地熊蜂内参基因的筛选和稳定性研究
Screening and stability analysis of internal reference genes in Bombus terrestris
夏中炎 刘福罡 杨 帆 张志浩 杨俊豪 杨慧鹏 李继莲
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DOI：10.7679/j.issn.2095-1353.2024.096
作者单位：中国农业科学院蜜蜂研究所，国家资源昆虫重点实验室
中文关键词：地熊蜂；内参基因；基因筛选；实时荧光定量PCR；稳定性分析
英文关键词：Bombus teresstris; internal-reference gene; gene selection; quantitative real-time PCR; stability analysis
中文摘要：【目的】 内参基因的选择在昆虫基因表达研究中尤为重要。本研究利用实时荧光定量PCR技术对5个候选内参基因在地熊蜂Bombus terrestris三型蜂的4种组织中的表达进行稳定性评价，筛选出地熊蜂相关基因表达定量研究的最佳内参基因。【方法】 本研究针对GenBank数据库中地熊蜂Actin5C、PLA2（磷脂酶A2）、S18、S28、EF-1a（延伸因子）共5个候选内参基因的核酸序列设计并筛选出特异性引物；标记刚羽化出房的熊蜂工蜂、蜂王和雄蜂，放回原蜂群饲养，分别取1、3、5、7和10日龄蜂的头、卵巢、腹神经索和储精囊4个组织，提取总RNA；反转录PCR克隆候选内参基因片段；以TA克隆将候选内参基因片段克隆进T载体；含有内参基因片段的T载体经测序验证后，作为模板，通过实时荧光定量PCR，优化候选内参基因的扩增条件并绘制候选内参基因荧光定量扩增的标准曲线，在此基础上测定候选内参基因在地熊蜂三型蜂4个组织中的表达量，并以NormFinder、BestKeeper、GeNorm、DCT以及RefFinder 5种软件评价候选内参基因在三型蜂4种组织中的稳定性。【结果】 不同软件对候选内参基因的稳定性排序具有一定差异，以RefFinder综合分析结果显示，蜂王和雄蜂头部、工蜂和雄蜂腹神经索、工蜂卵巢中最稳定的内参基因为PLA2，蜂王卵巢和工蜂头部最稳定的内参基因是Actin5C；蜂王腹神经索中EF-1a最稳定；雄蜂储精囊中最稳定的内参为S18。【结论】 熊蜂内参基因的稳定性因熊蜂级型及组织而异。因此，在进行熊蜂相关基因表达研究时需要根据样本特性选择更稳定的内参基因，以确保结果的可靠性。
英文摘要：[Aim] Selecting appropriate internal reference genes
is crucial for the analysis of gene expression in insects. Using real-time PCR,
this study assessed the expression stability of five candidate reference genes
across four tissue types in bumble bees, aiming to identify the most suitable
reference gene for quantitative gene expression analysis. [Methods] Specific primers were designed for the
five candidate internal reference genes of Bombus terrestris, including Actin5C, PLA2 (phospholipase A2), S18(ribosomal protein S18), S28(ribosomal
protein S28), and EF-1 (elongation factor 1 alpha). Newly emerged
workers, queens, and drones were labeled and reared in their original colonies.
Total RNA was extracted from samples of the head, ovary, ventral nerve cord,
and seminal vesicle taken at 1, 3, 5, 7, and 10 days of age. The fragments of
candidate reference genes were cloned into a pMD19-T vector before being
verified via sequencing. The amplification processes were then optimized and
the standard curves established. The levels of candidate internal reference
genes in queen, worker, and drone tissue samples were quantified using
real-time PCR. The expression stability of the internal candidate internal
reference genes was analyzed using NormFinder, BestKeeper, GeNorm, CT, and
RefFinder. [Results] The
expression stability of the candidate reference genes depended on the method of
analysis used. However, results from a comprehensive analysis using RefFinder
showed that PLA2 had the most stable expression level in the head of queens and
drones, ventral nerve cord of workers and drones, and ovaries of workers. Actin5C was most stably expressed in the ovaries of queens and workers, whereas EF-1 had the most stable expression level in the ventral nerve cord of queens.
Finally, S18 was the stably expressed gene in the seminal vesicle of
drones. [Conclusion] These
findings indicate that the quantitative stability of internal reference genes
varies among castes and tissues of bumble bees. Therefore, it is crucial to
select the most stable internal reference genes based on the sample tissue type
to ensure the reliability of the results.