蓖麻延伸因子基因的克隆与表达分析
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摘要
为探讨蓖麻延伸因子基因(RcEF-1α)作为参考基因进行相关研究的可行性,以蓖麻雌性花为试验材料,扩增RcEF-1α(GenBank登录号:XM_002518027. 3)的编码序列(CDS),并将其亚克隆至原核表达载体pET32a(+)构建成重组载体p ET32a(+)-RcEF-1α;将p ET32a(+)-RcEF-1α转化至大肠杆菌表达菌株BL21 (DE3)中进行优化表达,表达蛋白经纯化后进行Western blot验证。结果表明,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)可诱导一个分子量约66 kD的特异蛋白;采用纯化的表达蛋白免疫新西兰白兔制备抗RcEF-1α的多克隆抗血清,用多克隆抗血清进行Western blot验证,发现多克隆抗血清具有针对RcEF-1α的特异性,经ELISA检测,其效价为1∶51 200;以抗血清为一抗,通过Western blot分析RcEF-1α在时空条件一致且正常生长的根、茎、叶、雌花、雄花和果实中的表达量,结果显示,蓖麻各组织中RcEF-1α蛋白质的含量存在显著差异,其中雌花和雄花中的含量显著高于其他组织。RT-qPCR分析结果表明,蓖麻各组织中RcEF-1αmRNA差异不显著。本研究结果为蓖麻功能基因表达分析参考基因的筛选提供了一定的理论依据。
In order to evaluate the feasibility that the elongation factor-1α( EF-1α) gene of castor( Ricinus communis L.),named as RcEF-1α,acts as internal reference gene for the relevant researchs,using the female flower of castor as the experimental material. The coding sequence( CDS) of RcEF-1α was isolated from mRNA of pistillate flowers from castor by RT-PCR with gene specific primers designed according to mRNA sequence of RcEF-1α published in GenBank( Accession number: XM_002518027.3). Furthermore,a prokaryotic expression vector pET32 a( +)-RcEF-1α was constructed by double restriction-enzyme digestion and transformed into E. coli BL21( DE3). The protein was purified and verified by Western blot. The results showed that Isopropyl-β-D-thiopyran galactoglyceride( IPTG) could induce a specific protein with a molecular weight of about 66 kD. RcEF-1α proteins purified were used to immunize adult rabbits following standard protocols. Consequently,it was found by using Western blot analysis that polyclonal immune serum against RcEF-1α had high specificity. The results of ELISA showed that the titer of polyclonal immune serum was 1: 51200. The expression of RcEF-1α protein were analyzed using the prepared polyclonal immune serum through Western blot in different tissues growing normally with the same spatio-temporal conditions,including roots,stems,leaves,female flowers,male flowers and fruits. It was found that RcEF-1α were significantly more expressed in female flowers and male flowers than other tissues. Meanwhile,The relative expression levels of RcEF-1α were determined by fluorescent RT-PCR in these tissues. Statistical analysis showed that there was no significant difference in the transcription level of RcEF-1α from castor. So RcEF-1α gene was suitable as an internal control at transcriptional level for analysis of functional genes from castor,but not suitable at translational level. Study on RcEF-1α expression facilitated the elucidation of its role in the analysis of the relative expression of functional genes from castor.
In order to evaluate the feasibility that the elongation factor-1α( EF-1α) gene of castor( Ricinus communis L.),named as RcEF-1α,acts as internal reference gene for the relevant researchs,using the female flower of castor as the experimental material. The coding sequence( CDS) of RcEF-1α was isolated from mRNA of pistillate flowers from castor by RT-PCR with gene specific primers designed according to mRNA sequence of RcEF-1α published in GenBank( Accession number: XM_002518027.3). Furthermore,a prokaryotic expression vector pET32 a( +)-RcEF-1α was constructed by double restriction-enzyme digestion and transformed into E. coli BL21( DE3). The protein was purified and verified by Western blot. The results showed that Isopropyl-β-D-thiopyran galactoglyceride( IPTG) could induce a specific protein with a molecular weight of about 66 kD. RcEF-1α proteins purified were used to immunize adult rabbits following standard protocols. Consequently,it was found by using Western blot analysis that polyclonal immune serum against RcEF-1α had high specificity. The results of ELISA showed that the titer of polyclonal immune serum was 1: 51200. The expression of RcEF-1α protein were analyzed using the prepared polyclonal immune serum through Western blot in different tissues growing normally with the same spatio-temporal conditions,including roots,stems,leaves,female flowers,male flowers and fruits. It was found that RcEF-1α were significantly more expressed in female flowers and male flowers than other tissues. Meanwhile,The relative expression levels of RcEF-1α were determined by fluorescent RT-PCR in these tissues. Statistical analysis showed that there was no significant difference in the transcription level of RcEF-1α from castor. So RcEF-1α gene was suitable as an internal control at transcriptional level for analysis of functional genes from castor,but not suitable at translational level. Study on RcEF-1α expression facilitated the elucidation of its role in the analysis of the relative expression of functional genes from castor.
