| 潘畅，张宇宏，庞虹*,2016,孟氏隐唇瓢虫qRT-PCR分析中内参基因的筛选[J].环境昆虫学报,(2):261-270 |
| 孟氏隐唇瓢虫qRT-PCR分析中内参基因的筛选 |
| Selection of the reference genes for gene expression studies in Cryptolaemus montrouzieri ouzieri Mulsant by qRT-PCR |
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| DOI： |
| 中文关键词: 孟氏隐唇瓢虫 qRT-PCR 内参基因 表达稳定性 GeNorm软件 NormFinder软件 BestKeeper软件 |
| 英文关键词:Cryptolaemus montrouzieri ouzieri Mulsant qRT-PCR reference genes expression stability GeNorm NormFinder BestKeeper |
| 基金项目:国家重点基础研究发展计划（973）项目（2013CB127600）；国家自然科学基金面上项目（31171899，31572052）；国家自然科学基金项目（31301720） |
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| 中文摘要: |
| 选择合适的内参基因是qRT-PCR研究的关键。本文以孟氏隐唇瓢虫Cryptolaemus montrouzieri ouzieri Mulsant为研究材料，利用qRT-PCR技术，对孟氏隐唇瓢虫4个候选内参基因Actin、RPS23、GAPDH和β-tubulin的mRNA的表达量进行了分析，并用GeNorm、NormFinder和BestKeeper软件分析它们在孟氏隐唇瓢虫不同发育阶段及成虫不同组织中的表达稳定性。结果表明，以成虫不同组织为材料时，综合三种软件分析结果显示4个候选基因表达稳定性平均等级值排名为RPS23(rank=1)>β-tubulin(rank=2.3)>GAPDH(rank=3)>Actin(rank=3.7)，以不同发育时期虫体为材料时，综合分析结果显示4个候选内参基因表达稳定性平均等级值排名为RPS23(rank=1.7)>Actin(rank=2)>GAPDH(rank=2.7)>β-tubulin(rank=3.7)。综合分析在瓢虫不同发育阶段及成虫不同组织两种处理下，三种软件的评价效果，4个候选基因表达稳定性等级值的总平均排名为RPS23(rank=1.3)>Actin(rank=2.8)=GAPDH(rank=2.8)>β-tubulin (rank=3)。RPS23在瓢虫不同发育阶段及成虫不同组织中均显示出较高的表达稳定性及与其它基因之间极大的相关性，可以确定为孟氏隐唇虫不同发育阶段及成虫不同组织基因表达分析中一个稳定表达的基因，可作为单个内参基因或者其它内参基因的协同基因，本实验为开展孟氏隐唇瓢虫功能基因表达分析奠定了方法学基础。 |
| 英文摘要: |
| Selection of suitable reference genes was a critical step in qRT-PCR analysis In this study, by the method of qRT-PCR, the mRNA levels of four candidate reference genes such as Actin,RPS23,GAPDH and β-tubulin from Cryptolaemus montrouzieri ouzieri Mulsant were assessed. The stability of above four candidate reference genes in different developmental stages and different adult tissues of C.montrouzieri were respectively ranked based on three softwares (GeNorm, NormFinder and BestKeeper). The results showed that, through comprehensive analysis of above three softwares, the mean rank values of the expression stability of above four candidate reference genes were RPS23(rank=1)>β-tubulin (rank=2.3)> GAPDH (rank=3)> Actin(rank=3.7) from C. Montrouzieri in different adult tissues and RPS23(rank=1.7)> Actin (rank=2)> GAPDH (rank=2.7)>β-tubulin (rank=3.7) fromC.montrouzieri in different developmental stages. The average rank values of the expression stability of above four candidate reference genes were RPS23(rank=1.3)> Actin (rank=2.8)= GAPDH (rank=2.8)> β-tubulin (rank=3) in both experimental condition groups .In conclusion, RPS 23 can be determined to be a stable reference gene to be a single reference gene or cooperated reference gene in the gene expression of C.montrouzieri in different developmental stages or different adult tissues as it has the highest stability in both experimental conditions. The work done by this study provides methodological basis for further research on the expression of functional genes from C.montrouzieri |
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