Evaluation and validation of reference genes for SYBR Green qRT-PCR normalization in Sesamia inferens (Lepidoptera: Noctuidae)

다국어 초록 (Multilingual Abstract)

The pink stemborer, Sesamia inferens (Walker) (Lepidoptera: Noctuidae), has gradually become amajor threat to rice production in Asian rice growth area. Using reverse-transcription quantitative real-time PCR (qRT-PCR) to investigate gene expression of S. inferens under different conditions is a popular technique. However, numerous studies demonstrate that the experimental conditions affect the stability of reference genes. Thus, it is necessary to normalize qRT-PCR data under a specific condition using a stable reference gene as an internal control. In the present study, five algorithms (Delta Ctmethod, Bestkeeper, geNorm,Normfinder, and RefFinder) and seven candidate reference genes were evaluated to find out the most suitable reference genes under different biotic and abiotic conditions including different tissues and developmental stages, exposure to insecticides, different rice varieties, and different diets. Our results showed that the more stable series of reference genes were as follows: GAPDH, RPL10, and RPS3 for different tissues;GAPDH and RPS3 for developmental stages; RPS3 and EF1 for larval insecticide exposure; RPS3 and RPL10 for larvae reared on different rice varieties; RPS3 and ACTB for larvae fed on different diets. Hence, a combination of the best sets of reference genes for normalization of expression analysis will increase the accuracy and reliability of qRT-PCR data in S. inferens. In addition, these results establish the basis for selection of reference genes in other lepidopterous insect species.

참고문헌 (Reference)

1 van den Berg, J., "Vetiver grass (Vetiveria zizanioides (L.) Nash) as trap plant for Chilo partellus (Swinhoe)(Lepidoptera: Pyralidae) and Busseola fusca (Fuller)(Lepidoptera: Noctuidae)" Annales de la Société entomologique de FranceTaylor & Francis 449-454, 2006

2 Cheng, D., "Validation of reference genes in Solenopsis invicta in different developmental stages, castes and tissues" 8 : e57718-, 2013

3 Lourenço, A. P., "Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR" 39 : 372-385, 2008

4 Bansal, R., "Validation of reference genes for gene expression studies in Aphis glycines (Hemiptera: Aphididae)" 105 : 1432-1438, 2012

5 Gutierrez, L., "The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription‐polymerase chain reaction (RT‐PCR) analysis in plants" 6 : 609-618, 2008

6 Chai, H. -N., "The complete mitochondrial genome of the pink stem borer, Sesamia inferens, in comparison with four other noctuid moths" 13 : 10236-10256, 2012

7 Sun, W., "Suitable reference gene selection for different strains and developmental stages of the carmine spider mite, Tetranychus cinnabarinus, using quantitative real-time PCR" 10 : 208-, 2010

8 Hornakova, D., "Selection of reference genes for real-time polymerase chain reaction analysis in tissues from Bombus terrestris and Bombus lucorum of different ages" 397 : 118-120, 2010

9 Yuan, M., "Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reversetranscription quantitative PCR" 9 : e86503-, 2014

10 Gao, Y., "Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis" 105 : 11-15, 2010

11 Lu, Z., "Rice Planthoppers" Springer 161-178, 2015

12 Xu, L. N., "Review of history, present situation and prospect of pink stem borer in China" 27 : 244-248, 2011

13 de Boer, M. E., "Reference genes for QRT-PCR tested under various stress conditions in Folsomia candida and Orchesella cincta (Insecta, Collembola)" 10 : 54-, 2009

14 Li, R., "Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)" 8 : e53006-, 2013

15 Maroniche, G. A., "Reference gene selection for gene expression studies using RT-qPCR in virus-infected planthoppers" 8 : 308-, 2011

16 Huggett, J., "Real-time RT-PCR normalisation; strategies and considerations" 6 : 279-284, 2005

17 Zheng, X., "Potential function of Sudan grass and vetiver grass as trap crops for suppressing population of stripped stem borer Chilo suppressalis in rice" 25 : 299-303, 2009

18 Andersen, C. L., "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" 64 : 5245-5250, 2004

19 Wu, M., "Monitoring of insecticides resistance of Chilo suppressails (Walker) and Sesamia inferens (Walker) and susceptibility baseline of Chilo suppressails to flubendiamide" Nanjing Agrucultural University 2007

20 Rubia, E., "Mechanisms of compensation of rice plants to yellow stem borer Scirpophaga incertulas (Walker) injury" 15 : 335-340, 1996

21 Majerowicz, D., "Looking for reference genes for real‐time quantitative PCR experiments in Rhodnius prolixus (Hemiptera: Reduviidae)" 20 : 713-722, 2011

22 Jiang, M., "Interactions between the striped stem borer Chilo suppressalis (Walk.)(Lep., Pyralidae) larvae and rice plants in response to nitrogen fertilization" 76 : 124-128, 2003

23 Chen, M., "Insect-resistant genetically modified rice in China: from research to commercialization" 56 : 81-101, 2011

24 Klie, M., "Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida)" 4 : 518-526, 2011

25 Barsalobres-Cavallari, C. F., "Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions" 10 : 1-, 2009

26 Lu, Y., "Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae)" 8 : e68059-, 2013

27 Van Hiel, M. B., "Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions" 10 : 56-, 2009

28 Thellin, O., "Housekeeping genes as internal standards: use and limits" 75 : 291-295, 1999

29 Radonić, A., "Guideline to reference gene selection for quantitative real-time PCR" 313 : 856-862, 2004

30 Lu, Y., "Genome organization, phylogenies, expression patterns, and three-dimensional protein models of two acetylcholinesterase genes from the red flour beetle" 7 : e32288-, 2012

31 Barber, R. D., "GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues" 21 : 389-395, 2005

32 Wang, Y., "Expression of Cry1Ab protein in a marker-free transgenic Bt rice line and its efficacy in controlling a target pest, Chilo suppressalis (Lepidoptera: Crambidae)" 43 : 528-536, 2014

33 Cruz, F., "Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR" 23 : 607-616, 2009

34 Pathak, M., "Ecology of common insect pests of rice" 13 : 257-294, 1968

35 Pfaffl, M. W., "Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations" 26 : 509-515, 2004

36 Dale, D., "Biology and Management of Rice Insects" Wily Eastern Ltd 388-408, 1994

37 Chapuis, M. -P., "Assessment and validation of a suite of reverse transcriptionquantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust" 12 : 7-, 2011

38 Robledo, D., "Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot(Scophthalmus maximus)gonad dataset" 15 : 648-, 2014

39 Cheng, X., "Agrobacterium-transformed rice plants expressing synthetic cryIA (b) and cryIA (c) genes are highly toxic to striped stem borer and yellow stem borer" 95 : 2767-2772, 1998

40 Ruan, W., "Actin, a reliablemarker of internal control?" 385 : 1-5, 2007

41 Vandesompele, J., "Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes" 3 : 2002

42 Bustin, S. A., "Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays" 25 : 169-193, 2000

43 Fu, Q., "A semi-artificial diet of Sesamia inferens and preparation method"