低氮胁迫下高粱实时荧光定量PCR内参基因的筛选

发布时间：2018-10-24 22:02

【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,sb GAPDH)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。
[Abstract]:Real-time quantitative PCR is widely used to study the transcriptional changes of plant functional genes. The key to obtain accurate analysis results is to select appropriate internal reference genes. The absorption and utilization of nitrogen is an important determinant of sorghum (Sorghum bicolor) yield. In this study, seven common domestic reference genes, actin gene (Actin), polyubiquitin gene (ubiqutin conjugating enzyme,UBQ, glyceraldehyde 3-phosphate dehydrogenase gene (glyceralde-hyde-3-phosphate dehydrogenase,GAPDH), UBQ10, eukaryotic translation initiation factor 2B (eukaryotic translation initiation factor 2BF2B, glyceraldehyde 3-phosphate dehydrogenase geneticizer, were selected. Family (glyceralde-hyde-3-phosphate dehydrogenase,sb GAPDH) and eukaryote translation initiation factor 4a (eukaryotic translation initiation factor 4aEIF4a were used as candidate internal reference genes. Using sorghum roots and leaves as materials, the best internal reference genes of real-time fluorescence quantitative PCR (qRT-PCR) system for sorghum under low nitrogen stress were screened. The results of primer specificity showed that the primers of each candidate internal reference gene met the requirements of stability screening. The results of fluorescence quantitative PCR analysis showed that the expression level and stability of the seven candidate internal reference genes were different with different tissues and nitrogen treatments. The expression abundance of GAPDH in leaves was higher than that of other reference genes, but the abundance of UBQ10 in roots was the highest. The stability of candidate internal reference gene was evaluated by GeNorm software. The results showed that the stability of GAPDH and EIF4a expression in leaves was the highest, while the stability of sbGAPDH and EIF4a. in root system was the highest. The results of this study provide basic data for the analysis of differentially expressed genes related to low nitrogen stress in sorghum, and can be used as a reference for the similar studies of other crops.
【作者单位】：山西省农业科学院生物技术研究中心;农业部黄土高原作物基因资源创制重点实验室;晋中学院;山西农业大学农学院;
【基金】：山西省财政支农项目(No.2015TGSF-10) 山西省科技攻关项目(No.20140311003-4)
【分类号】：S514

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