基于全基因组鉴定华石斛优良内参基因

Genome-wide Identification of Superior Reference Genes in Den-drobium sinense

华石斛是海南岛特有的热带兰科植物,具有很高的观赏和药用价值。然而,未确定的内参基因限制了该物种基因表达分析。本研究利用之前华石斛转录组数据,筛选出164个变异系数低(CV≤0.2)和表达量中等(TPM在10~300之间)的候选内参基因。为了避免共表达现象对内参基因稳定性分析的影响,根据GO功能聚类分析结果,从不同的功能群中共筛选出24个候选基因。为避免模板中可能残留的基因组DNA在RT-qPCR扩增过程中产生假阳性,本研究设计了跨越内含子引物,并通过电泳筛选出符合要求的15个候选内参基因的扩增引物。为了确保RT-qPCR扩增的特异性,使用LightCycler 96进行熔解曲线分析。依据15个候选基因的RT-qPCR数据,同时综合多个算法分析,在干旱胁迫下,不同软件分别确定CLP1&SEC23、SEC23&CPY71、CLP1&SEC23和RNG1L&PECT为最佳内参基因,综合排序后选取SEC23和CLP1为华石斛干旱胁迫下最稳定内参基因。在华石斛不同组织中,不同软件分别确定了ADF11&IBR5、PRP19&CLP1、ADF11&CLP1、ACBP2&IBR5为最佳内参基因,综合分析后确定ADF11和ACBP2为不同组织中最稳定内参基因。结合上述干旱胁迫和不同组织分析数据,最终确定IBR5和CPY71为综合条件下较稳定的内参基因。内参基因验证表明,与传统的Actin内参相比,本研究筛选出的内参基因可获得更为精确的RT-qPCR校准结果,表明所筛选的内参基因具有极高的稳定性。综上所述,本研究为利用转录组数据鉴定稳定内参基因提供思路,首次在华石斛全基因组范围内鉴定稳定内参基因,为进一步研究基因表达和功能奠定基础。

Dendrobium sinense is a tropical plant endemic to Hainan Island with great ornamental and medicinal values.However,unascertained reference genes restrict the quantitative analysis of gene expression in the species.In this study,using our previous transcriptome data of D.sinense,the 164 candidate genes were identified with low variance(CV≤0.2)and moderate expressions(TPM between 10-300).By GO clustering,24 candidate genes were selected from dif-ferent functional groups to avoid the effect of co-expression on further stability analysis.In order to avoid the false positive of genomic DNA that may remain in the template during RT-qPCR amplification,primers across introns were designed in this study,and amplification primers that met the requirements of 15 candidate reference genes were screened by electrophoresis.To ensure the specificity of RT-qPCR amplification,LightCycler 96 was used for melting curve analysis.According to the RT-qPCR data of 15 candidate genes,four tools were used to evaluate the expression stability.The results of multiple algorithm analysis showed that CLP1&SEC23,SEC23&CPY71,CLP1&SEC23 and RNG1L&PECT were the best reference genes under drought stress.After comprehensive sorting,SEC23 and CLP1 were selected as the most stable reference genes under drought stress.In different tissues of D.sinense,ADF11&IBR5,PRP19&CLP1,ADF11&CLP1,ACBP2&IBR5 were identified as the best reference genes,respectively.After com-prehensive analysis,ADF11 and ACBP2 were identified as the most stable reference genes in different tissues.Com-bined with the above drought stress and different tissue analysis data,IBR5 and CPY71 were finally determined as stable reference genes under comprehensive conditions.The validation of internal reference genes showed that compared with the traditional Actin internal reference,the internal reference genes screened in this study could obtain more accurate RT-qPCR calibration results,indicating that the generated internal reference genes had extremely high stability.In summary,this study provides ideas for identifying stable reference genes using transcriptome data.For the first time,stable reference genes were identified in the whole genome of D.sinense,which laid a foundation for further study of gene expression and function.