二化螟实时荧光定量PCR内参基因筛选和表达稳定性评价

doi:10.16819/j.1001-7216.2019.8035

摘要/Abstract

摘要：

【目的】筛选特定试验条件下二化螟(Chilo suppressalis)稳定表达的内参基因,为二化螟基因表达研究奠定基础。【方法】根据二化螟转录组测序结果挑选出11个候选基因,利用实时荧光定量PCR测定其在二化螟不同发育历期、不同组织、温度处理、杀虫剂处理、取食不同饲料、取食不同水稻、dsRNA处理和混合样品的表达量,利用RefFinder、BestKeeper、GeNorm和NormFinder等软件和ΔC_t值对11个候选基因的稳定性进行评估。【结果】5种分析方法表明,在二化螟不同发育历期稳定性较高的内参基因是AK、RPL10和EF1,不同组织中稳定性较高的内参基因是EF1、TUB和ACTB,不同温度下较稳定的内参基因为TUB、RPL10和EF1,杀虫剂处理样品中较稳定的是TF4和ACTA,取食不同饲料较稳定的是TUB、TF4、EF1和RPL10,取食不同品种水稻较稳定的是TUB和EF1,dsRNA处理样品中较稳定的是TUB、AK、ACTB和EF1,混合样品中较稳定的是EF1、TUB和ACTB。【结论】为不同试验条件下选择合适的内参基因提供了参考,也有利于在二化螟基因表达研究中获得更加可靠准确的数据。

关键词: 二化螟, 内参基因, 荧光定量PCR, 基因表达

Abstract:

【Objective】We aim to screen reference genes stably expressed in striped stem borers (SSB), Chilo suppressalis, under certain conditions, to lay a foundation for the study on the gene expression in SSB. 【Method】According to the results of SSB transcriptome data, 11 candidate genes from SSB were selected and their expression stability was detected under different treatment conditions (including development duration, tissues, feeding on different diets and rice varieties, dsRNA and mixed samples under temperature stress, insecticide treatment), by using real-time fluorescence quantitative PCR. The stability of the 11 candidate genes was evaluated by RefFinder, BestKeeper, GeNorm, NormFinder and ΔC_t. 【Result】AK, RPL10 and EF1 were highly stable in different development durations; EF1, TUB and ACTB were highly stable in different tissues. TUB, RPL10 and EF1 were relatively stable under temperature stress. TF4 and ACTA were stable in insecticide treated samples. TUB, TF4, EF1 and RPL10 were stable in samples fed on different diets. TUB and EF1 were stable reference genes in samples fed on different rice varieties. TUB, AK, ACTB and EF1 were stable in the sample treated with dsRNA. EF1, TUB and ACTB were stable in mixed samples. 【Conclusion】 It provide a reference for selection of suitable reference gene under different test conditions, and also provide more reliable and accurate data for research in gene expression in rice SSB.

Key words: Chilo suppressalis, reference gene, qRT-PCR, gene expression

中图分类号:

徐红星, 王国荣, 鲁艳辉, 杨亚军, 郑许松, 田俊策, 吕仲贤. 二化螟实时荧光定量PCR内参基因筛选和表达稳定性评价[J]. 中国水稻科学, 2019, 33(1): 75-84.

Hongxing XU, Guorong WANG, Yanhui LU, Yajun YANG, Xusong ZHENG, Junce TIAN, Zhongxian LÜ. Screening Reference Genes and Evaluating of Their Expression Stability for qRT-PCR Normalization in Chilo suppressalis (Lepidoptera: Pyralididae)[J]. Chinese Journal OF Rice Science, 2019, 33(1): 75-84.

