舒氏猪齿鱼不同组织qPCR内参基因的筛选与验证

Screening and validation of reference genes for qPCR in different tissues of blackspot tuskfish (Choerodon schoenleinii)

  • 摘要:
    目的 筛选在舒氏猪齿鱼不同组织中稳定表达的内参基因。
    方法 选取18个候选内参基因eif2s2、ube2a、actc1、rpl7l1、eif1、eif3a、ppia、rps18、rps27a、snrpd2、taf13、tor1a、tubb4b、rps27l、dera、gapdh、actg1、hsp90ab1,通过实时荧光定量PCR(qPCR)方法对其在舒氏猪齿鱼性腺、肝脏、脾脏、肾脏、肠、心脏、脑、肌肉、皮肤和鳃组织的表达进行了检测,结合BestKeeper、NormFinder、geNorm及RefFinder系统评估了候选基因在不同组织中表达的稳定性。
    结果 18个候选内参基因中,除hsp90ab1扩增效率异常外,其余引物扩增效率为90%–103%,决定系数(R2)均大于0.98,具有良好的扩增特异性。CT值分析显示各基因在不同组织中的表达存在差异,其中eif2s2波动最小而rps27a波动最大。基于BestKeeper、NormFinder、geNorm及RefFinder综合分析,不同算法筛选结果总体一致,均表明eif3aeif2s2、tubb4brps27l稳定性较高,而actc1和gapdh稳定性较差。geNorm分析显示V4/5=0.143,表明在多组织条件下采用4个内参基因可满足标准化要求;在单一组织中,各组织均推荐使用2个内参基因。
    结论 tubb4beif2s2、eif3arps27l在10个组织中表达较为稳定,推荐作为舒氏猪齿鱼不同组织中qPCR实验的内参基因组合,另外,在单个组织中开展目标基因表达研究时,本研究推荐使用双内参组合。本研究为舒氏猪齿鱼基因表达分析及功能研究提供了可靠工具。

     

    Abstract: Blackspot tuskfish (Choerodon schoenleinii) is an economically important coral reef fish with high aquaculture value and a unique protogynous hermaphroditic reproductive pattern. Quantitative real-time PCR (qPCR) is widely used for gene expression analysis, but its accuracy depends on the use of stable reference genes. However, suitable reference genes for C. schoenleinii have not yet been systematically evaluated. To screen for stably expressed reference genes in different tissues of C. schoenleinii, eighteen candidate reference genes (eif2s2, ube2a, actc1, rpl7l1, eif1, eif3a, ppia, rps18, rps27a, snrpd2, taf13, tor1a, tubb4b, rps27l, dera, gapdh, actg1, and hsp90ab1) were selected. Their expression levels in gonad, liver, spleen, kidney, intestine, heart, brain, muscle, skin, and gill tissues were analyzed using qPCR, and expression stability was evaluated using BestKeeper, NormFinder, geNorm, and RefFinder. Among the 18 candidate genes, all except hsp90ab1 exhibited acceptable amplification efficiencies (90% - 103%) and high coefficients of determination (R2 > 0.98), indicating good amplification specificity. CT value analysis revealed variation in expression across tissues, with eif2s2 showing the least variation and rps27a the greatest. Comprehensive analysis using the four algorithms showed generally consistent results, identifying eif3a, eif2s2, tubb4b, and rps27l as the most stable genes, while actc1 and gapdh were the least stable. geNorm analysis indicated that V4/5 = 0.143, suggesting that four reference genes are sufficient for normalization across multiple tissues, whereas two reference genes are adequate for single-tissue analyses. Overall, tubb4b, eif2s2, eif3a, and rps27l were stably expressed across the ten tissues and are recommended as a reference gene combination for qPCR studies in different tissues of C. schoenleinii. For studies focusing on a single tissue, a dual-reference gene strategy is recommended. This study provides reliable tools for gene expression analysis and functional research in C. schoenleinii.

     

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