穿心莲茉莉酸甲酯及非生物胁迫下Real-time PCR内参基因的筛选

Screening of Reference Genes of Andrographis paniculata Under MeJA and Abiotic Stresses by Real-time Fluorescence-based Quantitative PCR

目的:筛选合适的穿心莲茉莉酸甲酯(MeJA)和非生物胁迫下实时荧光定量聚合酶链式反应(Real-time PCR)检测的内参基因。方法:利用高温、干旱、紫外和MeJA处理的穿心莲转录组数据,挑选到7个候选内参基因肌动蛋白1(ACT1),肌动蛋白2(ACT2),转录延伸因子(EF-1α),甘油醛-3-磷酸脱氢酶(GAPDH),微管蛋白(TUB),多聚泛素酶(UBQ)和18S核糖体RNA(18S),并以上述处理后的穿心莲叶片为实验材料,进行Real-time PCR验证。利用geNorm,NormFinder和BestKeeper3种内参稳定性评估软件进行分析,再利用Refinder软件进行综合分析。结果:3种软件基于不同指标进行稳定性评估,结果并不相同,综合分析得出候选内参基因在高温胁迫下表达稳定性顺序依次为UBQ>18S>EF-1α>ACT2>ACT1>GAPDH>TUB;干旱胁迫下表达稳定性顺序依次为ACT1>UBQ>EF-1α>18S>ACT2>GAPDH>TUB;UV胁迫下表达稳定性顺序依次为EF-1α>TUB>ACT2>UBQ>18S>GAPDH>ACT1;MeJA胁迫下表达稳定性顺序依次为ACT1>EF-1α>UBQ>ACT2>18S>TUB>GAPDH。其中18S基因表达丰度较高,不适合作为内参基因。结合转录组数据对4种胁迫中穿心莲内酯合成相关基因酶羟甲基戊二酰-辅酶A合成酶(HMGS)的相对表达水平验证,发现合适内参基因的Real-time PCR结果更可靠。结论:穿心莲高温、干旱、紫外和MeJA胁迫时,UBQ,ACT1和UBQ,EF-1α和TUB,ACT1和EF-1α分别为最适内参基因组合,为后期开展穿心莲高温、干旱、紫外和MeJA处理中基因的功能调控和表达研究提供了参考。

Objective: To screen the appropriate reference genes for real-time fluorescence-based quantitative polymerase chain reaction(Real-time PCR)analysis of the Andrographis paniculata under methyl jasmonate(MeJA)and various abiotic stresses. Method: The actin 1(ACT1),actin 2(ACT2),elongation factor(EF-1α),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),tubulin(TUB),polyubiquitin(UBQ),and 18 S rRNA(18 S)gene were selected as candidate reference genes based on the RNA-seq data of high temperature,drought,UV,and MeJA. The expression of seven candidate reference genes in the A. paniculata leaves was assessed by Real-time PCR,and the stability was analyzed by geNorm,NormFinder,BestKeeper,and Refinder.Result: The results of stability evaluated by geNorm,NormFinder,and BestKeeper were not the same due to different indicators. As analyzed by Refinder,for the stability of the expression,the genes were ranked as UBQ>18 S>EF-1α >ACT2>ACT1>GAPDH>TUB under high temperature stress,ACT1>UBQ>EF-1α >18 S>ACT2>GAPDH>TUB under drought stress,EF-1α >TUB>ACT2>UBQ>18 S>GAPDH>ACT1 under UV stress,and ACT1>EF-1α >UBQ>ACT2>18 S>TUB>GAPDH under MeJA stress. Among them,18 S gene was not suitable as an internal reference gene duo to its high expressive abundance. This study also verified the relative expression level of andrographolide synthesis-related gene hydroxy-methylglutaryl-CoA synthase(HMGS) in the four stresses on the basis of transcriptome data,and found that the Real-time PCR results of appropriate internal reference genes were accurate and reliable. Conclusion: UBQ-ACT1-UBQ,EF-1α-TUB,and ACT1-EF-1α were the suitable combinations under stresses of high temperature,drought,UV,and MeJA. This study is expected to provide references for the research on function regulation and expression of genes in A. paniculata under high temperature,drought,UV,and MeJA stresses.