西葫芦CpActin内参基因的分离及其作为内参基因的初步应用
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摘要
荧光定量PCR(q RT-PCR)是研究分子生物学中基因表达的一种新型核酸定量技术,具备快速高效、特异性强、重复性好、灵敏度高和自动化程度高等优点,因而根据相应的实验材料选择适宜的内参基因对于提高qRT-PCR分析的准确性显得尤为重要。Actin基因表达范围广且表达稳定,常常作为内参基因应用于qRT-PCR表达分析中。前期利用转录组测序(RNA-seq)技术获得了西葫芦低温弱光转录组数据库,本研究从中筛选到1条长达2543 bp的cDNA,并对其进行了序列分析。生物信息学分析结果表明,该序列包含1个开放读码框(ORF),大小为2064 bp,预测编码氨基酸数为682个,理论分子大小约为79.67 kD,蛋白质等电点为6.28。Wolf Psort分析发现,CpActin蛋白亚细胞定位于细胞核中。Motif Scan分析显示,CpActin蛋白质的氨基酸序列207～406位和558～682位分别为Actin的中端和C端保守区域。同源性分析表明,基因编码的蛋白质与同为南瓜属的中国南瓜和印度南瓜同源蛋白的相似性达到99%,具有高度的保守性。基于所获得的基因全长cDNA序列,设计了基因ORF全长引物对CpActin-F、CpActin-R,经PCR扩增得到一条特异、明亮的条带,经测序验证,序列与RNAseq数据库的c DNA序列一致,基因命名为CpActin,GenBank登录号为MH211008。在此基础上,设计了一对荧光定量PCR引物CpActin-Fq、CpActin-Rq,分析显示,该引物具有较高的特异性和扩增效率,且在西葫芦不同组织(根、茎、花和果)和不同胁迫处理[正常(25℃,300μmol/m~2·s)、低温处理(4℃,300μmol/m~2·s)、弱光处理(25℃,80μmol/m~2·s)、低温弱光处理(4℃,80μmol/m~2·s)、强光处理(25℃,2000μmol/m~2·s)和高温处理(38℃,300μmol/m~2·s)]叶片中均能稳定表达,适合在西葫芦基因qRT-PCR表达分析的研究中作为候选内参基因。
Quantitative real-time PCR(i.e.,qRT-PCR),as one of nucleic acid quantification technologies used for analyzing gene expression in molecular biology,has provided the advantage of high efficiency,high specificity,good repeatability,high sensitivity and high degree of automation.Therefore,selecting suitable internal reference genes based on the corresponding experimental materials is particularly important for ensuring the accuracy of qRT-PCR analysis.Since the Actin gene expresses consistently without tissue specificity,it has been often used as internal reference for normalizing the gene expression.Previously,we generated the early low-temperature and low-light transcriptome datasets of Cucurbita pepo L.using transcriptome sequencing(i.e.,RNA-seq)technology.Here we identified a 2543 bp cDNA from this database,with a size of 2064 bp open reading frame(i.e.,ORF)encoding 682 amino acids.This deduced protein showed a theoretical molecular weight of 79.67 kD and a protein isoelectric point(i.e.,PI)of 6.28.Wolf Psort analysis indicated that CpActin protein was located in the nucleus,and Motif Scan analysis showed that CpActin protein had the Mid-end and C-end domains of conserved Actin in the position of 207-406 and 558-682 sites,respectively.Cpactin shared 99% identity on amino acids with the homologous proteins from the relatives Cucurbita moschata Duchesne and Cucurbita maxima Duchesne,proving that this protein was highly conservative.By using a pair of primers that was designed for amplifying the full-length ORF,we re-generated and verified the CpActin fragment(Genbank ID:MH211008).What′s more,a pair of qRT-PCR primers CpActin-Fq and CpActin-Rq were designed.The analysis showed that the pair primers has high specificity and amplification efficiency,which can be stably expressed in different tissues(i.e.,root,stem,flower,fruit)and different stress treatments [i.e.,normal(25 ℃,300 μmol/m~2· s),low temperature treatment(4 ℃,300 μmol/m~2· s),low light treatment(25 ℃,80 μmol/m~2· s),low temperature and low light treatment(4 ℃,80 μmol/m~2· s),high light treatment(25 ℃,2000 μmol/m~2· s)and high temperature treatment(38 ℃,300 μmol/m~2· s)] leaves of Cucurbita pepo L..And it was suitable for use as a candidate internal reference gene in the study of qRT-PCR expression analysis of Cucurbita pepo L..Thus,this study provided a reference gene CpActin being suitable for qRT-PCR analysis of important functional genes of Cucurbita pepo L..
