黄脊竹蝗荧光定量PCR内参基因的鉴定与筛选

doi:10.11707/j.1001-7488.20220108

摘要/Abstract

摘要：

目的: 基于黄脊竹蝗若虫不同龄、成虫不同性别及不同组织筛选稳定表达的内参基因, 为黄脊竹蝗后续转录调控研究提供参考。方法: 依据黄脊竹蝗转录组测序结果, 以TUB、RPS27A、RPL10、AK、GAPDH、EF1A、RPS3、Actin和18S rRNA常用的9种昆虫内参基因作为候选, 本地Blast后获得碱基序列并设计引物。利用GeNorm、NormFinder和BestKeeper软件分析这9种内参基因的稳定性, 筛选出在若虫不同龄、成虫不同性别与不同组织(触角、头、唇须、肌肉组织、精/卵巢)中表达量最为稳定的内参基因。结果: 9种候选内参基因在黄脊竹蝗体内均为首次验证并报道, 且与其他昆虫相应基因同源性高(高于70%), 差异值小; 9种候选内参基因的引物均具有良好的扩增效率(均在90%~120%); 在所有样品中, 18S rRNA表达水平最高(C_t = 10.78 ± 2.58, P < 0.05), Actin表达水平差异值最大(C_t = 23.55 ± 3.84, P < 0.05)。黄脊竹蝗不同龄若虫的内参基因稳定性分析时, 3个软件排在前2位的内参基因均为RPS3与RPL10; 基于不同组织分析时, GeNorm与BestKeeper软件表明RPL10是最合适内参基因, 而NormFinder软件则表明GAPDH为最合适内参基因; 在黄脊竹蝗成虫不同性别分析时, 3种软件评估结果均表明GAPDH和RPL10为合适内参基因。结论: 筛选出RPL10为在不同条件下于黄脊竹蝗体内表达稳定并具有差异性的基因, 可作为黄脊竹蝗转录组学及功能基因组学研究的内参基因。

关键词: 黄脊竹蝗, 内参基因, 基因筛选, 荧光定量PCR

Abstract:

Objective: To provide a reference for further transcriptomics studies of the yellow-spined locust, Ceracris kiangsu, the reference genes were screened based on different instars, different genders of adults and different tissues of C. kiangsu. Method: According to the transcriptome sequencing result, nine internal reference genes commonly used in insects, TUB, RPS27A, RPL10, AK, GAPDH, EF1A, RPS3, Actin, and 18S rRNA were chosen as candidates. The base sequences were obtained, and primers were designed after local Blast. GeNorm, NormFinder and BestKeeper were used to analyze the stability of the nine reference genes, and then the most suitable reference genes were screened according to the expression levels in different instars, genders of aduits, and tissues (antennae, head, labial palp, muscle tissue, testis/ovary) of adults. Result: Nine candidate reference genes of C. kiangsu were verified and reported for the first time, and they showed high homology (more than 70%) with the corresponding genes of other insects. The primers for the nine candidate reference genes all had good amplification efficiency (within 90%- 120%). In all samples, the expression level of 18S rRNA was the highest (C_t value = 10.78 ± 2.58, P < 0.05), and the difference in Actin expression level was the largest (C_t= 23.55 ± 3.84, P < 0.05). In analyzing the stability of the reference genes of C. kiangsu at different stages of nymphs, the top two of the three software were RPS3 and RPL10. Based on the analysis of different tissues, RPL10 was the most suitable reference gene suggested by GeNorm and BestKeeper, while GAPDH was most suitable one according the analysis of NormFinder. In terms of the analysis of different genders of the C. kiangsu adults, GAPDH and RPL10 were proved to be the two most suitable reference genes by the three software. Conclusion: RPL10 with stable and different expression under various conditions in C. kiangsu has bee screened as reference gene, which can be used as an internal reference gene for the research of transcriptomics and functional genomics for C. kiangsu.

Key words: Ceracris kiangsu, reference gene, gene screening, RT-qPCR

中图分类号:

S718.7

方林鑫,李志红,张守科,张威,舒金平,王浩杰. 黄脊竹蝗荧光定量PCR内参基因的鉴定与筛选[J]. 林业科学, 2022, 58(1): 70-77.

Linxin Fang,Zhihong Li,Shouke Zhang,Wei Zhang,Jinping Shu,Haojie Wang. Identification and Selection of the Internal Reference Genes of Ceracris kiangsu (Orthoptera: Arcypteridae) by RT-qPCR[J]. Scientia Silvae Sinicae, 2022, 58(1): 70-77.

