›› 2017, Vol. 60 ›› Issue (11): 1266-1277.doi: 10.16380/j.kcxb.2017.11.004

• 研究论文 • 上一篇    下一篇


杨苓, 胡晓静, 徐志峰, 何林, 肖伟*   

  1. (西南大学植物保护学院, 重庆 400716)
  • 出版日期:2017-11-20 发布日期:2017-11-20

Screening of reference genes for qRT-PCR in Conogethes punctiferails (Lepidoptera: Crambidae)

 YANG Ling, HU Xiao-Jing, XU Zhi-Feng, HE Lin, XIAO Wei   

  1. (College of Plant Protection, Southwest University, Chongqing 400716, China)
  • Online:2017-11-20 Published:2017-11-20

摘要: 【目的】筛选特定条件下桃蛀螟Conogethes punctiferails稳定表达的内参基因,为桃蛀螟基因定量研究奠定基础。【方法】参考文献报道,并根据桃蛀螟转录组测序结果筛选出β-肌动蛋白基因ACT、甘油醛-3-磷酸脱氢酶基因GAPDH、核糖体蛋白49基因RP49、α-微管蛋白基因α-tub、核糖体蛋白L13基因RPL13和液泡型ATP合酶基因V-ATPase等6个候选基因。再利用实时荧光定量PCR(qRT-PCR)技术测定6个候选基因在桃蛀螟不同发育时期和成虫不同组织中的表达量;然后通过ΔCt法,GeNorm, NormFinder, BestKeeper和RefFinder 5个软件对6个候选基因的稳定性进行评估;最后以桃蛀螟嗅觉受体基因(olfactory receptor co-receptor, Orco)和性信息素结合蛋白基因 (pheromone binding protein1, PBP1)为目的基因,验证6个候选基因分别在桃蛀螟不同发育时期和成虫不同组织中的的稳定性。【结果】ΔCt法, GeNorm, NormFinder和BestKeeper分析结果表明,4个软件对6个候选基因稳定性的排序相似,在桃蛀螟不同发育时期和成虫不同组织中稳定性较高的3个基因为RP49, RPL13和GAPDH;同时4个软件均判定ACT的稳定性最低。进一步利用RefFinder进行综合分析,结果显示,在桃蛀螟成虫不同组织中,最稳定的基因为RPL13,其次为RP49;而在不同发育时期,最稳定的基因为RP49,其次为GAPDH。另外经GeNorm软件计算得出,在桃蛀螟不同发育时期和成虫不同组织中桃蛀螟内参基因的最佳数目为2。最后,以嗅觉受体基因 OrcoPBP1为目的基因,对筛选出的候选基因的稳定性进行验证,发现当使用RPL13和RP49以及RP49和GAPDH作为内参基因时,OrcoPBP1的表达量规律一致,并与桃蛀螟生活习性及前人研究结果一致;而使用ACT作为内参基因计算得到的OrcoPBP1表达量则不规律。【结论】在不同发育时期桃蛀螟基因定量研究中建议以RP49和GAPDH作为内参基因,而在桃蛀螟成虫不同组织中基因定量研究中建议以RPL13和RP49作为内参基因。

关键词: 桃蛀螟, 内参基因, 基因筛选, 表达稳定性, 实时荧光定量PCR

Abstract: 【Aim】 This study aims to screen and verify stably expressed genes under given conditions as reference genes for quantitative real-time PCR (qRT-PCR) in Conogethes punctiferalis, so as to provide the basis for quantitative studies of genes of this moth. 【Methods】 Based on transcriptomics sequencing results in C. punctiferalis and reported reference genes in other insect species, six candidate genes including β-actin gene (ACT), glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH), ribosomal protein 49 gene (RP49), alpha tubulin gene (α-tub), ribosomal protein L13 gene (RPL13) and vacuolar-type ATPase gene (V-ATPase) were cloned, and their expression levels in different developmental stages and different adult tissues were measured by qRT-PCR. Then the stabilities of these candidate genes were evaluated by a series of programs including ΔCt method, GeNorm, NormFinder, BestKeeper and an online program, RefFinder. Finally, the stabilities of selected reference genes were validated with an olfactory receptor co-receptor gene (Orco) and pheromone binding protein 1 gene (PBP1). 【Results】 Analyzed by four programs including ΔCt method, GeNorm, NormFinder and BestKeeper, similar rankings of six candidate genes were obtained, among which RP49, RPL13 and GAPDH were the most stable genes in different developmental stages and different adult tissues, and ACT was ranked as the least stable gene despite of experimental conditions by all programs. Comprehensive analysis with RefFinder further showed that in different adult tissues, RPL13 was the most stable gene, followed by RP49, and in different developmental stages, RP49 was the most stable gene, followed by GAPDH. Additionally, the optimal number of reference genes was calculated by GeNorm as 2. Finally, the stabilities of selected reference genes were validated with Orco and PBP1 as target genes. The results indicated that when two pairs of genes, RPL13 and RP49, and RP49 and GAPDH, were respectively used as reference genes, Orco and PBP1 showed reliable expression patterns, which were consistent with life habits of C. punctiferalis and the results of the previous research. The expression patterns of Orco and PBP1, however, were irregular when ACT was used as the reference gene. 【Conclusion】 In C. punctiferalis, two pairs of genes, RPL13 and RP49, and RP49 and GAPDH, are recommended as reference genes in different adult tissues and different developmental stages, respectively.

Key words: Conogethes punctiferails, reference genes, gene screening, expression stability, quantitative real-time PCR