美国白蛾内参基因的鉴定及筛选

doi:10.11707/j.1001-7488.20190912

摘要/Abstract

摘要： [目的]为筛选美国白蛾在不同温度处理、不同发育阶段、幼虫不同组织中稳定的内参基因，为美国白蛾相关的基因定量研究奠定基础。[方法]在美国白蛾转录组测序结果中筛选出8个候选内参基因GAPDH、ACT、RPL12、RPL18a、RPS16、EF1α、EF1β、18S rRNA，获得碱基序列。将以上序列进行克隆、测序、比对最终证明其为美国白蛾的基因且碱基序列正确，之后将其上传到NCBI获得登录号。设计以上8个候选内参基因的实时荧光定量PCR（qRT-PCR）引物，利用qRT-PCR技术测定8个候选内参基因在美国白蛾不同发育阶段（幼虫1~6龄）、不同温度处理下（-10、-5、0、25、35、40、45℃处理2 h）、幼虫的不同组织（头、肠道、脂肪体、马氏管、表皮）中的表达量，记录C_t值；然后通过ΔC_t法、GeNorm、NormFinder和BestKeeper共4种方法对8个候选内参基因在不同条件下的C_t值进行统计分析，最后综合RelFinder选出最稳定的内参基因。通过GeNorm计算V_n/（n+1）最合适内参基因的数目。[结果]C_t分析表明，在不同发育阶段和在不同温度处理下表达最稳定的是RPL12；在幼虫不同组织中，最稳定的是RPS16。GeNorm分析结果与ΔC_t分析结果相同。NormFinder分析表明，在不同的发育阶段和不同温度处理下最稳定的候选内参基因分别是RPL18a和EF1α；在幼虫组织中，最稳定的是ACT。BestKeeper分析认为，不同发育阶段，ACT、GAPDH不适合作为内参基因；不同温度处理下，ACT、RPL18a、RPL12、RPS16、EF1β不适合作为内参基因；在不同幼虫组织中，RPL18a、EF1α、EF1β、18S rRNA不适合作为内参基因。最后RelFinder综合评价显示，在不同的发育阶段最稳定的内参基因组合是RPL12和EF1β，最不稳定的是ACT；在不同的温度处理下，最稳定的内参基因组合是EF1α和GAPDH，最不稳定的是ACT；在不同组织中，最稳定的组合是ACT和RPS16，最不稳定的是RPL18a。经GeNorm软件计算，最合适的内参基因数目应该是2个。[结论]在美国白蛾不同发育阶段的基因定量研究中，建议以RPL12和EF1β作为内参基因；在不同温度处理的基因定量研究中，建议以EF1α和GAPDH作为内参基因；在幼虫不同组织的基因定量研究中，建议以ACT和RPS16作为内参基因。此外，亦初步说明了昆虫内参基因在不同试验条件下的不稳定性。

关键词: 内参基因, 基因筛选, qRT-PCR

Abstract: [Objective] In this study, the stably expressed genes under given conditions, including different temperature stresses, different developmental stages and different tissues of larva, were screened and verified as internal reference genes for quantitative real-time PCR (qRT-PCR) in Hyphantria cunea, to provide a basis for quantitative studies of genes of the moth.[Method] Based on transcriptomics sequencing results in H. cunea (the data was constructed by our laboratory and not published), eight candidate internal reference genes of GAPDH, ACT, RPL12, RPL18a, RPS16, EF1α, EF1β and 18S rRNA were selected, and their base sequences were obtained. The above sequences were cloned, sequenced and compared, and finally proved to be the gene of H. cunea and the base sequence was correct. Then the sequence was uploaded to NCBI to obtain GenBank accession No. Real-time fluorescent quantitative PCR primers (qRT-PCR) of the eight candidate reference genes were designed. The qRT-PCR technique was applied to measure the eight candidate reference genes at different developmental stages (1-6 age of larvae), under the different temperature stress (-10, -5, 0, 25, 35, 40, 45℃ with 2 h), and in different tissues of larva (head, the gut, fat body, malpighian tube, skin), and the C_t values were recorded. The C_t values of the eight candidate reference genes under different conditions were analyzed by four methods of ΔC_t, GeNorm, NormFinder and BestKeeper, finally. The most stable reference genes were chosen by RelFinder. The value of V_n/(n+1) was calculated by GeNorm to determine the most suitable number of reference genes.[Result] The ΔC_t showed that the most stable reference gene was RPL12 at different development stages, the most stable reference gene was RPL12 under different temperature stresses, and the most stable reference gene was RPS16 in different tissues of larva. The results obtained by GeNorm analysis were similar to those obtained by the C_t analysis. NormFinder showed that RPL18a and EF1α were the most stable reference genes at different developmental stages and under different temperatures, respectively. ACT was the most table reference gene in different tissues of larva. According to BestKeeper analysis, ACT and GAPDH were not suitable as reference genes at different development stages, ACT, RPL18a, RPL12, RPS16 and EF1β were not suitable as reference genes in different temperature treatments, and RPL18a, EF1α, EF1β, and 18S rRNA were not suitable as reference genes in different tissues of larva. At last, RelFinder showed that the most stable reference gene combination was RPL12 and EF1β, while the most unstable reference gene was ACT at different development stages; the most stable reference gene combination was EF1, GAPDH, while ACT was the most unstable reference gene in different temperature conditions; the most stable reference gene combination was ACT and RPS16, while the most unstable candidate reference gene was RPL18a. The most suitable number of reference genes should be 2 by GeNorm.[Conclusion] In H. cunea, three pairs of genes, RPL12 and EF1β, EF1α and GAPDH, and ACT and RPS16, are recommended as reference genes in different developmental stages, different temperature stresses and different tissues of larvae.