图/表 7

表1

表2

图1

图2

图3

表3

表4

参考文献 0

	陈孝仁, 张博月, 邢玉平, 等. 恶疫霉实时定量RT-PCR分析中内参基因的选择. 中国农学通报, 2012, 28 (30): 201- 207.
	Chen X R , Zhang B Y , Xing Y P , et al. Selection of internal control genes for real-time RT-PCR data normalization in Phytophthora cactorum. Chinese Agricultural Science Bulletin, 2012, 28 (30): 201- 207.
	崔淼, 刘晓健, 李涛, 等. 五龄飞蝗不同发育时间实时定量PCR内参基因的筛选. 应用昆虫学报, 2014, 51 (3): 733- 740.
	Cui M , Liu X J , Li T , et al. Selection of reference genes on different days during the development of the fifth-instar nymph of Locusta migratoria with quantitative real-time PCR. Chinese Journal of Applied Entomology, 2014, 51 (3): 733- 740.
	杜畅. 2013. 石蚕ST突变体近等基因系与其回交亲本的蛋白质组学差异分析. 合肥: 安徽农业大学.
	Du C. 2013. Comparative proteomic analysis between the near-isogenic line of silkworm stony (st) mutant and its backcross parent. Hefei: Anhui Agricultural University. [in Chinese]
	冯波, 郭前爽, 毛必鹏, 等. 松墨天牛化学感受组织荧光定量PCR内参基因的鉴定与筛选. 昆虫学报, 2016, 59 (4): 427- 437.
	Feng B , Guo Q S , Mao B P , et al. Identification and selection of valid reference genes for assaying gene expression in the chemosensory tissues of Monochamus alternatus (Coleoptera: Cerambycidae) by RT-qPCR. Acta Entomologica Sinica, 2016, 59 (4): 427- 437.
	郭长宁. 2014. 金纹细蛾内参基因克隆分析及稳定性评价. 杨凌: 西北农林科技大学.
	Guo C N. 2014. Cloning and stability analysis of reference gene from Phyllonorycter ringoniella. Yangling: Northwest Agricultural and Forestry University. [in Chinese]
	贾冰, 马亿, 庞保平, 等. 牧草盲蝽实时定量PCR内参基因的筛选. 昆虫学报, 2019, 62 (12): 1379- 1391.
	Jia B , Ma Y , Pang B P , et al. Screening of reference genes for quantitative real-time PCR in Lygus pratensis (Hemiptera: Miridae). Acta Entomologica Sinica, 2019, 62 (12): 1379- 1391.
	刘小强, 蒋红波, 李慧敏, 等. 赤拟谷盗章鱼胺受体3(TcOctβR3)cDNA克隆, 表达及功能. 中国农业科学, 2018, 51 (7): 1315- 1324.
	Liu X Q , Jiang H B , Li H M , et al. The cDNA cloning, expression profiling and functional characterization of octopamine receptor 3 (TcOctβR3) in Tribolium castaneum. Scientia Agricultura Sinica, 2018, 51 (7): 1315- 1324.
	马飞. 2012. 极大节旋藻实时荧光定量PCR内参基因的选择. 青岛: 中国海洋大学.
	Ma F. 2012. Reference genes for Arthrospira maxima when using quantitative real-time PCR. Qingdao: Ocean University of China. [in Chinese]
	史彩华, 张友军. 内参基因在昆虫实时荧光定量PCR中的研究进展. 应用昆虫学报, 2016, 53 (2): 237- 246.
	Shi C H , Zhang Y J . Advances in reference gene for real-time quantitative reverse transcription PCR (qRT-PCR) of insects research. Chinese Journal of Applied Entomology, 2016, 53 (2): 237- 246.
	陶蓉, 李慧, 孙宇航, 等. 美国白蛾内参基因的鉴定及筛选. 林业科学, 2019, 55 (9): 111- 120.
	Tao R , Li H , Sun Y H , et al. Identification and screening of internal reference genes of Hyphantria cunea (Lepidoptera: Arctiidae). Scientia Silvae Sinicae, 2019, 55 (9): 111- 120.
	滕莹. 2012. 黄脊竹蝗"趋尿"的行为及化学机制研究. 北京: 中国林业科学研究院.
	Teng Y. 2012. Study on the behavioral and chemical mechanisms for "urine-puddling" of yellow-spined bamboo locust. Beijing: Chinese Academy of Forestry. [in Chinese]
	王金星, 张利军, 廖资亿, 等. 刺槐实时定量PCR分析中内参基因的选择. 林业科学, 2014, 50 (9): 167- 172.
	Wang J X , Zhang L J , Liao Z Y , et al. The selection of reference genes for real-time quantitative PCR normalization in black locust (Robinia pseudoacacia). Scientia Silvae Sinicae, 2014, 50 (9): 167- 172.
	徐红星, 王国荣, 鲁艳辉, 等. 二化螟实时荧光定量PCR内参基因筛选和表达稳定性评价. 中国水稻科学, 2019, 33 (1): 75- 84.
	Xu H X , Wang G R , Lu Y H , et al. Screening reference genes and evaluating of their expression stability for qRT-PCR normalization in Chilo suppressalis (Lepidoptera: Pyralididae). Chinese Journal of Rice Science, 2019, 33 (1): 75- 84.
	徐天森, 王浩杰. 中国竹子主要害虫. 北京: 中国林业出版社, 2004.
	Xu T S , Wang H J . Main pests of bamboo in China. Beijing: China Forestry Publishing House, 2004.
	张守科, 张威, 舒金平, 等. 黄脊竹蝗取食偏好与竹叶品质的关系. 生态学杂志, 2017, 36 (8): 2266- 2273.
	Zhang S K , Zhang W , Shu J P , et al. Relationship between the feeding preference of yellow-spined bamboo locust, Ceracris kiangsu and bamboo leaf quality. Chinese Journal of Ecology, 2017, 36 (8): 2266- 2273.
	张威, 张守科, 舒金平, 等. 昆虫趋泥行为的研究进展. 林业科学研究, 2018, 31 (1): 150- 157.
	Zhang W , Zhang S K , Shu J P , et al. A review on mud-puddling behavior of herbivorous arthropods. Forest Research, 2018, 31 (1): 150- 157.
	Andersen C L , Jensen J L , Ørntoft T F . Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research, 2004, 64 (15): 5245- 5250. doi: 10.1158/0008-5472.CAN-04-0496
	Cao S N , Zhang X W , Ye N H , et al. Evaluation of putative internal reference genes for gene expression normalization in Nannochloropsis sp. by quantitative real-time RT-PCR. Biochemical and Biophysical Research Communications, 2012, 424 (1): 118- 123. doi: 10.1016/j.bbrc.2012.06.086
