非生物胁迫下黄缨菊实时荧光定量PCR内参基因的筛选与验证

Screening and Validation of Reference Genes for Xanthopappus subacaulis by Real-time Quantitative PCR under Abiotic Stress

为了筛选基因分析中黄缨菊(Xanthopappus subacaulis)稳定表达的内参基因,本研究以冷、盐和干旱胁迫下黄缨菊的根、茎和叶为材料,依据三代转录组数据选取8个常用内参基因(18S rRNA,GAPDH,PAL,UBQ,TIP,RPS,SAND,TUA)作为候选基因,利用qRT-PCR技术并结合geNorm,NormFinder,BestKeeper和RefFinder软件综合评价候选内参基因的表达稳定性。结果表明,冷胁迫下黄缨菊根、茎、叶中最佳稳定性的内参基因分别为SAND,TIP和GAPDH;干旱胁迫下最稳定的内参基因分别是SAND,TUA和UBQ;盐胁迫下稳定性最好的内参基因分别为RPS,RPS和SAND。此外,分别以干旱胁迫处理下黄缨菊根、茎、叶中2个稳定性最佳和1个稳定性最差的基因作为内参基因,衡量耐逆基因WRKY表达量的准确性,表明不同胁迫下筛选出的黄缨菊不同组织中内参基因的稳定性结果均是可靠的。研究结果为今后黄缨菊耐逆基因的表达分析提供了基因资源和理论依据。

To screen the steadily expressed reference genes in the gene analysis,we analyzed the reference genes that were stable expression in Xanthopappus subacaulis.We used the root,stem and leaf of Xanthopappus subacaulis as experimental materials under different abiotic stressed,and screened eight candidate internal reference genes based on the full-length transcript sequencing.Meanwhile,we utilized qRT-PCR technology and combined geNorm,NormFinder,BestKeeper,and RefFinder software to comprehensively evaluate the expression stability of candidate reference genes.The results showed that SAND,TIP and GAPDH were the most stable reference genes in root,stem and leaf of X.subacaulis under cold stress,respectively.SAND,TUA and UBQ were the most stable in roots,stems and leaves under drought stress,respectively.RPS,RPS and SAND were the most stable in roots,stems and leaves under salt stress.In addition,we chose two genes with the best stability and one gene with the worst stability in the roots,stems and leaves as internal reference genes under drought treatment to evaluate the accuracy of expression level of the stress-tolerant gene WRKY,the results indicated that the stability of internal reference genes in different tissues of X.subacaulis were reliable under different stresses.The study provides the gene resources and theoretical basis for the expression analysis of stress-tolerant genes of X.subacaulis in the future.