Shao Chenxi, Lao Wenhao, Liang Yingmei
The Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, China.
Museum of Beijing Forestry University, Beijing Forestry University, Beijing 100083, China.
J Fungi (Basel). 2022 Aug 9;8(8):830. doi: 10.3390/jof8080830.
Apple rust disease caused by is the one of the major threats to the development of the apple industry in China, but the pathogenic molecular mechanism of the disease remains unclear. It is imperative to screen out appropriate reference genes during the interaction between and apple leaves to analyze the gene expression patterns during the pathogenesis of . , , , , , , , and were selected as candidate reference genes based on the transcriptomic dataset of . The expression levels were tested by real-time quantitative PCR during time-course infection of apple leaves and the expression stabilities were evaluated by Ct method as well as by three software (NormFinder, geNorm and BestKeeper) and one web-based analysis software (RefFinder). The expression stability of the candidate reference genes was further validated by using the effector candidate gene as the target gene in RT-qPCR. According to the results by Ct and BestKeeper, , and were the most stable reference genes, while , and were the most stable reference genes based on the NormFinder analysis result. The geNorm recommended the most stable genes , and as reference genes. Comprehensive analysis results of the RefFinder indicated , and were the most suitable genes. Based on these results, , and were considered as reference genes for analyzing the gene expression profiles of in different infection stages, and the results were consistent with the transcriptome data. All the results suggest that the combination of , and proved to be acceptable reference genes during the interaction between and apple leaves.
由[病原体名称未给出]引起的苹果锈病是中国苹果产业发展的主要威胁之一，但该病的致病分子机制仍不清楚。在[病原体名称未给出]与苹果叶片相互作用过程中筛选出合适的内参基因对于分析[病原体名称未给出]致病过程中的基因表达模式至关重要。基于[数据集名称未给出]的转录组数据集，[基因名称未给出]被选为候选内参基因。在苹果叶片的时间进程感染过程中通过实时定量PCR检测其表达水平，并通过Ct法以及三个软件（NormFinder、geNorm和BestKeeper）和一个基于网络的分析软件（RefFinder）评估其表达稳定性。通过在RT-qPCR中使用效应子候选基因[基因名称未给出]作为靶基因进一步验证候选内参基因的表达稳定性。根据Ct和BestKeeper的结果，[基因名称未给出]是最稳定的内参基因，而基于NormFinder分析结果[基因名称未给出]是最稳定的内参基因。geNorm推荐最稳定的基因[基因名称未给出]作为内参基因。RefFinder的综合分析结果表明[基因名称未给出]是最合适的基因。基于这些结果，[基因名称未给出]被认为是分析[病原体名称未给出]在不同感染阶段基因表达谱的内参基因，结果与转录组数据一致。所有结果表明，在[病原体名称未给出]与苹果叶片相互作用过程中，[基因名称未给出]的组合被证明是可接受的内参基因。