Key words: reference genes, gene screening, qRT-PCR

中图分类号:

S718.7

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.

Tao Rong, Li Hui, Sun Yuhang, Yu Xiaohang, Zhu Han, Hao Dejun. Indentification and Screening of Internal Reference Genes of Hyphantria cunea (Lepidoptera: Arctiidae)[J]. Scientia Silvae Sinicae, 2019, 55(9): 111-120.

参考文献

陈立华, 张月华, 何庆玲, 等. 2014. 蓖麻蚕基因转录表达分析的内参基因筛选. 河北农业大学学报, 37(6):78-84.
(Chen L H, Zhang Y H, He Q L, et al. 2014. Selection of reference genes for transcription expression analysis in Philosamia cynthia ricini. Journal of Agricultural University of Hebei,37(6):78-84.[in Chinese])
曹利军, 杨帆, 唐思莹, 等. 2014. 适合三种鳞翅目昆虫的一种人工饲料配方. 应用昆虫学报, 51(5):1376-1386.
(Cao L J, Yang F, Tang S Y, et al. 2014. Development of an artificial diet for three lepidopteran insects. Chinese Bulletin of Entomology, 51(5):1376-1386.[in Chinese])
陈芳, 陆永跃. 2014. 热胁迫下棉花粉蚧内参基因的筛选. 昆虫学报, 57(10):1146-1154.
(Cheng F, Lu Y Y. 2014. Selection of reference genes in Phenacoccus solenopsis (Hemiptera:Pseudococcidae) under heat stress. Acta Entomologica Sinica, 57(10):1146-1154.[in Chinese])
冯波, 郭前爽, 毛必鹏, 等. 2016. 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, 59(4):427-437.
(Feng B, Guo Q S, Mao B P, et al. 2016. Identification and selection of valid reference genes for assaying gene expression in the chemosensory tissues of Monochamus alternatus (Coleoptera:Cerambycidae) by RT-qPCR. Acta EntomologicaSinica, 59(4):427-437.[in Chinese])
符伟, 谢文, 张卓, 等. 2012. Bt毒素诱导下小菜蛾实时定量PCR内参基因的筛选. 昆虫学报, 55(12):1406-1412.
(Fu W, Xie W, Zhang Z, et al. 2012. Selection of valid reference genes for gene expression studies by quantitative real-time PCR in Plutella xylostella (Lepidoptera:Plutellidae) after exposure to Bt toxin. Acta Entomologica Sinica, 55(12):1406-1412.[in Chinese])
李柯, 阴环, 奚耕思, 等. 2010. 黏虫 β-actin 基因 cDNA 的克隆、序列分析及表达量检测. 昆虫知识, 47 (6):1089-1094.
(Li K, Yin H, Xi G S, et al. 2010. Molecular cloning, sequence analysis and expression detection of β-actin gene in the oriental armyworm, Mythimna separata. Chinese Bulletin of Entomology, 47(6):1089-1094.[in Chinese])
刘慧慧, 张永安, 王玉珠, 等. 2017. 美国白蛾Wnt-1基因的基因组编辑. 林业科学, 53(3):119-127.
(Liu H H, Zhang Y A, Wang Y Z. et al. 2017. Genome editing of Wnt-1 in Fall webworm (Hyphantria cunea). Scientia Silvae Sinicae, 53(3):119-127.[in Chinese])
刘宁. 2017. 亚洲玉米螟滞育期间耐寒相关基因的表达分析. 沈阳农业大学,硕士学位论文.
(Liu N. 2017. The expression analysis of could-resistant genes in the Asian corn borer during diapause. Shenyang:MS thesis of Shenyang Agricultural University.[in Chinese])
吴文凯, 刘成前, 周志刚, 等. 2009. 用于莱茵衣藻荧光定量PCR分析的内参基因选择. 植物生理学报, 45(7):667-672.
(Wu W K, Liu C Q, Zhou Z G, et al. 2009. The selection of reference genes in Chlamydomonas reinhardtii P. A. dangeard by real-time quantitative PCR. Plant Physiology Communications, 45(7):667-672.[in Chinese])
闫晓平, 赵丹, 郭巍, 等. 2017. 美国白蛾几丁质脱乙酰酶的克隆、表达及酶学性质. 中国农业科学, 50(5):849-858.
(Yan X P, Zhao D, Guo W, et al. 2017. Cloning, expression and enzymatic characterization of chitin deacetylases from Hyphantria cunea. Scientia Agricultura Sinica, 50(5):849-858.[in Chinese])
杨忠岐, 张永安. 2007. 重大外来入侵害虫——美国白蛾生物防治技术研究. 应用昆虫学报, 44(4):465-471.
(Yang Z Q, Zhang Y A. 2007. Researches on techniques for biocontrol of the fall webworm, Hyphantria cunea, a severe invasive insect pest to China. Chin Bull Entomol, 44(4):465-471.[in Chinese])
杨苓, 胡晓静, 徐志峰, 等. 2017.桃蛀螟实时荧光定量PCR内参基因的筛选.昆虫学报, 60(11):1266-1277.
(Yang L, Hu X J, Xu Z F, et al. 2017. Screening of reference genes for qRT-PCR in Conogethes punctiferails (Lepidoptera:Crambidae). Acta Entomologica Sinica, 60(11):1266-1277.[in Chinese])
Andersen C L, Jensen J L, Orntoft T F. 2004. 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 Res, 64:5245-5250.
de Jonge H J M, Fehrmann R S N, de Bont E S J M, et al. 2007. Evidence based selection of housekeeping genes. PLoS ONE, 2(9):e898.
