铜胁迫下天蓝苜蓿根组织实时定量PCR内参基因的选择  

Reference Gene Selection for Real-time Quantitative PCR in Black Medic(Medicago lupulina L.) Root Tissue Under Copper Stress

作　　者：孙亚丽[1] 张德辉[1] 赵亮[1] 夏聪聪[1] 丑敏霞[1] 

机构地区：[1]西北农林科技大学生命科学学院/旱区作物逆境生物学国家重点实验室,杨凌712100

出　　处：《农业生物技术学报》2014年第10期1223-1231,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31172252)

摘　　要：豆科植物-根瘤菌共生体系因固氮能力卓越,肥土效应明显,常被作为先锋植物进行环境修复。利用qRT-PCR分析铜胁迫下天蓝苜蓿(Medicago lupulina L.)与根瘤菌(Sinorhizobium meliloti,CCNWSX0020)共生固氮过程中差异基因时,由于环境中铜离子差异可能使持家基因表达不稳定,因此需要筛选合适的持家基因作为内参。本研究选取8个天蓝苜蓿的持家基因(肌动蛋白(beta actin,ACT)、组蛋白(histone H2A,H2A)、核糖体蛋白18S(ribosomal 18S,18S)、微管蛋白(tubulin beta,TUB)、泛素连接酶(ubiquitin,UBI)、延伸因子1(elongation factor 1,EF1)、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)和亲环蛋白(cyclophilin,CYP))作为候选内参基因,人工模拟不同水平铜(浓度分别为50、100、150和200 mg/kg)污染,以接种根瘤菌后30 d的天蓝苜蓿根为材料,筛选最佳内参基因。经引物特异性及PCR扩增效率检测,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,8个候选内参基因的表达水平和稳定性随铜离子浓度不同而呈现差异。在线评估所有样品中候选内参基因表达稳定性,结合geNorm软件分析最佳内参基因组合数目,结果发现,最适内参基因组合为GAPDH和EF1;分别在线评估不同铜浓度处理下候选内参基因表达稳定性,结果表明,低浓度铜(≤50 mg/kg)处理下最佳内参基因为GAPDH,中高浓度铜(≥100 mg/kg)处理时最佳内参基因为UBI。本研究结果为铜胁迫下天蓝苜蓿共生结瘤过程中差异基因的表达分析提供了基础资料,同时为其他重金属胁迫下内参基因的筛选提供了参考和借鉴。Legumes are often recognised as the pioneer plants in tailing areas because of their outstanding symbiotic nitrogen fixation.As the copper ions in the environment may lead to unstable expression of housekeeping genes,screening reference genes is important to identify the differentially expressed genes in black medic （Medicago lupulina L.） during the symbiotic nodulation with Sinorhizobium meliloti CCNWSX0020 under copper stress using qRT-PCR.In this study,the eight housekeeping genes（beta actin （ACT）,histone H2A（H2A）,ribosomal 18S（18S）,tubulin beta（TUB）,ubiquitin（UBI）,elongation factor 1（EF1）,glyceraldehyde-3-phosphate dehydrogenase（GAPDH） and cyclophilin（CYP）） from black medic were selected and used to screen the optimal reference genes.The genes were generated from the roots of black medic inoculated rhizobia at 30 d which were planted under the different levels of copper （50,100,150 and 200 mg/kg,respectively）.The analyses of primers specificity and PCR amplification efficiency showed that all the primers were accordance with the requirements of the stability screening.The results of qRT-PCR indicated that the expression level and stability of candidate genes were various along with the different concentration of copper ion.With geNorm software and the online estimation of gene expression stabilities in all samples,the optimal combination of reference genes was found to be GAPDH and EF1.The expressions of candidate genes under different levels of copper were also online assessed separately and the results displayed that the best reference gene was GAPDH at low concentration of copper （≤50 mg/kg） and UBI at moderate or high copper level （≥100 mg/kg）.The conclusions provide the appropriate reference genes for characterizing the genes related to symbiotic nodulation and copper stress of Medicago lupulina L.,and basic data for the plant reference genes under other heavy metal stress.

关 键 词：天蓝苜蓿 内参基因 荧光定量PCR geNorm软件 

分 类 号：Q75[生物学—分子生物学]