	Dzaki N , Ramli K N , Azlan A , et al. Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti. Scientific Reports, 2017, 7, 43618.
	Ferdous A S , Islam M T , Alam S S , et al. Identification of stable reference genes for quantitative PCR in jute under different experimental conditions: an essential assessment for gene expression analysis. Australian Journal of Crop Science, 2015, 9 (7): 646- 655.
	Huggett J , Dheda K , Bustin S , et al. Real-time RT-PCR normalisation; strategies and considerations. Genes and Immunity, 2005, 6 (4): 279- 284. doi: 10.1038/sj.gene.6364190
	Ibanez F , Tamborindeguy C . Selection of reference genes for expression analysis in the potato psyllid, Bactericera cockerelli. Insect Molecular Biology, 2016, 25 (3): 227- 238. doi: 10.1111/imb.12219
	Janská A , Hodek J , Svoboda P , et al. The choice of reference gene set for assessing gene expression in barley (Hordeum vulgare L.) under low temperature and drought stress. Molecular Genetics and Genomics, 2013, 288 (11): 639- 649. doi: 10.1007/s00438-013-0774-4
	Kozera B , Rapacz M . Reference genes in real-time PCR. Journal of Applied Genetics, 2013, 54 (4): 391- 406.
	Liang P, Guo Y J, Zhou X G, et al. 2014. Expression profiling in Bemisia tabaci under insecticide treatment: indicating the necessity for custom reference gene selection. PLoS One, 9(1): e87514.
	Lü J , Yang C X , Zhang Y J , et al. Selection of reference genes for the normalization of RT-qPCR data in gene expression studies in insects: a systematic review. Frontiers in Physiology, 2018, 9, 1560. doi: 10.3389/fphys.2018.01560
	Lu Y H , Yuan M , Gao X W , et al. Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae). PLoS One, 2013, 8 (7): e68059. doi: 10.1371/journal.pone.0068059
	Lu Y H , Zheng X S , Liang Q , et al. Evaluation and validation of reference genes for SYBR Green qRT-PCR normalization in Sesamia inferens (Lepidoptera: Noctuidae). Journal of Asia-Pacific Entomology, 2015, 18 (4): 669- 675.
	Mughal B B , Leemans M , Spirhanzlova P , et al. Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis. Scientific Reports, 2018, 8 (1): 496.
	Pfaffl M W , Tichopad A , Prgomet C , et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnology Letters, 2004, 26 (6): 509- 515.
	Qu C, Wang R, Che W N, et al. 2018. Selection and evaluation of reference genes for expression analysis using quantitative real-time PCR in the Asian Ladybird Harmonia axyridis (Coleoptera: Coccinellidae). PLoS ONE, 13(6): e0192521.
	Radonić A , Thulke S , Mackay I M , et al. Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications, 2004, 313 (4): 856- 862.
	Sękalska B , Ciechanowiez A , Dolegowska B , et al. Optimized RT-PCR method for assaying expression of monocyte chemotactic protein type 1 (MCP-1) in rabbit aorta. Biochemical Genetics, 2006, 44 (3/4): 133- 143.
	Shakeel M , Rodriguez A , Tahir U B , et al. Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects. Biotechnology Letters, 2018, 40 (2): 227- 236.
	Shivhare R , Lata C . Selection of suitable reference genes for assessing gene expression in pearl millet under different abiotic stresses and their combinations. Scientific Reports, 2016, 6 (1): 23036.
	Shu B S , Zhang J J , Cui G F , et al. Evaluation of reference genes for real-time quantitative PCR analysis in larvae of Spodoptera litura exposed to azadirachtin stress conditions. Frontiers in Physiology, 2018, 9, 372.
	Vandesompele J, De Preter K, Pattyn F, et al. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3(7): research0034.
	Wan H J , Zhao Z G , Qian C T , et al. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Analytical Biochemistry, 2010, 399 (2): 257- 261.
	Wang J X , Abbas M , Wen Y Z , et al. Selection and validation of reference genes for quantitative gene expression analyses in black locust (Robinia pseudoacacia L.) using real-time quantitative PCR. PLoS ONE, 2018, 13 (3): e0193076.
	Yan J W , Yuan F R , Long G Y , et al. Selection of reference genes for quantitative real-time RT-PCR analysis in citrus. Molecular Biology Reports, 2012, 39 (2): 1831- 1838.
	Yuan M , Lu Y H , Zhu X , et al. Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR. PLoS ONE, 2014, 9 (1): e86503.
	Zhang S D , An S H , Li Z , et al. Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera (Lepidoptera: Noctuidae). Gene, 2015, 555 (2): 393- 402.

