不同浓度Cd2+胁迫下烟草实时荧光定量PCR内参基因的筛选  

Screening of internal reference genes for real-time fluorescence quantitative PCR under different concentrations of Cd2+ stress in tobacco

作　　者：池俊玲 赵一博 郭江波 张龙 刘汉阳 辛翠花 CHI Jun-ling;ZHAO Yi-bo;GUO Jiang-bo;ZHANG Long;LIU Han-yang;XIN Cui-hua(School of Life Science and Technology,Inner Mongolia University of Science and Technology,Baotou,Inner Mongolia 014010,China)

机构地区：[1]内蒙古科技大学生命科学与技术学院

出　　处：《南方农业学报》2019年第10期2133-2140,共8页Journal of Southern Agriculture

基　　金：国家自然科学基金项目（31660064,31660414）;内蒙古自然科学基金项目（2018MS03063,2018MS03025）

摘　　要：【目的】筛选出镉(Cd)胁迫下烟草的最佳内参基因,为后续研究与Cd^2+相关的功能性基因提供理论依据。【方法】分别以0、250和500 mg/L氯化镉(CdCl2)溶液对本生烟草进行胁迫处理,利用实时荧光定量PCR(qRT-PCR)检测不同浓度Cd^2+胁迫处理下肌动蛋白基因(ACT)、微管蛋白基因(TUB)、蛋白磷酸酶2基因(PP2A)、18S rRNA、甘油醛-3-磷酸脱氢酶基因(GAPDH)及转录延伸因子基因(EF1a)6个候选内参基因的表达情况,并利用geNorm和Norm-Finder综合评价Ct各内参基因表达的稳定性。【结果】以不同浓度Cd^2+胁迫处理的本生烟草cDNA为模板均可PCR扩增出ACT、TUB、18S rRNA、PP2A、GAPDH和EF1a等6个内参基因。6个内参基因的qRT-PCR扩增曲线走势正常,平行性好,无引物二聚体和非特异性条带产生,且绘制的标准曲线上各点基本分布在一条直线上,扩增效率在要求范围(90.00%~105.00%)之内,符合下一步评价要求,且斜率的回归系数(R2)>0.9800,说明线性关系较好。GAPDH和EF1a基因的表达丰度较高,18S rRNA处于中等水平,ACT、TUB和PP2A基因的表达丰度较低。geNorm分析结果显示,6个内参基因的表达稳定性排序为EF1a基因>GAPDH基因>ACT基因>TUB基因>PP2A基因>18S rRNA;Norm-Finder分析结果显示,6个内参基因的表达稳定性排序为EF1a基因>TUB基因>GAPDH基因=PP2A基因>ACT基因>18S rRNA。【结论】EF1a基因在不同浓度Cd^2+胁迫处理下表达水平较高,稳定性最好,可作为Cd胁迫下本生烟草的最佳内参基因。【Objective】The optimal reference genes for tobacco under cadmium(Cd)stress were screened,which laid the foundation for the subsequent study of functional genes related to heavy metal Cd^2+.【Method】The tobacco was treated with 0,250 and 500 mg/L cadmium chloride(CdCl2)solution,and real-time quantitative PCR were used to detect the expression levels of six commonly used internal reference genes,including actin(ACT),tubulin(TUB),protein phosphatase 2(PP2A),18S rRNA,glyceraldehyde-3-phosphate dehydrogenase(GAPDH)and elongation factor 1-ɑ(EF1a),under different concentrations of Cd^2+stress.And the stability of expression of these internal reference genes were evaluated by the geNorm and NormFinder.【Result】Six internal reference genes of ACT,TUB,18S rRNA,PP2A,GAPDH and EF1a could be amplified by PCR using the tobacco cDNA treated with different concentrations of Cd^2+stress.The qRT-PCR amplification curves of the six internal reference genes showed normal trend,geed parallelism,no primer dimer and non-specfic bands,and the points on the standard curve were basically distributed on a straight line.The amplification efficiency was within the required range(90.00%-105.00%),it met the requirements of the next evaluation,and the slope regression coefficient R^2>0.9800,indicating a good linear relationship.The expression abundances of GAPDH and EF1a were high and more stable,18S rRNA was in the middle,and the expression abundances of ACT,TUB and PP2A were low.The expression stability of EF1a was the highest in the geNorm program under different concentrations of Cd^2+stress,followed by GAPDH,TUB,PP2A,ACT and 18SrRNA.The NormFinder analysis showed that the expression stability rank was EF1a>TUB>GAPDH=PP2A>ACT>18S rRNA.【Conclusion】EF1a has higher expression under different concentrations of Cd^2+stress,and is the most stable one.It can be used as the optimal reference gene for tobacco under different concentrations of Cd stress.

关 键 词：烟草 镉胁迫 实时荧光定量PCR(qRT-PCR) 内参基因 筛选 

分 类 号：S572[农业科学—烟草工业]