图/表 6

参考文献 43

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基因名（缩写） Gene name (Gene symbol)	引物序列 Sequence (5′-3′)	基因功能 Putative function
Beta微管蛋白	F: CTCCGACTTACAGTTAGAGC	细胞骨架结构蛋白 Cytoskeleton structural protein	104.2	0.994
Beta-tubulin(TUB)	R: AGTACTGAATCGACAAGCTC	细胞骨架结构蛋白 Cytoskeleton structural protein
延长因子-1α	F: CTGGGTATTGGACAAACTGA	核糖体的组成结构 Structural constituent of ribosome	107.0	0.997
Elongation factor-1α(EF1)	R: GAGGTTCCTGTGATCATGTT	核糖体的组成结构 Structural constituent of ribosome
转录因子4	F: ATTGCTGTGATAAAGAAGAAC	催化GTP结合到核糖体的受体 Catalyze GTP binding to the acceptor of ribosome	98.7	0.993
Transcription factor 4(TF4)	R: AGAAGGTGGTGGACTCAAC
甘油醛-3-磷酸脱氢酶Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)	F: GGGTATTCTTGACTACAC	糖酵解酶Glycolytic enzyme	105.9	0.995
	R: CTGGATGTACTTGATGAG	糖酵解酶Glycolytic enzyme
肌动蛋白A1	F: GTCGCTTCCCAAATTACATC	参与细胞的运动、结构和完整性Involved in cell motility, structure and integrity	103.5	0.987
Actin A1(ACTA)	R: CTCCATATCGTTCCAGTCG
Beta肌动蛋白	F: GATCATGTTTGAGACCTT	参与细胞的运动、结构和完整性Involved in cell motility, structure and integrity	97.6	0.992
Beta-actin(ACTB)	R: GATCTTCATGAGGTAGTC
精氨酸激酶	F: CTGAAGAAGTACCTTACC	细胞能量代谢关键酶Key enzyme F cellular energy metabolism	99.4	0.996
Argininase kinase(AK)	R: CAATCCAGCAGAGTTGAG	细胞能量代谢关键酶Key enzyme F cellular energy metabolism
核糖体蛋白S2	F: CAACGATGAGGTCTTGAAGA	核糖体的组成结构 Structural constituent of ribosome	102.1	0.990
Ribosomal protein S2(RPS2)	R: CGATCTTGTTACCCCAGTAG	核糖体的组成结构 Structural constituent of ribosome
核糖体蛋白S3	F: CGGAGATCATCATTATGG	核糖体的组成结构 Structural constituent of ribosome	103.5	0.989
Ribosomal protein S3(RPS3)	R: GAGTTTGTATCTGAGAGAC	核糖体的组成结构 Structural constituent of ribosome
核糖体蛋白S5	F: TACTGCCATAATCAACTCCG	核糖体的组成结构 Structural constituent of ribosome	105.4	0.992
Ribosomal protein S5(RPS5)	R: TTAGATGAACCCTTAGCAGC	核糖体的组成结构 Structural constituent of ribosome
核糖体蛋白L10	F: GACTTGGGTAAGAAGAAG	核糖体的组成结构 Structural constituent of ribosome	101.4	0.989
Ribosomal protein L10(RPL10)	R: GATGACATGGAATGGATG	核糖体的组成结构 Structural constituent of ribosome

基因名（缩写） Gene name (Gene symbol)	引物序列 Sequence (5′-3′)	基因功能 Putative function
Beta微管蛋白	F: CTCCGACTTACAGTTAGAGC	细胞骨架结构蛋白 Cytoskeleton structural protein	104.2	0.994
Beta-tubulin(TUB)	R: AGTACTGAATCGACAAGCTC	细胞骨架结构蛋白 Cytoskeleton structural protein
延长因子-1α	F: CTGGGTATTGGACAAACTGA	核糖体的组成结构 Structural constituent of ribosome	107.0	0.997
Elongation factor-1α(EF1)	R: GAGGTTCCTGTGATCATGTT	核糖体的组成结构 Structural constituent of ribosome
转录因子4	F: ATTGCTGTGATAAAGAAGAAC	催化GTP结合到核糖体的受体 Catalyze GTP binding to the acceptor of ribosome	98.7	0.993
Transcription factor 4(TF4)	R: AGAAGGTGGTGGACTCAAC
甘油醛-3-磷酸脱氢酶Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)	F: GGGTATTCTTGACTACAC	糖酵解酶Glycolytic enzyme	105.9	0.995
	R: CTGGATGTACTTGATGAG	糖酵解酶Glycolytic enzyme
肌动蛋白A1	F: GTCGCTTCCCAAATTACATC	参与细胞的运动、结构和完整性Involved in cell motility, structure and integrity	103.5	0.987
Actin A1(ACTA)	R: CTCCATATCGTTCCAGTCG
Beta肌动蛋白	F: GATCATGTTTGAGACCTT	参与细胞的运动、结构和完整性Involved in cell motility, structure and integrity	97.6	0.992
Beta-actin(ACTB)	R: GATCTTCATGAGGTAGTC
精氨酸激酶	F: CTGAAGAAGTACCTTACC	细胞能量代谢关键酶Key enzyme F cellular energy metabolism	99.4	0.996
Argininase kinase(AK)	R: CAATCCAGCAGAGTTGAG	细胞能量代谢关键酶Key enzyme F cellular energy metabolism
核糖体蛋白S2	F: CAACGATGAGGTCTTGAAGA	核糖体的组成结构 Structural constituent of ribosome	102.1	0.990
Ribosomal protein S2(RPS2)	R: CGATCTTGTTACCCCAGTAG	核糖体的组成结构 Structural constituent of ribosome
核糖体蛋白S3	F: CGGAGATCATCATTATGG	核糖体的组成结构 Structural constituent of ribosome	103.5	0.989
Ribosomal protein S3(RPS3)	R: GAGTTTGTATCTGAGAGAC	核糖体的组成结构 Structural constituent of ribosome
核糖体蛋白S5	F: TACTGCCATAATCAACTCCG	核糖体的组成结构 Structural constituent of ribosome	105.4	0.992
Ribosomal protein S5(RPS5)	R: TTAGATGAACCCTTAGCAGC	核糖体的组成结构 Structural constituent of ribosome
核糖体蛋白L10	F: GACTTGGGTAAGAAGAAG	核糖体的组成结构 Structural constituent of ribosome	101.4	0.989
Ribosomal protein L10(RPL10)	R: GATGACATGGAATGGATG	核糖体的组成结构 Structural constituent of ribosome