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以文心兰叶片为材料,根据Gen Bank中登录的植物肌动蛋白基因同源核苷酸保守序列设计简并引物,利用RT-PCR技术得到了2个动蛋白基因(Actin)片段。序列分析结果表明,2个Actin基因片段长度均为1062 bp,2条序列一致性为90.87%,分别命名为OnA CT1和OnA CT2,并在Gen Bank注册,登录号分别为JN981136和JN981137。2条序列编码完全相同的354个氨基酸,具有Actin蛋白的特征序列;进化树分析表明,文心兰Actin蛋白与萼脊兰和蕙兰的亲缘关系最近。采用实时荧光定量(qR T-PCR)和半定量RT-PCR(Semi-quantitative RT-PCR)方法进行表达分析,结果显示该基因在营养生长和生殖生长阶段的不同组织中表达相对稳定,可以作为发育阶段相关基因表达研究的内参基因。
The degenerate primers were designed according to the conserved sequences of plant actin gene homologs registered in GenBank. Two Actin fragments were obtained by RT-PCR. Sequence analysis showed that the two Actin gene fragments were both 1062 bp in length and 90.87% in sequence identity with the two sequences, which were named OnA CT1 and OnA CT2 respectively and registered in Gen Bank with accession numbers JN981136 and JN981137.2 The deduced amino acid sequence of 354 amino acids had the characteristic sequence of Actin protein. Phylogenetic tree analysis showed that Actin protein was closely related to Calyx and Cymbidium. The results of qR T-PCR and Semi-quantitative RT-PCR showed that the gene expression was relatively stable in different tissues of vegetative and reproductive growth stages and could be used as Development of internal genes related to gene expression studies.