氨氮胁迫下菲律宾蛤仔肝胰腺内参基因的筛选
Screening of Reference Genes in Hepatopancreas of Clam Ruditapes philippinarum Exposed to Ammonia Nitrogen
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摘要: 利用荧光定量PCR技术对目标基因进行转录水平分析，选择合适的内参基因作为对照是其前提条件。随着氨氮胁迫菲律宾蛤仔毒性效应的深入研究，需要准确评价氨氮对蛤仔肝胰腺组织基因表达的影响。然而，不同浓度氨氮条件下的菲律宾蛤仔肝胰腺组织的内参基因的筛选尚未报道。本研究以2种不同氨氮浓度暴露菲律宾蛤仔14 d的肝胰腺组织cDNA为qRT-PCR的模板，利用geNorm、NormFinder、BestKeeper和RefFinder软件评估β-肌动蛋白（Actin）、延伸因子1-α（EF-1α）、β-微管蛋白（Tubu）、18S核糖体RNA （18S）、泛素（Ubi）和亲环蛋白A （CyPA）6个内参基因的表达稳定性。结果表明，肝胰腺组织中备选内参基因的表达稳定性依次为：Actin>18S>CyPA>Ubi>Tubu>EF-1α，即Actin为表达最稳定的内参基因。本研究为进一步分析氨氮胁迫下菲律宾蛤仔肝胰腺组织的基因表达情况，提供了可靠的内参基因。Abstract: It is necessary to screen a suitable reference gene to quantify the expression levels of targeted genes by fluorescence quantitative PCR technology. With the further toxic study of ammonia nitrogen on clam Ruditapes philippinarum, it is necessary to accurately evaluate the expression profiles of related genes in clam hepatopancreas. However, there's no related report about the expression stability of reference genes in hepatopancreas of R. philippinarum. In this study, clams was exposed to two different concentrations of ammonia nitrogen for 14 d, and the cDNAs of hepatopancreas at different time points were used as qRT-PCR templates. GeNorm, NormFinder, BestKeeper and RefFinder statistical softwares were used to evaluate the expression stabilities of six candidate reference genes, namely beta-Actin (Actin), elongation factor 1-alpha (EF-1α), beta-tubulin (Tubu), 18S rRNA (18S), ubiquitin (Ubi) and cyclophilin A (CyPA). Final results indicated that the expression stability order of the six candidate reference genes in the hepatopancreas were Actin>18S>CyPA>Ubi>Tubu>EF-1α, which means Actin is the most stable reference gene and provides a reliable basis for further study on expression levels of other genes in the hepatopancreas of R. philippinarum under ammonia nitrogen stress.
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Key words:
- ammonia nitrogen /
- Ruditapes philippinarum /
- reference gene /
- screening
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