Quantitative real-time PCR(i.e.,qRT-PCR),as one of nucleic acid quantification technologies used for analyzing gene expression in molecular biology,has provided the advantage of high efficiency,high specificity,good repeatability,high sensitivity and high degree of automation.Therefore,selecting suitable internal reference genes based on the corresponding experimental materials is particularly important for ensuring the accuracy of qRT-PCR analysis.Since the Actin gene expresses consistently without tissue specificity,it has been often used as internal reference for normalizing the gene expression.Previously,we generated the early low-temperature and low-light transcriptome datasets of Cucurbita pepo L.using transcriptome sequencing(i.e.,RNA-seq)technology.Here we identified a 2543 bp cDNA from this database,with a size of 2064 bp open reading frame(i.e.,ORF)encoding 682 amino acids.This deduced protein showed a theoretical molecular weight of 79.67 kD and a protein isoelectric point(i.e.,PI)of 6.28.Wolf Psort analysis indicated that CpActin protein was located in the nucleus,and Motif Scan analysis showed that CpActin protein had the Mid-end and C-end domains of conserved Actin in the position of 207-406 and 558-682 sites,respectively.Cpactin shared 99% identity on amino acids with the homologous proteins from the relatives Cucurbita moschata Duchesne and Cucurbita maxima Duchesne,proving that this protein was highly conservative.By using a pair of primers that was designed for amplifying the full-length ORF,we re-generated and verified the CpActin fragment(Genbank ID:MH211008).What′s more,a pair of qRT-PCR primers CpActin-Fq and CpActin-Rq were designed.The analysis showed that the pair primers has high specificity and amplification efficiency,which can be stably expressed in different tissues(i.e.,root,stem,flower,fruit)and different stress treatments [i.e.,normal(25 ℃,300 μmol/m~2· s),low temperature treatment(4 ℃,300 μmol/m~2· s),low light treatment(25 ℃,80 μmol/m~2· s),low temperature and low light treatment(4 ℃,80 μmol/m~2· s),high light treatment(25 ℃,2000 μmol/m~2· s)and high temperature treatment(38 ℃,300 μmol/m~2· s)] leaves of Cucurbita pepo L..And it was suitable for use as a candidate internal reference gene in the study of qRT-PCR expression analysis of Cucurbita pepo L..Thus,this study provided a reference gene CpActin being suitable for qRT-PCR analysis of important functional genes of Cucurbita pepo L..
