Selection and Validation of Reference Genes for Quantitative Real-time PCR in Caulerpa lentillifera Under Stress Conditions

doi:10.13560/j.cnki.biotech.bull.1985.2020-1585

Abstract

Abstract:

In order to screen internal reference genes stably expressing in Caulerpa lentillifera for the analysis of real-time fluorescence quantitative PCR,the △Ct,BestKeeper,geNorm and NormFinder software were used to comprehensively compare the expression stabilities of 5 candidate reference genes,using the stolons and erect branches of C. lentillifera under various stress conditions,and the selected reference genes are verified. The expression stabilities of different reference genes in C. lentillifera differed largely. The expression stabilities of ClACT and ClGAPDH were quite good in different tissues and under different stress conditions,while the expression stability of ClTUB was poor. The combination of ClACT and ClGAPDH can be used as the internal reference gene and more accurate results can be acquired when RT-qPCR is used to analyze the gene expression of C. lentillifera under stress.

Key words: Caulerpa lentillifera, reference genes, RT-qPCR, stress

LI Tianjingwei, ZOU Xiao-xiao, ZHU Jun, BAO Shi-xiang. Selection and Validation of Reference Genes for Quantitative Real-time PCR in Caulerpa lentillifera Under Stress Conditions[J]. Biotechnology Bulletin, 2021, 37(10): 266-276.

