[关键词]
[摘要]
目的 克隆地黄中介体亚基基因RgMed6,分析其编码的蛋白序列特征和基因表达特性。方法 利用地黄转录组数据库进行同源比对,获得拟南芥Med6的同源基因片段,通过序列拼接获得RgMed6的全长开放阅读框(ORF),在NCBI上进行BLASTp获得RgMed6的同源蛋白,用MAFFT进行多序列联配,应用MEGA 6.0构建系统进化树,以实时荧光定量PCR技术检测RgMed6在地黄不同组织以及逆境胁迫下的相对表达量。结果 获得1条924 bp的cDNA序列,包含1个771bp的完整ORF,编码256个氨基酸,具有Med6蛋白的典型结构域和1个潜在的核定位信号序列。RgMed6在检测的5个地黄组织(器官)中均有表达,其中在地黄叶片里表达强度最大,其次为花蕾,在茎中表达量最低。在连作地黄块根中RgMed6的表达量降低,NaCl胁迫处理下RgMed6的表达量升高。结论 首次克隆了地黄中介体亚基基因RgMed6,为阐明其在地黄生长发育和逆境胁迫中的分子功能奠定基础。
[Key word]
[Abstract]
Objective To clone RgMed6 gene, which coded a subunit of mediator complex, from Rehmannia glutinosa, and to analyze the characteristics of protein sequence and gene expression. Methods The transcriptional EST database of R. glutinosa was used to search analogs of AtMed6 gene by BLAST, the full-length open reading frame (ORF) of RgMed6 was obtained by assembling the ESTs. BLASTp, the online analysis tool of NCBI was used to get the homologous sequences of RgMed6, and MAFFT has been performed to analyze the multiple sequence alignment. Phylogenetic tree has been constructed using MEGA 6.0 software. Quantitative RT-PCR has been applied to detecting the transcription level of RgMed6 in five tissues as well as in tuberous roots or leaves under three stresses. Results The cDNA sequence of RgMed6 containing 924 bp was obtained. The ORF of RgMed6 was 771 bp encoding 256 amino acids, which had typical structural domains and a potential nuclear localization signal (NLS). RgMed6 showed the highest expression level in leaves, followed by buds, but very weak in stems. The transcription level of RgMed6 mRNA was reduced under continuous cropping conditions in tuberous roots while it increased under salinity stress in leaves. Conclusion RgMed6, a mediator subunit gene from R. glutinosa has been obtained for the first time, which can lay the foundation for further studies about its molecular function in development and responses to stress.
[中图分类号]
[基金项目]
国家自然科学基金资助项目(81473299,81274022);中国博士后科学基金项目(2013M541977)