Saraiva Kátia D C, Fernandes de Melo Dirce, Morais Vanessa D, Vasconcelos Ilka M, Costa José H
Department of Biochemistry and Molecular Biology, Federal University of Ceara, Campus do Pici, Cx., Postal 6033, Fortaleza, Ceará, 60451-970, Brazil.
Plant Cell Rep. 2014 Sep;33(9):1453-65. doi: 10.1007/s00299-014-1628-1. Epub 2014 May 13.
The EF1α genes were stable in the large majority of soybean tissues during development and in specific tissues/conditions under stress. Quantitative real-time PCR (qPCR) analysis strongly depends on transcript normalization using stable reference genes. Reference genes are generally encoded by multigene families and are used in qPCR normalization; however, little effort has been made to verify the stability of different gene members within a family. Here, the expression stability of members of the soybean EF1α gene family (named EF1α 1a1, 1a2, 1b, 2a, 2b and 3) was evaluated in different tissues during plant development and stress exposure (SA and PEG). Four genes (UKN1, SKIP 16, EF1β and MTP) already established as stably expressed were also used in the comparative analysis. GeNorm analyses revealed different combinations of reference genes as stable in soybean tissues during development. The EF1α genes were the most stable in cotyledons (EF1α 3 and EF1α 1b), epicotyls (EF1α 1a2, EF1α 2b and EF1α 1a1), hypocotyls (EF1α 1a1 and EF1β), pods (EF1α 2a and EF1α 2b) and roots (EF1α 2a and UKN1) and less stable in tissues such as trifoliate and unifoliate leaves and germinating seeds. Under stress conditions, no suitable combination including only EF1α genes was found; however, some genes were relatively stable in leaves (EF1α 1a2) and roots (EF1α 1a1) treated with SA as well as in roots treated with PEG (EF1α 2b). EF1α 2a was the most stably expressed EF1α gene in all soybean tissues under stress. Taken together, our data provide guidelines for the selection of EF1α genes for use as reference genes in qPCR expression analyses during plant development and under stress conditions.
在大豆发育过程中的绝大多数组织以及胁迫下的特定组织/条件下，EF1α基因是稳定的。定量实时PCR（qPCR）分析强烈依赖于使用稳定的内参基因进行转录本标准化。内参基因通常由多基因家族编码，并用于qPCR标准化；然而，对于一个家族中不同基因成员的稳定性验证工作做得很少。在此，对大豆EF1α基因家族成员（命名为EF1α 1a1、1a2、1b、2a、2b和3）在植物发育和胁迫处理（SA和PEG）期间的不同组织中的表达稳定性进行了评估。四个已确定稳定表达的基因（UKN1、SKIP 16、EF1β和MTP）也用于比较分析。GeNorm分析揭示了在大豆发育过程中不同组合的内参基因在组织中是稳定的。EF1α基因在子叶（EF1α 3和EF1α 1b）、上胚轴（EF1α 1a2、EF1α 2b和EF1α 1a1）、下胚轴（EF1α 1a1和EF1β）、豆荚（EF1α 2a和EF1α 2b）和根（EF1α 2a和UKN1）中最稳定，而在三出复叶和单叶以及萌发种子等组织中稳定性较差。在胁迫条件下，未发现仅包含EF1α基因的合适组合；然而，一些基因在SA处理的叶片（EF1α 1a2）和根（EF1α 1a1）以及PEG处理的根（EF1α 2b）中相对稳定。EF1α 2a是胁迫下所有大豆组织中表达最稳定的EF1α基因。综上所述，我们的数据为在植物发育和胁迫条件下的qPCR表达分析中选择用作内参基因的EF1α基因提供了指导。