Figures/Tables 10

References 37

[1]	Wong ML, Medrano JF. Real-time PCR for mRNA quantitation[J]. BioTechniques, 2005, 39(1):75-85. doi: 10.2144/05391RV01 URL
[2]	Artico S, Nardeli SM, Brilhante O, et al. Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data[J]. BMC Plant Biology, 2010, 10(1):49. doi: 10.1186/1471-2229-10-49 URL
[3]	Die JV, Román B, Nadal S, et al. Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions[J]. Planta, 2010, 232(1):145-153. doi: 10.1007/s00425-010-1158-1 URL
[4]	Schmidt GW, Delaney SK. Stable internal reference genes for normalization of real-time RT-PCR in tobacco(Nicotiana tabacum)during development and abiotic stress[J]. Molecular Genetics & Genomics, 2010, 283(3):233-241.
[5]	Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR[J]. Nature Protocols, 2006, 1(3):1559-1582. doi: 10.1038/nprot.2006.236 URL
[6]	Nicot N, Hausman JF, Hoffmann L, et al. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress[J]. Journal of Experimental Botany, 2005, 56(421):2907-2914. doi: 10.1093/jxb/eri285 URL
[7]	Expósito-Rodríguez M, Borges AA, Borges-Pérez A, et al. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process[J]. BMC Plant Biology, 2008, 8:131. doi: 10.1186/1471-2229-8-131 pmid: 19102748
[8]	Gutierrez L, Guénin M, Mauriat S, et al. 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, 2008, 6:609-18. doi: 10.1111/j.1467-7652.2008.00346.x pmid: 18433420
[9]	Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biology, 2002, 3(7):research0034. 1-0034. 11.
[10]	Andersen CL, Jensen JL, Ørntoft TF. 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, 2004, 64(15):5245-5250. doi: 10.1158/0008-5472.CAN-04-0496 URL
[11]	Pfaffl MW, 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[J]. Biotechnology Letters, 2004, 26(6):509-515. doi: 10.1023/B:BILE.0000019559.84305.47 URL
[12]	Liu Q, Wei C, Zhang MF, et al. Evaluation of putative reference genes for quantitative real-time PCR normalization in Lilium regale during development and under stress[J]. PeerJ, 2016, 4:e1837. doi: 10.7717/peerj.1837 URL
[13]	Li MY, Wang F, Jiang Q, et al. Validation and comparison of reference genes for qPCR normalization of celery(Apium graveolens)at different development stages[J]. Frontiers in Plant Science, 2016, 7:313.
[14]	Ma R, Xu S, Zhao YC, et al. Selection and validation of appropriate reference genes for quantitative real-time PCR analysis of gene expression in Lycoris aurea[J]. Frontiers in Plant Science, 2016, 7:536.
[15]	Leida C, Moser C, Esteras C, et al. Variability of candidate genes, genetic structure and association with sugar accumulation and climacteric behavior in a broad germplasm collection of melon(Cucumis melo L.)[J]. BMC Genetics, 2015, 16:28. doi: 10.1186/s12863-015-0183-2 URL
[16]	Wu JY, Zhang HN, Liu LQ, et al. 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, 2016, 7:780.
[17]	Wang WL, Wu XJ, Wang C, et al. Exploring valid internal-control genes in Porphyra yezoensis(Bangiaceae)during stress response conditions[J]. Journal of Oceanology and Limnology, 2014, 32(4):783-791.
[18]	Wu X, Niu J, Huang A, et al. Selection of internal control gene for expression studies in Porphyra Haitanensis(Rhodophyta)at different life-history stages1[J]. Journal of Phycology, 2012, 48(4):1040-1044. doi: 10.1111/jpy.2012.48.issue-4 URL
[19]	Ji NJ, Li L, Lin LX, et al. Screening for suitable reference genes for quantitative real-time PCR in Heterosigma akashiwo(Raphidophyceae)[J]. PLoS One, 2015, 10(7):e0132183. doi: 10.1371/journal.pone.0132183 URL
[20]	Deng YY, Hu ZX, Ma ZP, et al. Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR[J]. Acta Oceanologica Sinica, 2016, 35(8):106-112.
[21]	Saito H, Xue C, Yamashiro R, et al. High polyunsaturated fatty acid levels in two subtropical macroalgae, Cladosiphon okamuranus and Caulerpa lentillifera.[J]. Journal of Phycology, 2010, 46(4):665-673. doi: 10.1111/j.1529-8817.2010.00848.x URL
[22]	Niwano Y, Beppu F, Shimada T, et al. Extensive screening for plant foodstuffs in Okinawa, Japan with anti-obese activity on adipocytes in vitro[J]. Plant Foods for Human Nutrition, 2009, 64(1):6-10. doi: 10.1007/s11130-008-0102-z URL
[23]	邢诒炫, 曾俊, 吴翔宇, 等. 三种热带经济海藻养殖现状与应用前景[J]. 海洋湖沼通报, 2019(6):112-120.
	Xing ZX, Zeng J, Wu XY, et al. Cultivation status and application prospect of three tropical economic eeaweeds[J]. Transactions of Oceanology and Limnology, 2019(6):112-120.
[24]	周文川, 赵秋龙, 雷美华, 等. 光照等环境因子对长茎葡萄蕨藻生长的影响[J]. 海洋与渔业, 2017(6):70-72.
	Zhou WC, Zhao QL, Lei MH, et al. Effects of light and other environmental factors on the growth of Caulerpa lentillifera[J]. Ocean And Fishery, 2017(6):70-72.
[25]	苏醒. 两种底栖海藻的光强适应性及其高光胁迫应答研究[D]. 海口:海南大学, 2017.
	Su X. Studies on light intensity adaptability and high light stress response of two benthic seaweeds[D]. Haikou:Hainan University, 2017.
[26]	Quackenbush J. Microarray data normalization and transformation[J]. Nature Genetics, 2002, 32:496-501. doi: 10.1038/ng1032 URL
[27]	Thellin O, Zorzi W, Lakaye B, et al. Housekeeping genes as internal standards:use and limits[J]. Journal of Biotechnology, 1999, 75(2-3):291-295. pmid: 10617337
[28]	Dheda K. Validation of housekeeping genes for normalizing RNA expression in real-time PCR[J]. Biotechniques, 2004, 37(1):118-119.
[29]	Remans T, Smeets K, Opdenakker K, et al. Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations[J]. Planta, 2008, 227(6):1343-1349. doi: 10.1007/s00425-008-0706-4 pmid: 18273637
[30]	He YH, Yan HL, Hua WP, et al. Selection and validation of reference genes for quantitative real-time PCR in gentiana macrophylla[J]. Frontiers in Plant Science, 2016, 7:945.
[31]	邵惠. 条斑紫菜(Pyropia/Porphyra yezoensis)实时荧光定量PCR内参基因的筛选[D]. 青岛:中国海洋大学, 2012.
	Shao H. Screening of reference genes for real-time fluorescent quantitative PCR of Porphyra yezoensis[D]. Qingdao:Ocean University of China, 2012.
[32]	娄素琳, 林鑫, 黄思敏, 等. 莱茵衣藻CrDRBs基因的克隆和生物信息学分析[J]. 深圳大学学报:理工版, 2018, 35(5):87-92.
	Lou SL, Lin X, Huang SM, et al. Cloning and bioinformatics analysis of CrDRBs in Chlamydomonas reinhardtii[J]. Journal of Shenzhen University:Science and Engineering, 2018, 35(5):87-92.
[33]	张计育, 黄胜男, 王涛, 等. 金魁猕猴桃RT-qPCR内参基因的筛选[J]. 上海农业学报, 2018, 34(1):84-88.
	Zhang JY, Huang SN, Wang T, et al. Screening of reference genes for reverse transcription quantitative real - time PCR in Actinidia deliciosa[J]. Journal of Shanghai Agriculture, 2018, 34(1):84-88.
[34]	Chen H, He XH, Luo C, et al. Cloning and expression of longan carbonic anhydrase gene under low temperature stress[J]. Acta Horticulturae Sinica, 2012, 39(2):243-252.
[35]	Liu CL, Wu GT, Huang XH, et al. Validation of housekeeping genes for gene expression studies in an ice alga Chlamydomonas during freezing acclimation[J]. Extremophiles, 2012, 16(3):419-425. doi: 10.1007/s00792-012-0441-4 URL
[36]	Kim H, Saha P, Farcuh M, et al. RNA-Seq analysis of spatiotemporal gene expression patterns during fruit development revealed reference genes for transcript normalization in plums[J]. Plant Molecular Biology Reporter, 2015, 33(6):1634-1649. doi: 10.1007/s11105-015-0860-3 URL
[37]	Wang LJ, Wang YC, Zhou P. Validation of reference genes for quantitative real-time PCR during Chinese wolfberry fruit development[J]. Plant Physiology & Biochemistry, 2013, 70:304-310.

