低氮胁迫下高粱实时荧光定量PCR内参基因的筛选

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摘要摘要实时定量PCR技术广泛应用于植物功能基因转录水平变化的研究，选择适当的内参基因是获得准确分析结果的关键。氮元素的吸收和利用是影响高粱(Sorghum bicolor)产量的重要决定因素。本研究选取7个常用内参基因肌动蛋白基因(Actin)、多聚泛素酶基因(ubiqutin conjugating enzyme, UBQ)、甘油醛-3-磷酸脱氢酶基因(glyceralde-hyde-3-phosphate dehydrogenase, GAPDH)、UBQ10、真核生物翻译起始因子2B(eukaryotic translation initiation factor 2B, EIF2B)、甘油醛-3-磷酸脱氢酶基因家族(glyceralde-hyde-3-phosphate dehydrogenase, sbGAPDH)和真核生物翻译起始因子4a(eukaryotic translation initiation factor 4a, EIF4a)作为候选内参基因，以高粱根和叶为材料，筛选低氮胁迫下高粱实时荧光定量 PCR(qRT-PCR)实验体系的最佳内参基因。经引物特异性检测，各候选内参基因引物均符合稳定性筛选要求。荧光定量PCR分析结果表明，7个候选内参基因的表达水平和稳定性因组织和氮处理的不同而呈现差异。叶片中GAPDH较其他内参基因表达丰度高；而在根系中UBQ10表达丰度最高。应用 GeNorm 软件程序对候选内参基因稳定性进行评价。结果表明，内参基因GAPDH和EIF4a在叶片中的表达稳定性最高，而根系中表达稳定度最高的为sbGAPDH和EIF4a。本研究结果为高粱中低氮胁迫相关差异基因的表达分析提供了基础资料，同时可为其他作物的同类研究提供参考。

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	马建华
	孙毅
	王玉国
	段永红

关键词 ：高粱, 低氮胁迫, 内参基因, 荧光定量PCR

Abstract：Abstract Quantitative Real-time PCR is popularly used to study gene transcription levels in plants. Validation of candi-date reference genes is the key step to ensure accurate quantification. Nitrogen element assimilation and utilization play a critical role in sorghum (Sorghum bicolor) yield. The study was to select the most reliable reference genes for quantitative Real-time PCR (qRT-PCR) analysis of target sorghum genes under low nitrogen stress. We chose 7 commonly used housekeeping genes including Actin, ubiqutin conjugating enzyme (UBQ), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), UBQ10, eukaryotic translation initiation factor 2B (EIF2B), glyceraldehyde-3-phosphate dehydrogenase (sbGAPDH) and eukaryotic translation initiation factor 4a (EIF4a) to systematically assess their expression levels in leaves and roots of sorghum under low nitrogen stress by qRT-PCR. The results of primers specificity analysis showed that all the primers were in line with the requirements of the stability screening. The consequences of qRT- PCR indicated that the stability and expression level of candidate genes varied in the different tissues and under different treatments. The expression level of GAPDH was the highest in sorghum leaves, and that of UBQ10 was the highest in roots. We also analyzed and evaluated the stability of candidate reference genes by geNorm software. The results showed that GAPDH and EIF4a were the most suitable reference genes for normalizing target gene expression in sorghum leaves under low nitrogen stress, whereas sbGAPDH and EIF4a were recommended as the optimum combination in roots. In conclusion, this study has provided the basic data for expression analysis of target sorghum genes under low nitrogen stress and reference data for similar studies of other plants.

Key words： Sorghum Low nitrogen stress Reference gene Quantitative Real-time PCR (qRT-PCR)

收稿日期: 2016-11-11 出版日期: 2017-05-02

基金资助:山西省财政支农项目;山西省科技攻关项目

通讯作者: 孙毅 E-mail: sunyi692003@163.com

引用本文:

马建华孙毅王玉国段永红. 低氮胁迫下高粱实时荧光定量PCR内参基因的筛选[J]. , 2017, 25(5): 805-812.

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http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I5/805

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