|
|
|
| Screening and Verification of Reference Genes for Real-time Fluorescence Quantitative PCR in Cornus hongkongensis subsp. tonkinensis Under Salt Stress |
| SUN Da-Wei1,2, YUAN Jia-Qiu1,2, CAI Mei1,2, FU Xiang-Xiang1,2* |
1 College of Forestry, Nanjing Forestry University, Nanjing 210037, China;
2 Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China |
|
|
|
|
Abstract For analyzing the expression of genes accurately by quantitative real-time PCR (qRT-PCR), screening of stabilized reference genes is of great important. Based on transcriptome data, 9 candidate genes including 18S ribosome RNA (18S rRNA), initiation factor 2 (IF2), elongation factor-1α 1 (EF-1-α1), EF-1-α2, adenine phosphoribosyltransferase (APT), cycloidea1 (cyc1), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), GAPDH2 and β -tubulin (TUB) were amplified by qRT-PCR in leaves and roots of Cornus hongkongensis subsp. tonkinensis suffering from different salt stress time. The expression stability of 9 candidate genes were measured by GeNorm, NormFinder and BestKeeper. Suitable reference gene was finally screened based on comprehensive evaluation of expression stability with RefFinder. Then the selected reference genes were verified by target genes superoxide dismutase (SOD) and catalase (CAT). The results showed that 18S rRNA and GAPDH were the optimal candidate reference genes in various organs under salt stress, whereas EF-1- α2 was the least stable gene. However, the Ct value of 18S rRNA was higher, indicated that the expression of 18S rRNA was lower than that of GAPDH in various organs. The results also showed that the relative expression levels of target genes were consistent with the transcriptome sequencing results when using GAPDH as reference gene. This study determined that the most suitable reference gene was GAPDH for qRT-PCR, which could provide the reference for further study on the expression of related gene in Cornus hongkongensis subsp. tonkinensis under salt stress.
|
|
Received: 25 March 2022
|
|
|
|
Corresponding Authors:
*xxfu@njfu.edu.cn
|
|
|
|
[1] 樊有存, 张红岩, 韩芊, 等. 2021. 盐胁迫下蚕豆不同组织实时荧光定量 PCR 内参基因的筛选[J]. 青海大学学报, 39(01): 16-23.
(Fan Y C, Zhang H Y, Han Q, et al. 2021. Selection of reference genes in the different tissues of faba bean under the salt stress via real-time quantitative PCR[J]. Journal of Qinghai University, 39(01): 16-23.)
[2] 方彦, 张森, 吴显坤, 等. 2020. 大花四照花研究进展[J]. 上海农业学报, 36(04): 156-162.
(Fang Y, Zhang Sen, Wu X K, et al. 2020. A review of the research on Cornus flori- da L.[J]. Acta Agriculturae Shanghai, 36(04): 156-162.)
[3] 洑香香, 徐杰, 刘国华. 2015. 观赏型四照花种质资源及其开发利用[J]. 林业科技开发, 29(03): 1-6.
(Fu X X, Xu J, Liu G H. 2015. Development and utilization of germplasm resources of ornamental Cornus[J]. China Forestry Science Technology, 29(3): 1-6.)
[4] 韩维栋. 1993. 四照花类群种质资源及其开发利用[J]. 中国野生植物资源, 13(01): 37-40.
(Han W D. 1993. Development and utilization of germplasm resources of Cor- nus group[J]. Chinese Wild Plant Resources, 13(1): 37-40.)
[5] 何永明, 曾小春. 2010. 开花期水稻颖花实时定量 RT-PCR 分析中内参基因的选择[J]. 江西农业大学学报, 34(6): 1086-1092.
(He Y M, Zeng X C. 2010. Reference gene selection for quantitative real-time RT-PCR normalization in rice florets during anthesis[J]. Acta Agriculturae Universitatis Jiangxiensis, 34(6): 1086-1092.)
[6] 李静宇, 孙铭阳, 徐世强, 等. 2022. 穿心莲茉莉酸甲酯及非生物胁迫下 real-time PCR 内参基因的筛选[J]. 中国实验方剂学杂志, 28(05): 133-140.
