[关键词]
[摘要]
目的 克隆山茱萸转录因子CobHLH7的cDNA序列。方法 以转录组数据中unigene c15204_g1序列为参考,在其开放阅读框两端设计特异引物,提取山茱萸果实总RNA,并反转录成cDNA,以此为模板,经RT-PCR扩增、连接pMD19-T克隆载体并测序,获得CobHLH7的cDNA序列。通过Protparatam、ProtScale、SOPMA等软件,对其进行生物信息学分析。结果 CobHLH7的cDNA长度为941 bp,编码266个氨基酸,其相对分子质量为29 220,等电点为6.32。经SOPMA在线软件预测,该蛋白为不稳定蛋白,其高级结构以α-螺旋和无规卷曲为主,含有少量的β-转角和延伸带。多序列比对和亲缘关系比较显示,CobHLH7氨基酸序列与马铃薯和模式植物烟草的bHLH类转录因子亚家族的ILR3同源性较高。结论 在首次对山茱萸叶片和果实转录组测序的基础上,对筛选并预测的可能与环烯醚萜苷合成相关的转录因子CobHLH7进行克隆与生物信息学分析,为进一步研究其功能奠定基础。
[Key word]
[Abstract]
Objective Cornus officinalis is a commonly used medicinal material in our country. In the study, the CobHLH7 was cloned and analyzed, which might be closely related to the iridoid glycosides synthesis, based on the transcriptome sequencing of Cornus officinalis fruits. Methods The special primers were designed from the both sides of open reading frame of unigene c15204_g1. The total RNA was extracted and reversed transcribed into cDNA. The transcription factor CobHLH7 was cloned through RT-PCR method, and the pMD19-T cloning vector were used for sequencing. A series of bioinformatics analysis of CobHLH7 was performed by software Protparatam, ProtScale, and SOPMA etc. Results The cDNA of CobHLH7 gene was 941bp in length, encoding 266 amino acids with a molecular weight of 29 220 and isoelectric point of 6.32. The analysis of bioinformatics through SOPMA showed that the protein was a neutral unstable protein. Its advanced structure mainly was alpha helix and random coil, and the content of beta turn and extended strand were less. Multiple sequence alignments and phylogenetic trees showed that CobHLH7 protein has high homology with ILR3 of Solanum tuberosum, ILR3-like of model organism Nicotiana attenuate. Conclusion For the first time, the CobHLH7 cDNA sequence was cloned and analyzed successfully from C. officinalis, which would lay the foundation for studying its biological functions deeply.
[中图分类号]
R282.12
[基金项目]
国家自然科学基金资助项目(U1404829);西藏自治区重点科技计划项目(2015-ZD-NM-0)