处理组 Treatment group	处理条件 Treatment conditions	组织部位Tissues
对照组	温度27℃,光照40 μmol/（s · m²）,盐度30‰	直立枝
对照组	温度27℃,光照40 μmol/（s · m²）,盐度30‰	匍匐茎
温度胁迫	温度15℃,光照40 μmol/（s · m²）,盐度30‰	直立枝
	温度15℃,光照40 μmol/（s · m²）,盐度30‰	匍匐茎
	温度37℃,光照40 μmol/（s · m²）,盐度30‰	直立枝
	温度37℃,光照40 μmol/（s · m²）,盐度30‰	匍匐茎
光照胁迫	温度27℃,光照120 μmol/（s · m²）,盐度30‰	直立枝
	温度27℃,光照120 μmol/（s · m²）,盐度30‰	匍匐茎
	温度27℃,光照360 μmol/（s · m²）,盐度30‰	直立枝
	温度27℃,光照360 μmol/（s · m²）,盐度30‰	匍匐茎
盐度胁迫	温度27℃,光照360 μmol/（s · m²）,盐度15‰	直立枝
	温度27℃,光照360 μmol/（s · m²）,盐度15‰	匍匐茎
	温度27℃,光照360 μmol/（s · m²）,盐度40‰	直立枝
	温度27℃,光照360 μmol/（s · m²）,盐度40‰	匍匐茎

处理组 Treatment group	处理条件 Treatment conditions	组织部位Tissues
对照组	温度27℃,光照40 μmol/（s · m²）,盐度30‰	直立枝
对照组	温度27℃,光照40 μmol/（s · m²）,盐度30‰	匍匐茎
温度胁迫	温度15℃,光照40 μmol/（s · m²）,盐度30‰	直立枝
	温度15℃,光照40 μmol/（s · m²）,盐度30‰	匍匐茎
	温度37℃,光照40 μmol/（s · m²）,盐度30‰	直立枝
	温度37℃,光照40 μmol/（s · m²）,盐度30‰	匍匐茎
光照胁迫	温度27℃,光照120 μmol/（s · m²）,盐度30‰	直立枝
	温度27℃,光照120 μmol/（s · m²）,盐度30‰	匍匐茎
	温度27℃,光照360 μmol/（s · m²）,盐度30‰	直立枝
	温度27℃,光照360 μmol/（s · m²）,盐度30‰	匍匐茎
盐度胁迫	温度27℃,光照360 μmol/（s · m²）,盐度15‰	直立枝
	温度27℃,光照360 μmol/（s · m²）,盐度15‰	匍匐茎
	温度27℃,光照360 μmol/（s · m²）,盐度40‰	直立枝
	温度27℃,光照360 μmol/（s · m²）,盐度40‰	匍匐茎

候选基因Candidate gene	基因登录号 Accession No.	引物序列（上游/下游,5'-3'） Primer sequence（forward/revers, 5'-3'）	片段大小 Fragment length/bp	Tm/℃	E/%
ClACT	MW039598	GGTGGTGAACGATTCCGAT TTCTGACATCTCTTTACTGACTCTC	203	50	93.76
Cl18S	JN034412.1	ATCTAAGGAAGGCAGCAGG TCTATCGCCAGAAGTCCAAC	241	50	94.80
ClGAPDH	MW036245	GAAGAAGGACTGGAGAGGA GTAGGAACTCTAAACGCCA	135	50	95.36
ClEF1a	MW021590	AGCTGGAATTTCAAAAGACG AGAGATAGGAACAAAGGGGA	211	50	102.00
ClTUB	MW039599	ATTGGGAAGGAGATGGTGGA GACTTGCGGAGACGGATAAA	204	54	90.36

候选基因Candidate gene	基因登录号 Accession No.	引物序列（上游/下游,5'-3'） Primer sequence（forward/revers, 5'-3'）	片段大小 Fragment length/bp	Tm/℃	E/%
ClACT	MW039598	GGTGGTGAACGATTCCGAT TTCTGACATCTCTTTACTGACTCTC	203	50	93.76
Cl18S	JN034412.1	ATCTAAGGAAGGCAGCAGG TCTATCGCCAGAAGTCCAAC	241	50	94.80
ClGAPDH	MW036245	GAAGAAGGACTGGAGAGGA GTAGGAACTCTAAACGCCA	135	50	95.36
ClEF1a	MW021590	AGCTGGAATTTCAAAAGACG AGAGATAGGAACAAAGGGGA	211	50	102.00
ClTUB	MW039599	ATTGGGAAGGAGATGGTGGA GACTTGCGGAGACGGATAAA	204	54	90.36

基因 Gene	基因登录号 Accession No.	引物序列（上游/下游,5'-3'） Primer sequence (forward/reverse, 5'-3')	片段大小 Fragment length/bp	Tm/℃
ClHSP90	MW088997	ATGGAGGCATTGTCCGCTGTTGCTCATCGTCGTTATGGTGT	123	55
ClSSD	MW088998	CACTCGTGATGAGGATGGAAAGCTGGCTCCTTGCTCTTATCGTA	98	55