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目的克隆地黄中介体亚基基因Rg Med6,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RgM ed6的全长开放阅读框(ORF),在NCBI上进行BLASTp获得RgM ed6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Rg Med6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c DNA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。Rg Med6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RgM ed6的表达量降低,Na Cl胁迫处理下Rg Med6的表达量升高。结论首次克隆了地黄中介体亚基基因Rg Med6,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。
Objective To clone Rehmannia glutinosa intermedium subunit gene Rg Med6 and analyze the characteristics of its encoded protein sequence and gene expression. Methods Homology comparison of Rehmannia glutinosa transcriptome database was used to obtain the homologous gene fragment of Arabidopsis Med6. The full length open reading frame (ORF) of RgM ed6 was obtained by sequence splicing. The homology of RgM ed6 was obtained by BLASTp on NCBI MFA 6.0 was used to construct the phylogenetic tree. Real-time quantitative PCR was used to detect the relative expression of Rg Med6 in different tissues and under stress of Rehmannia glutinosa. Results A 924 bp c DNA sequence was obtained, which contained a complete 771 bp ORF encoding a protein of 256 amino acids with a typical domain of Med6 protein and a potential nuclear localization signal sequence. Rg Med6 was detected in all the five Rehmannia glutinosa tissues (organs), among which the expression intensity was highest in Rehmannia glutinosa leaves, followed by flower buds and the lowest in stems. The expression level of RgM-ed6 was reduced in the root tuber of Rehmannia glutinosa, and the expression of Rg-Med6 was increased under NaCl stress. Conclusions The rhG intermediin subunit gene Rg Med6 was cloned for the first time to lay the foundation for clarifying its molecular function in the growth and development of Rehmannia glutinosa.