基因名称 Gene name	比对物种 Hit species	比对基因登录号 Hit_accession No.	E值 E value	比对长度 Hit_length/bp	一致性 Identity (%)
TUB	松褐天牛 Monochamus alternatus	KU521361.1	0	1 165	79.08
RPS27A		KU521362.1	4E-98	344	84.16
RPL10		KU521363.1	3E-125	432	76.62
AK		KU521364.1	0	791	76.58
GAPDH		KU521365.1	0	619	74.47
RPS3		KU521367.1	2E-116	404	74.29
Actin		KU521368.1	0	1 229	83.98
18S rRNA		KU521369.1	0	2 627	89.61
EF1A	双栉果蝇 Drosophila bipectinata	XM017244340.1	0	624	70.71

基因 Gene	上游引物(5’ - 3’) Forward primer (5’ - 3’)	下游引物(3’ - 5’) Reverse primer (3’ - 5’)	PCR产物长度 Amplicon size/DP	斜率 Slope	判定系数 Correlationcoefficient (R²)	扩增效率 Amplificationefficiency (E)(%)
TUB	AGGTGGCGATGACAGCTTTA	AGCTTGCGAATACGGTCCAG	246	-3.043	0.988	113.119
RPS27A	ATGGCCGAACATTGTCCGAT	AGCCATGAAGACTCCAGCAC	245	-3.334	0.993	99.499
RPL10	GGTGCTGTGCCTTTCTGTTG	GGTGGTCCCATCGTTGACTT	156	-3.340	0.993	99.251
AK	ACTTGGGACAGGTTTACCGC	CCTGTCAGCTGCAAGTTTCG	170	-3.492	0.989	93.361
GAPDH	GACCCGTTCATTGGGTTGGA	GACTGTCACCATCAGCCTTCA	97	-3.273	0.997	102.083
EF1A	CTCCAGGTTTTGTCCTTTGCG	ATTACCGCCGAGTAACCAGC	118	-3.113	0.998	109.524
RPS3	GTTGATACTGCTACCCGCCA	GGCTTCTTGGGACCACGTTT	107	-3.117	0.997	109.325
Actin	GATCGGTGGTTCCATCCTGG	CACGTTTCAGTCTTGCGAGC	136	-3.416	0.994	96.219
18S rRNA	CGCGCTACACTGAAGGAATC	AAGGGCAGGGACGTAATCAA	164	-3.431	0.991	95.641

