Selection of reliable reference genes for expression studies by reverse transcription quantitative real-time PCR in litchi under different experimental conditions
Selection of reliable reference genes for expression studies by reverse transcription quantitative real-time PCR in litchi under different experimental conditions
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DOI：
10.1007/s00299-010-0992-8
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发表时间：
2011-02
影响因子：
6.2
通讯作者：
H. Zhong;Jian-wen Chen;Caiqin Li;Lei Chen;Jianyang Wu;Jian-ye Chen;Wang-jin Lu;Jianguo Li
中科院分区：
文献类型：
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作者：
H. Zhong;Jian-wen Chen;Caiqin Li;Lei Chen;Jianyang Wu;Jian-ye Chen;Wang-jin Lu;Jianguo Li
逆转录定量实时PCR（RT-qPCR）是一种用于定量基因表达的灵敏技术，其依赖于用于数据标准化的参考基因的稳定性。在果树中对参考基因的研究很少，在荔枝中则没有。本研究选取了肌动蛋白（ACTIN）、甘油醛-3-磷酸脱氢酶（GADH）、延伸因子1-α（EF-1α）、多聚泛素酶（UBQ）、α-微管蛋白（TUA）、β-微管蛋白（TUB）和RNA聚合酶-II转录因子（RPII）等7个常用的候选参考基因，对其在荔枝中的表达稳定性进行了研究。对78个不同品种、不同组织、不同器官、不同发育时期的番茄样品进行了分析，并对不同处理的番茄样品进行了NAA、遮荫、环剥+去叶等处理。结果表明：GAPDH是所有供试材料、不同器官和NAA处理中最适合的参考基因; ACTIN在品种和果实发育期稳定表达; RPII和UBQ在组织中表达稳定性较好; EF-1α在遮荫和环剥+脱叶处理中表达最稳定。此外，将两个基因组合作为参考基因，可提高RT-qPCR检测荔枝基因表达的可靠性。GAPDH +EF-1α或GAPDH + ACT IN组合对所有受检样品均较好。此外，利用已验证的参考基因，进一步定量了目的基因LcARF 13在不同实验条件下的表达。这些结果首次为不同实验条件下参考基因的选择提供了指导，也为RT-qPCR在荔枝中更准确、更广泛的应用奠定了基础。
Reverse transcription quantitative real-time PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Only a few studies on reference genes have been done in fruit trees and none in litchi. In the present study, seven frequently used candidate reference genes, including actin (ACTIN), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), elongation factor 1-alpha (EF-1α), poly ubiquitin enzyme (UBQ), α-tubulin (TUA), β-tubulin (TUB) and RNA polymerase-II transcription factor (RPII), were evaluated for their expression stability in litchi. A total of 78 samples, including different varieties, tissues, organs, developmental stages and treatments, such as NAA, shading and girdling plus defoliation, were addressed in this analysis. Our results showed thatGAPDHwas the most suitable reference gene among all the tested samples, different organs and NAA treatment.ACTINwas stably expressed in varieties and fruit developmental stages.RPIIandUBQexhibited better expression stability in tissues.EF-1αwas the most stable gene in shading and girdling plus defoliation treatments. Moreover, using combination of two genes as reference genes might improve the reliability of gene expression by RT-qPCR in litchi. A better combination wasGAPDH+EF-1αorGAPDH+ACTINfor all the examined samples. In addition, the validated reference genes were further relied on to quantify the expression of an interested gene,LcARF13under different experimental conditions. These results first provide guidelines for reference genes selection under different experimental conditions and also a foundation for more accurate and widespread use of RT-qPCR in litchi.