Identification of suitable reference genes for studying gene expression in cucumber plants subjected to abiotic stress and growth regulators
Identification of suitable reference genes for studying gene expression in cucumber plants subjected to abiotic stress and growth regulators
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DOI：
10.1007/s11032-010-9487-0
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发表时间：
2011-10-01
影响因子：
3.1
通讯作者：
Papierniak, A.
中科院分区：
文献类型：
--
作者：
Migocka, M.;Papierniak, A.
已经证明，使用半定量技术(例如Northern杂交)被认为是有效参考基因的基因在使用高灵敏的实时聚合酶链式反应(QPCR)或微阵列时似乎不那么可靠。因此，验证参考基因的表达稳定性已成为使用此类分析方法进行任何研究的重要组成部分。到目前为止，还没有参考基因被证实用于黄瓜基因的表达研究。由于这种广泛种植的作物的基因组最近已经测序，迫切需要有合适的参考基因来分析新的黄瓜基因的表达。为了对黄瓜靶基因的表达进行规范化研究，本研究测定了12个参比基因在不同黄瓜组织中以及在不同胁迫和生长调节剂作用下的稳定性。这些基因包括黄瓜常用的参考基因，如肌动蛋白、EF、亲环素、泛素和微管蛋白，以及新发现的候选参考基因，这些基因编码的是笼状蛋白复合体亚单位(CACs)、F-box蛋白、PPA2激活剂(液泡膜固有蛋白，TIP41)、有丝分裂蛋白(YSL8)、蛋白磷酸酶2(PDF2)、解旋酶(HEL)和At4g33380的蛋白同源基因。利用BestKeeper、geNorm和NormFinder三个常用的基于Excel的小程序对实时定量PCR数据进行分析，证实了参考基因的表达稳定性取决于实验参数。此外，他们还发现，除EF外，最稳定的黄瓜基因主要包括新的参考基因：CACS、F-box和TIP41，而常用的内部对照基因表现出多种(肌动蛋白、亲环素、泛素)或更低的稳定性(微管蛋白)。因此，这项研究的作者认为，新的黄瓜参考基因将在未来的黄瓜植物表达研究中更好地标准化和量化转录水平。
It has been shown that genes considered to be valid reference genes using semi-quantitative techniques (e.g. northern blot) appear to be less reliable when highly sensitive real-time PCR (qPCR) or microarrays are used. Therefore, the validation of expression stability of reference genes has become an important component of any study using such types of assay. No reference genes have been validated for expression studies of cucumber genes to date. Since the genome of this widely cultivated crop has been recently sequenced, the availability of suitable reference genes for expression analyses of the new cucumber genes is urgently required. For the purpose of normalization in studying expression of cucumber target genes, the stability of twelve reference genes in different cucumber tissues and under various stresses and growth regulators were determined in this study. These included commonly used cucumber reference genes, such as actin, EF, cyclophilin, ubiquitin and tubulin and the newly identified candidates for reference genes that encode clathrin adaptor complex subunit (CACS), F-box protein, PPA2 activator (tonoplast intrinsic protein, TIP41), mitosis protein (YSL8), protein phosphatase 2 (PDF2), helicase (HEL) and protein homolog of At4g33380. Analyses of quantitative real-time PCR data by three commonly used Excel-based applets, BestKeeper, geNorm and NormFinder, confirmed that expression stability of reference genes depends on the experimental parameters. In addition, they revealed that, except for EF, the most stable cucumber genes included mainly the new reference genes: CACS, F-box and TIP41, whereas the commonly used internal controls demonstrated various (actin, cyclophilin, ubiquitin) or much lower stability (tubulin). Hence, the authors of this study assume that the novel cucumber reference genes will enable better normalization and quantification of transcript levels in future expression studies on cucumber plants.