银杏bHLH91转录因子基因的克隆及表达分析

Cloning and expression analysis of a bHLH91 transcription factor gene from Ginkgo biloba

关键词:  银杏, bHLH, 转录因子, 基因克隆, 表达分析

Key words:  Ginkgo biloba, bHLH, transcription factors, gene clone, express analysis

摘要:

bHLH转录因子在植物的生长发育、胁迫应答和次生代谢中具有重要的调控作用。该研究通过PCR技术从银杏(Ginkgo biloba)叶中分离得到了一个bHLH基因的cDNA序列,并将其命名为GbbHLH91。序列分析结果显示扩增的GbbHLH91基因cDNA序列长度为1 425 bp,开放阅读框是1 065 bp,编码354个氨基酸,分子量为 40.1 kDa,等电点为8.20。系统进化分析结果显示,从用于进化树构建的bHLH蛋白质聚类情况来看,银杏GbbHLH91蛋白与裸子植物油松(Pinus tabuliformis)bHLH蛋白亲缘关系最近,且与被子植物无油樟(Amborella trichopoda)bHLH蛋白相似性达到60%,表明该基因在进化过程中相对比较保守。实时荧光定量PCR分析发现银杏bHLH91基因在银杏的各个组织中均有表达,其中在银杏叶中表达量最高,在根和茎中基因的表达量次之,在银杏雌花和果中表达量较少,在雄花中的表达水平最低; GbbHLH91基因在不同发育时期的银杏叶片中,表达量也存在一定的差异,其中在4月中旬该基因的表达水平达到最高,而后随着叶片的生长发育,该基因的表达水平呈现下降趋势。该研究结果为进一步验证GbbHLH91基因的功能奠定了前期基础。

Abstract:

bHLH transcription factor plays an important role in plant growth, stress response and secondary metabolism. In order to study the function of bHLH transcription factor gene in Ginkgo biloba, we isolated a bHLH gene from G. biloba and carried out bioinformatics and expression analyses. In this study, a cDNA sequence of bHLH gene was isolated from G. biloba by PCR, which was designated as GbbHLH91. DNA sequencing and sequence analysis showed that the amplified GbbHLH91 gene was 1 425 bp, GbbHLH91 gene contained a complete open reading frame, and the open reading frame of GbbHLH91 was 1 065 bp, encoding 354 amino acids. The predicted GbbHLH91 protein had a molecular mass of 40.1 kDa with isoelectric point value of 8.20. Homology analysis with BLASTP and Align X indicated that the putative GbbHLH91 share a high identical with other known bHLH proteins from different plant species. Phylogenetic analysis showed that the GbbHLH91 protein was closely related to bHLH protein from Pinus tabuliformis, and was 60% identity to bHLH from Amborella trichopoda, which indicated the bHLH gene was relatively conserved during evolution. Real-time PCR assay found that GbbHLH91 gene was expressed in all tested tissues of Ginkgo biloba, while expression level in leaf was the highest, followed by root and stem, the GbbHLH91 gene was lowly expressed in female flowers and fruits of G. biloba, and the lowest in male flower. The expression level of GbbHLH91 gene in G. biloba leaves at different developmental stages was also different. The expression level of GbbHLH91 gene was the highest in mid-April, and then the expression level of the gene showed a decreasing trend with the growth and development of G. biloba leaves. These results provide a preliminary basis for further validation of the GbbHLH91 gene function.