(Li J Y, Sun M Y, Xu S Q, et al. 2022. Screening of reference genes under MeJA and abiotic stresses of Andrographis paniculata by real-time fluorescence quantitative PCR[J]. Chinese Journal of Experimental Traditional Medical Formulae, 28(05): 133-140.)
[7] 李铁铮, 王金铃, 刘晓, 等. 2021. 管花肉苁蓉实时荧光定量 PCR 分析中内参基因的选择和验证[J]. 植物生理学报, 57(04): 969-981.
(Li T Z, Wang J L, Liu X, et al. 2021. Selection and validation of appropriate reference genes for qRT-PCR analysis in Cistanche tubulosa[J]. Plant Physiology Journal, 57(04): 969-981.)
[8] 刘圆, 王丽鸳, 韦康, 等. 2016. 不同氮处理茶树实时定量PCR 内参基因筛选和验证[J]. 茶叶科学, 36(01): 92-101.
(Liu Y, Wang L Y, WEI K, et al. 2016. Screening and validation of reference genes for quantitative real- time PCR analysis in tea plant (Camellia sinensis) under different nitrogen nutrition[J]. Journal of Tea Science, 36(01): 92-101.)
[9] 马青江, 孙操稳, 张乐英, 等. 2019. 东亚四照花群体中国潜在适生区预测研究[J]. 南京林业大学学报(自然科学版), 43(05): 135-140.
(Ma Q J, Sun C W, Zhang L Y, et al. 2019. Identification of potential distribution region for east asian dogwoods (Cornus) in China[J]. Journal of Nanjing Forestry University (Natural Sciences Edition), 43(05): 135-140.)
[10] 瞿印权, 陈小玲, 陈必芹, 等. 2021. 青钱柳实时荧光定量PCR 内参基因的筛选[J]. 分子植物育种, 19(24): 8133-8140.
(Qu Y Q, Chen X L, Chen B Q, et al. 2021. Selection of reference genes for quantitative real-time PCR in Cyclocarya paliurus[J]. Molecular Plant Breeding, 19(24): 8133-8140.)
[11] 苏西娅, 石元豹, 杨晓明, 等. 2019. 银杏实时荧光定量 PCR 分析中内参基因的选择与验证[J]. 植物生理学报, 55(06): 875-882.
(Su X Y, Shi Y B, Yang X M, et al. 2019. Selection and validation of reference genes for quantitative real-time PCR analysis in Ginkgo biloba[J]. Plant Physiology Journal, 55(06): 875-882.)
[12] 涂冬萍, 莫长明, 马小军, 等. 2015. 罗汉果实时荧光定量PCR 内参基因的选择[J]. 中国中药杂志, 40(02): 204-209.
(Tu D P, Mo C P, Ma X J, et al. 2015. Selection of reference genes of Siraitia grosvenorii by real-time PCR[J]. China Journal of Chinese Materia Medica, 40(02): 204-209.)
[13] 赵艺蕊, 黄春颖, 王克涛, 等. 2022. 山核桃实时荧光定量 PCR 分析中内参基因的筛选与验证[J]. 果树学报, 39(01): 10-21.
(Zhao Y R, Huang C Y, Wang K T, et al. 2022. Screening and verification of internal reference genes for real time quantitative PCR analysis in Carya cathayensis[J]. Journal of Fruit Science, 39(01): 10-21.)
[14] 周良云, 莫歌, 王升, 等. 2014. 基于实时荧光定量 PCR 对镉处理下黄花蒿内参基因稳定性的分析[J]. 中国中药杂志, 39(5): 777-784.
(Zhou L Y, Mo G, Wang S, et al. 2014. Stability analysis of reference gene based on real- time PCR in Artemisia annua under cadmium treatment[J]. China Journal of Chinese Materia Medica, 39(5): 777-784.)
[15] Andersen C L, Jensen J L, Ørntoft 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[J]. Cancer Research, 64(15): 5245-5250.
[16] Bustin S A, Benes V, Nolan T, et al. 2005. Quantitative real- time RT-PCR- a perspective[J]. Journal of Molecular Endocrinology, 34(3): 597-601.