项目Item	TUB	RPS27A	RPL10	AK	GAPDH	EF1A	RPS3	Actin	18S rRNA
不同组织Different tissues	1.336	1.014	0.739	2.281	0.320	1.985	0.852	2.570	1.429
不同性别Different genders	1.776	0.631	0.458	2.287	0.318	2.917	1.233	2.470	0.683
不同龄Different instars	0.284	0.530	0.237	0.468	0.512	0.491	0.107	0.963	0.412

项目Item	SD-P	TUB	RPS27A	RPL10	AK	GAPDH	EF1A	RPS3	Actin	18S rRNA
不同组织 Different tissues	SD(± CP)	1.22	2.45	1.19	4.23	1.98	1.46	2.24	4.35	1.89
不同组织 Different tissues	P	0.247	0.001	0.001	0.001	0.001	0.647	0.001	0.001	0.001
不同性别 Different genders	SD(± CP)	2.77	2.58	1.85	4.13	2.07	2.44	2.62	3.79	2.27
不同性别 Different genders	P	0.005	0.001	0.001	0.001	0.001	0.153	0.001	0.001	0.001
不同龄期 Different instars	SD(± CP)	0.48	0.66	0.51	1.65	1.00	0.33	0.59	1.87	0.70
不同龄期 Different instars	P	0.159	0.146	0.032	0.011	0.072	0.605	0.006	0.002	0.443

[1]	陶蓉, 李慧, 孙宇航, 于晓航, 朱晗, 郝德君. 美国白蛾内参基因的鉴定及筛选[J]. 林业科学, 2019, 55(9): 111-120.
[2]	章小铃, 王留强, 孙佩, 吴立栓, 樊玮, 张进, 卢孟柱, 胡建军. 杨树不同种质花粉萌发和致敏蛋白的差异[J]. 林业科学, 2017, 53(2): 54-64.
[3]	李真, 王留强, 卢孟柱. 毛白杨PtoWOX11/12a对杨树扦插苗生长发育的影响[J]. 林业科学, 2017, 53(11): 69-76.
[4]	储文渊, 王玉娇, 朱东悦, 陈竹, 严涵薇, 项艳. 盐和干旱胁迫下杨树新内参基因的筛选[J]. 林业科学, 2017, 53(10): 70-79.
[5]	刘文哲, 牛明月, 李秀云, 林二培, 黄华宏, 童再康. 光皮桦实时荧光定量PCR内参基因的筛选[J]. 林业科学, 2016, 52(8): 29-37.
[6]	成龙平, 胡海涛, 郭卫东, 杨莉, 王长春, 杨玲. 牛奶子实时定量PCR分析中内参基因的评价与验证[J]. 林业科学, 2015, 51(5): 135-144.
[7]	王金星, 张利军, 廖资亿, 张运根, 邱乾栋, 孙鹏, 孙宇涵, 胡瑞阳, 卢楠, 李云. 刺槐实时定量PCR分析中内参基因的选择[J]. 林业科学, 2014, 50(9): 167-172.
[8]	陈新;王贵禧;梁丽松;马庆华. 平榛冷适应相关基因CBF的克隆及时空表达特性分析[J]. 林业科学, 2012, 48(1): 167-172.
[9]	魏志刚;钱婷婷;张凯旋;刘关君;刘桂丰;杨传平. 外源GA₃对白桦成花基因影响的定量PCR分析[J]. 林业科学, 2011, 47(7): 187-192.
[10]	杨帆;丁菲杜天真. 盐胁迫下构树DREB转录因子基因表达的实时荧光定量PCR分析[J]. 林业科学, 2010, 46(4): 146-150.
[11]	段续伟;邓舒;沈元月;冯永庆;秦岭. 板栗CmAPs基因的克隆及在芽变短雄花序中的表达分析[J]. 林业科学, 2010, 46(12): 49-55.
[12]	文樵夫;沈红香;姚允聪;田佶;宋婷婷. 苹果属观赏海棠McDFR的克隆及不同叶色品种间的表达差异[J]. 林业科学, 2010, 46(11): 16-24.
[13]	白根本沈昕王沙生. 胡杨盐诱导基因与盐抑制基因的差减杂交显示研究[J]. 林业科学, 2003, 39(2): 168-170.
[14]	张太佐. 红头芫菁防治竹蝗的研究[J]. , 1994, 30(4): 370-375.
[15]	黄脊竹蝗研究课题组. 黄脊竹蝗防治指标的研究[J]. , 1992, 28(5): 459-465.