Huis R, Hawkins S, Neutelings G. 2010. Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biol, 10(1):71.
Hunter T, Garrels J I. 1977. Characterization of the mRNAs for α-, β- and γ-actin. Cell, 12:67-781.
Ladror D T, Frey B L, Scalf M, et al. 2014. Methylation of yeast ribosomal protein S2 is elevated during stationary phase growth conditions. Biochem Biophys Res Commun, 445:535-541.
Li R M, Xie W, Wang S L, et al. 2013. Reference gene selection for qRT-PCR analysis in the sweet potato whitefly, Bemisia tabaci(Hemiptera:Aleyrodidae). PLoS ONE, 8:e53006.
Lourenco A P, Mackert A, Cristino A D S, et al. 2008. Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR. Apidologie, 39 (3):372-385.
Lu Y H, Yuan M, Gao X W, et al. 2013. Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura (Lepidoptera:Noctuidae). PLoS ONE, 8(7):e68059.
Ma K S, Li F, Liang P Z, et al. 2016. Identification and validation of reference genes for the normalization of gene expression data in qRT-PCR analysis in Aphis gossypii (Hemiptera:Aphididae). J Insect Sci, 16(1):17.
Matta B P, Bitner-Mathe B C, Alves-Ferreira M. 2011. Getting real with real-time q-PCR:a case study of reference gene selection for morphological variation in Drosophila melanogaster wings. Development Genes and Evolution, 221(1):49-57.
Pfaffl M W, Tichopad A, Prgomet C, et al. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity:BestKeeper-Excel-based tool using pair-wise correlations. Biotechnol Lett, 26:509-515.
Radonic A, Thulke S, Mackay I M, et al. 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys. Res Commun, 313(4):856-862.
Scharlaken B, de Graaf D C, Goossens K, et al. 2008. Reference gene selection for insect expression studies using quantitative real-time PCR:the head of the honeybee, Apis mellifera, after a bacterial challenge. Journal of Insect Science, 8(33):1-10.
Shakeel M, Zhu X, Kang T H, et al. 2015. Selection and evaluation of reference genes for quantitative gene expression studies in cotton bollworm, Helicoverpa armigera (Lepidoptera:Noctuidae). J Asia-Pac Entomol, 18:123-130.
Silver N, Best S, Jiang J, et al. 2006. Selection of housekeeping genes for gene expression studies in human reticulocytes using realtime PCR. BMC Mol Biol, 7:33.
Sun H, Meng Y, Cui G, et al. 2009. Selection of housekeeping genes for gene expression studies on the development of fruit bearing shoots in Chinese jujube (Ziziphus jujuba Mill). Mol Biol Rep, 36(8):2183-2190.
Thellin O, Zorzi W, Lakaye B, et al. 1999. Housekeeping genes as internal standards:use and limits. J Biotechnol, 75(2/3):291-295.
Vandesompele J, Preter K D, Pattyn F, et al. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol, 37:1-11.
Xie F L, Sun G L, Stiller J W, et al. 2011. Genome-wide functional analysis of the cotton transcriptome by creating an integrated EST database. PLoS ONE, 6(11):e26980.
Yan J, Yuan F, Long G, et al. 2012. Selection of reference genes for quantitative real-time RT-PCR analysis in citrus. Molecular Biology Reports, 39(2):1831-1838.
Yang C X, Pan H P, Liu Y, et al. 2014. Selection of reference genes for expression analysis using quantitative real-time PCR in the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera:Aphidiae). PLoS ONE, 9:e110454.
Yuan M, Lu Y H, Zhu X, et al. 2014. 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, 9(1):e86503.
Zhai Y, Lin Q, Zhou X, et al. 2014. Identification and validation of reference genes for quantitative real-time PCR in Drosophila suzukii (Diptera:Drosophilidae). PLoS ONE, 9 (9):e106800.
Zhang S, An S, Li Z, et al. 2015. Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera(Lepidoptera:Noctuidae). Gene, 555(2):393-402.

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