引文
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[3]Cruz F,Kalaoun S,Nobile P,Colombo C,Almeida J,Barros L M G,Romano E,Grossi-de-Sa M F,Vaslin M,Alves-Ferreira M.Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR.Molecular Breeding,2009,23(4):607-616
[4]Andersen C L,Jensen J L,Orntoft T F.Normalization of Realtimequantitativereversetranscription-PCRdata:Amodelbased variance estimation approach to identify genes suited for normalization,appliedtobladderandcoloncancerdatasets.Cancer Research,2004,64(15):5245-5250
[5]朱海生,陈敏氡,温庆放,蓝新隆,李永平,王彬,张前荣,吴卫东.丝瓜18S rRNA基因克隆及其作为内参基因的应用.核农学报,2016,30(1):35-41ZhuHS,ChenMD,WenQF,LanXL,Li YP,WangB,ZhangQR,Wu WD.Cloningof18SrRNAgenefromLuffa cylindrical and its application as an internal standard.Journal of Nuclear Agricultural Sciences,2016,30(1):35-41
[6]Chen J H,Sun Y,Sun F,Xia X L,Yin W L.Tobacco plants ectopicallyexpressingtheAmmopiptanthusmongolicusAmCBL1genedisplayenhancedtolerancetomultipleabiotic stresses.Plant Growth Regulation,2011,63(3):259-269
[7]Zhou L,Wang Y,Peng Z H.Molecular characterization and expressionanalysisofchalconesynthasegeneduringflower developmentintreepeony(Paeoniasuffruticosa).African Journal of Biotechnology,2011,10(8):1275-1284
[8]王彦杰,董丽,张超,王晓庆.牡丹实时定量PCR分析中内参基因的选择.农业生物技术学报,2012,20(5):521-528WangYJ,DongL,ZhangC,WangXQ.Referencegene selectionforreal-timequantitativePCRnormalizationintree peony(PaeoniasuffruticosaAndr.).Journalof Agricultural Biotechnology,2012,20(5):521-528
[9]陈颖,王刚,赵俊霞.高等植物体内的肌动蛋白.生物学通报,2003(1):13-15Chen Y,Wang G,Zhao J X.Actin in higher plants.Bulletin of Biology,2003(1):13-15
[10]Dominguez R,Holmes K C.Actin structure and function.Annual Review ofBiophysics,2011,40:69-186
[11]Vidali L,McKenna S T,Hepler P K.Actin polymerization is essential for pollen tube growth.Molecular Biology of the Cell,2001,12(8):2534-2545
[12]Nadella K S,Saji M,Jacob N K.Regulation of actin function by protein kinase A-mediated phosphorylation of limk1.European MolecularBiologyOrganizationReport,2009,10(6):599-605
[13]Kandasamy M K,McKinney E C,Meagher R B.A single vgetativeactinisovariantoverexpressedunderthecontrol ofmultipleregulatorysequencesissufficientfornormal Arabidopsis development.Plant Cell,2009,21:701-718
[14]Reece K S,McEkoy D,Wu R.Genomic nucleotide sequence of four rice(Oryza saiva)Actin genes.Plant Molecular Biology,1990,14(41):621-624
[15]Yasuda H,Kanda K,Koiwa H.Localization of actin filaments onmitoticapparatusintobaccoBY-2cells.Planta,2005,222(1):118-129
[16]范丙友,李芳,张文婷,史国安.芍药肌动蛋白基因的克隆及表达分析.中草药,2013,44(15):2136-2142FanB Y,LiF,Zhang W T,ShiG A.Cloningandexpression analysisonactingenefromPaeonialactiflora.Chinese Traditional and Herbal Drugs,2013,44(15):2136-2142
[17]梁云,袁素霞,冯慧颖,徐雷锋,袁迎迎,刘春,明军.百合肌动蛋白基因lilyActin的克隆与表达分析.园艺学报,2013,40(7):1318-1326Liang Y,YuanSX,FengH Y,XuLF,Yuan Y Y,LiuC,Ming J.Cloning and expression analysis of actin gene(lily Actin)from Lily.Acta Horticulturae Sinica,2013,40(7):1318-1326
[18]侯双利,韩梅,刘翠晶,杨利民.人参β-actin基因实时荧光定量PCR方法的建立.中草药,2014,45(17):2530-2533HouSL,HanM,LiuCJ,YangLM.Developmentofrealtime fluorescence quantitative RT-PCR assay forβ-actin gene of Panax ginseng.Chinese Traditional and Herbal Drugs,2014,45(17):2530-2533
[19]徐纪明,向太和.全雌性黄瓜中3个肌动蛋白基因片段的克隆和表达分析.细胞生物学杂志,2008,30,(1):125-130XuJM,XiangTH.Cloningandcharacterizationofthree actingenesingynoeciouslinecucumber(CucumissativusL.).Chinese Journal of Cell Biology,2008,30(1):125-130
[20]吕品,李娜,刘慧,赵文恩.西瓜八氢番茄红素合成酶基因全长的克隆与表达分析.郑州大学学报:理学版,2014,46(2):104-108Lv P,Li N,Liu H,Zhao W E.Cloning and expression analysis of phytoene synthase gene in watermelon(Citrullus lanatus).JournalofZhengzhouUniversity:NaturalScienceEdition,2014,46(2):104-108
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