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根据珙桐转录组测序结果,筛选到一个与原花青素合成相关的基因,通过克隆该基因片段并进行序列分析确定该基因编码一个珙桐MYB转录因子,将其命名为DiMYB1(GenBank登录号KR996175)。该基因开放阅读框全长为924 bp,编码产物包含307个氨基酸。对其编码产物的基本理化性质、亲水性和疏水性、保守结构域、亚细胞定位等方面进行了生物信息学分析和预测。对DiMYB1基因编码产物的氨基酸序列进行聚类分析表明,其与拟南芥MYB123转录因子的同源性最高。应用qRT-PCR分析该基因的表达模式发现,DiMYB1基因在珙桐紫色幼叶中的表达量最高,其次是雄蕊,在芽、茎、成熟叶片、苞片、果肉和种子中只有微量表达。另外,DiMYB1基因的表达量在珙桐败育种子中显著高于正常种子,且在中期败育种子中表达量达到最高。构建原核表达载体pET-28a(+)-DiMYB1,并在大肠杆菌BL21(DE3)中获得高效表达。本研究通过对DiMYB1基因进行克隆与表达分析,为探索该基因在珙桐逆境胁迫、花青素合成和种子发育过程中的调控功能奠定基础。
According to the sequencing results of A. involucrata transcriptome, a gene related to proanthocyanidin synthesis was screened. The gene was cloned and sequenced. The gene was identified as DiMYB1 (GenBank accession number KR996175). The full length of the open reading frame of this gene is 924 bp, and the encoded product contains 307 amino acids. Bioinformatics analysis and prediction were made on the basic physical and chemical properties, hydrophilicity and hydrophobicity, conserved domains and subcellular localization of the encoded products. Cluster analysis of the amino acid sequence of the DiMYB1 gene showed that it had the highest homology with Arabidopsis MYB123 transcription factor. The expression pattern of DiMYB1 in young leaves of Davidia involucrata was the highest, followed by the stamens by qRT-PCR. Expression of DiMYB1 was only slightly expressed in shoots, stems, mature leaves, bracts, flesh and seeds. In addition, the expression level of DiMYB1 gene was significantly higher than that of normal seed in Davidia involucrata seed, and reached the highest level in aborted seeds. The prokaryotic expression vector pET-28a (+) - DiMYB1 was constructed and highly expressed in E. coli BL21 (DE3). In this study, we cloned and expressed the DiMYB1 gene, which laid the foundation for exploring the regulatory function of DiMYB1 in the stresses of Davidia involucrate, anthocyanin synthesis and seed development.