[17] Dheda K, Huggett J F, Bustin S A, et al. 2004. Validation of house-keeping genes for normalizing RNA expression in real-time PCR[J]. BioTechniques, 37(1): 112-119.
[18] Fan C, Qiu Z, Zeng B, et al. 2017. Selection of reference genes for quantitative real-time PCR in Casuarina equi-setifolia under salt stress[J]. Biologia Plantarum, 61(3): 463-472.
[19] Fleige S, Pfaffl M W. 2006. RNA integrity and the effect on the real-time qRT-PCR performance[J]. Molecular Aspects of Medicine, 27(23): 126-139.
[20] Fu X X, Liu H N, Xu J, et al. 2014. Primary metabolite mobilization and hormonal regulation during seed dormancy release in Cornus japonica var. chinensis[J]. Scandinavian Journal of Forestry Research, 29(6): 542-551.
[21] Fu X X, Liu H N, Zhou X L, et al. 2013. Seed dormancy mechanism and dormancy breaking techniques for Cor- nus kousa var. chinensis[J]. Seed Science and Technology, 41(3): 458-463.
[22] Gutierrez L, Mauriat M, Guénin S, et al. 2008. The lack of a systematic validation of reference genes: A serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants[J]. Plant Biotechnology Journal, 6(6): 609-618.
[23] Hou S, Zhao T, Yang D, et al. 2021. Selection and validation of reference genes for quantitative RT-PCR analysis in Corylus heterophylla Fisch. × Corylus avellana L.[J]. Plants, 10(1): 159.
[24] Khan M I, Shin J H, Shin T S, et al. 2018. Anthocyanins from Cornus kousa ethanolic extract attenuate obesity in association with anti-angiogenic activities in 3T3-L1 cells by down-regulating adipogeneses and lipogenesis[J]. PLOS ONE, 13(12): e0208556.
[25] Kong Q, Yuan J, Gao L, et al. 2014. Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon[J]. PLOS ONE, 9(2): e90612.
[26] Li Z, Lu H, He Z, et al. 2019. The lack of a systematic validation of reference genes: A serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT- PCR) analysis in plants[J]. PLOS ONE, 14(12): e0225926.
[27] Lu Q, Xu J, Fu X X, et al. 2019. Physiological and growth responses of two dogwoods to short-term drought stress and re-watering[J]. Acta Ecologica Sinica, 40(2): 172-177.
[28] Lu Q, Yang L, Wang H, et al. 2021. Calcium ion richness in Cornus hongkongensis subsp. elegans (W. P. Fang et Y. T. Hsieh) Q. Y. Xiang could enhance its salinity tolerance[J]. Forests, 12(11): 1522.
[29] Mafra V, Kubo K S, Alves-Ferreira M, et al. 2012. Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions[J]. PLOS ONE, 7(2): e31263.
[30] 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[J]. Biotechnology Letters, 26(6): 509-515.
[31] Qu R, Miao Y, Cui Y, et al. 2019. Selection of reference genes for the quantitative real-time PCR normalization of gene expression in Isatis indigotica Fortune[J]. BMC Molecular Biology, 20(1): 1-12.
[32] Radonic A, Thulke S, Mackayl M, et al. 2004. Guideline for reference selection for quantitative real-time PCR[J]. Biochemical and Biophysical Research Communications, 313(4): 856-862.
[33] Vandesompele J, Preter K, Pattyn F, et al. 2002. Accurate normalization of quantitative real-time PCR data by geometric averaging multiple internal control genes.[J]. Genome Biology, 3(7): research0034.
[34] Wang H, Cao F, Zhang W, et al. 2013. Cloning and expression of stearoyl-ACP desaturase and two oleate desaturases genes from Ginkgo biloba L.[J]. Plant Molecular Biology Reporter, 31(3): 633-648.
[35] Wu J Y, Zhang H G, Liu L Q, et al. 2016. Validation of reference genes for RT-qPCR studies of gene expression in preharvest and postharvest longan fruits under different experimental conditions[J]. Frontiers in Plant Science, 7: 780.
[36] Zhang L L, Zhang Q K, Jiang Y, et al. 2018. Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum[J]. Plant Methods, 14: 110. |
|
|
|
