Screening of Reference Genes of Andrographis paniculata Under MeJA and Abiotic Stresses by Real-time Fluorescence-based Quantitative PCR
10.13422/j.cnki.syfjx.20220313
- VernacularTitle: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
- Author:
Jing-yu LI
1
;
Ming-yang SUN
1
;
Shi-qiang XU
1
;
Yu MEI
1
;
Yan GU
1
;
Fang ZHOU
1
;
Wei SUN
2
;
Ji-hua WANG
1
Author Information
1. Crop Research Institute/Guangdong Provincial Key Laboratory of Crop Genetic Improvement，Guangdong Academy of Agricultural Sciences，Guangzhou 510640，China
2. Institute of Chinese Meteria Medica，China Academy of Chinese Medical Sciences， Beijing 100700，China
- Publication Type:Journal Article
- Keywords:
Andrographis paniculata;
internal reference gene;
real-time fluorescence-based quantitative polymerase chain reaction（Real-time PCR）
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2022;28(5):133-140
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo 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. MethodThe actin 1（ACT1），actin 2（ACT2），elongation factor（EF-1α），glyceraldehyde-3-phosphate dehydrogenase（GAPDH），tubulin（TUB），polyubiquitin（UBQ）， and 18S rRNA（18S）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. ResultThe 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>18S>EF-1α>ACT2>ACT1>GAPDH>TUB under high temperature stress， ACT1>UBQ>EF-1α>18S>ACT2>GAPDH>TUB under drought stress， EF-1α>TUB>ACT2>UBQ>18S>GAPDH>ACT1 under UV stress， and ACT1>EF-1α>UBQ>ACT2>18S>TUB>GAPDH under MeJA stress. Among them，18S 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. ConclusionUBQ-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.
