence of MYB14 was obtained by transcriptome sequencing and bioinformatics analysis. Accordingly, specific primers for the cloning were designed, bioinformatics on the open reading frame sequence established, and overexpression vector constructed. Result In the TBP-like subfamily of MYB-related family of genes, MYB14 had an 831 bp ORF encoding 276 amino acids with one MYB binding site. It was a non-transmembrane hydrophilic protein with 57 amino acid phosphorylation sites in the cytoplasm. Its secondary structure was irregular crimp and α-helix. The predicted secondary and tertiary structures were highly consistent. The phylogenetic tree on the gene of longan showed a homology with that of cannabis. Conclusion MYB14 of Longan was successfully cloned with the constructed expression vector, PBI121-MYB14, which provided reference for the further study on the function of MYB14. -
Key words:
- Longan /
- MYB-related /
- gene cloning /
- expression vector construction
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图 1 基因克隆结果检测
注：M：Maker DL2000；1：目的条带。a：cDNA为模板的MYB14 PCR扩增产物。b：pMD18-T MYB14质粒为模板的菌液PCR电泳结果。
Figure 1. Detection of cloned gene
Note: M: Maker DL2000; 1: target band. a: PCR amplification products of MYB14 using cDNA as template. b: electrophoresis of microbial PCR using pMD18-T MYB14 plasmid as template.
图 2 MYB14基因ORF序列及编码蛋白质
注：*为终止子
Figure 2. ORF sequence and encoding protein of MYB14
Note: * was terminator.
图 3 NCBI中龙眼MYB14氨基酸序列保守结构域
Figure 3. Conserved domain in longan MYB14 amino acid sequence by NCBI
图 5 不同物种MYB蛋白三级结构预测
注：A：龙眼MYB14三级结构；B：柑橘MYB组蛋白三级结构；C：大麻类MYB组蛋白三级结构；D：山茶端粒重复结合因子三级结构。
Figure 5. Predicted tertiary structure of MYB proteins from different species
Note: A: predicted tertiary structures of MYB14 in longan; B: that of Citrus sinensis single MYB histone 4-like; C: that of Cannabis sativa single MYB histone 3-like; and, D: that of Camellia sinensis telomere repeat-binding factor 4-like.
图 6 龙眼MYB14蛋白与其他物种MYB蛋白的系统进化树比较
Figure 6. Phylogenetic trees of longan MYB14 and MYB proteins of other species
图 7 不同物种同源基因的氨基酸序列比对结果
Figure 7. Homologous amino acid sequence alignment on genes of different species
图 8 载体构建结果检测
注：M：Maker DL2000；1：目的条带。a：MYB14 PCR扩增条带。b:Npt II PCR 扩增条带。
Figure 8. Detection of constructed vector
Note: M: Maker DL2000; 1: Target band. a: MYB14 PCR amplification band. b: Npt II PCR amplification band.
表 1 引物序列
Table 1. Sequence of primers
引物名称 Primer name 序列（5′-3′）Sequences（5′-3′） 引物用途 Application MYB14 F GCTTATTTTCGTAGCAGAGGCA 基因克隆 Gene Cloning MYB14 R1 CTCTACTTGATGCGAACCATAT MYB14 R2 ACTGTAGTCCGTTACTGTGAGA CBD MYB14-F CACGGGGGACTCTAGAATGGGAAATCCGAAGCAGAAAT（下划线为Xba I酶切位点） 载体构建 Construct Expression Vector CBD MYB14-R TCCTTTACCCATCCCGGGCTATGCCATGATCACAATTTCA（下划线为Sma I酶切位点） MF47 CGCCAGGGTTTTCCCAGTCACGAC 克隆检测 Clone detection MR48 AGCGGATAACAATTTCACACAGGA 
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