引文
[1]Martins P K,MafraⅤ,de Souza W R,Ribeiro A P,Vinecky F,Basso M F,de Cunha B A D B,Kobayashi A K,Molinari H B C.Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis[J].Scientific Reports,2016,6(1):e28348
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[3]Janska A,Hodek J,Svoboda P,Zamecnik J,Prasil I T,Vlasakova E,Milella L,Ovesna J.The choice of reference gene set for assessing gene expression in barley(Hordeum vulgare L.)under low temperature and drought stress[J].Molecular Genetics&Genomics Mgg,2013,288(11):639-649
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[5]Nguyen D Q,Eamens A L,Grof C P L.Reference geneidentification for reliable normalisation of quantitative RT-PCR data in Setaria viridis[J].Plant Methods,2018,14(1):24-35
[6]赵亚运,张玉,曹明月,黄红丽,李娟,薛良义.大黄鱼microRNA荧光定量PCR中内参基因的选择与评价[J].核农学报,2017,31(7):1299-1309
[7]Joseph J T,Poolakkalody N J,Shah J M.Plant reference genes for development and stress response studies[J].Journal of Biosciences,2018,43(1):173-187
[8]Lilly S T,Drummond R S,Pearson M N,MacD iarmid R M.Identification and validation of reference genes for normalization of transcripts from virus infected Arabidopsis thaliana[J].Molecular Plant Microbe Interactions.2011,24(3):294-304
[9]覃迎姿,黄先益,叶兴枝,王爱勤,李杨瑞.植物延伸因子eE F1A研究进展[J].广西农业科学,2009,40(5):472-477
[10]蒋素华,王默霏,宋彩霞,梁芳,李艳辉,崔波.萼脊兰EF1α基因片段的克隆及序列分析[J].贵州农业科学,2016,44(5):5-8
[11]Pittman Y R,Kandl K,Lewis M,Valente L,Kinzy T G.Coordination of eukaryotic translation elongation factors 1A(eE F1A)function in action organization and translation elongation by the guanine nucleotide exchange factor eE F1Bα[J].Journal of Biological Chemistry,2009,284(7):4739-4747
[12]Bhadula S K,Ristic Z.Heat-stress induced synthesis of chloroplast protein synthesis elongation factor(EF-Tu)in a heat-tolerant maize line[J].Planta,2001,212(3):359-366
[13]Momcilovic I,Ristic Z.Expression of chloroplast protein synthesis elongation factor,EF-Tu,in two lines of maize with contrasting tolerance to heat stress during early stages of plant development[J].Journal of Plant Physiology,2007,164(1):90-99
[14]Infante C,Asensio E,Ca1avate J P,Manchado M.Molecular characterization and expression analysis of five different elongation factor 1 alpha genes in the flatfish Senegalese sole(Solea senegalensis Kaup):differential gene expression and thyroid hormones dependence during metamorphosis[J].BMC Molecular Biology,2008,9(1):19
[15]Wan H,Zhao Z,Qian C,Sui Y,Malik A A,Chen J.Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber[J].Analytical Biochemistry,2010,399(2):257-261
[16]Mallona I,Lischewski S,Weiss J,Hause B,Egeacortines M.Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida[J].BMC Molecular Biology,2010,10(4):4-14
[17]Qi J,Yu S,Zhang F,Shen X,Zhao X,Yu Y,Zhang D.Reference gene selection for real-time quantitative polymerase chain reaction of mRNA transcript levels in Chinese cabbage(Brassica rapa ssp.pekinensis)[J].Plant Molecular Biology Reporter,2010,28(4):597-604
[18]王志敏,牛义,许俊强,孙梓健,丁泽琴,杨修勤,汤青林,宋明.结球甘蓝花粉蛋白延伸因子基因的克隆及其序列分析[J].园艺学报,2013,40(10):1990-1998
[19]彭亚兰,王友绍.红树植物桐花树EF1A基因的克隆与表达分析[J].生态科学,2014,33(4):704-712
[20]Aguilar F,Montandon P E,Stutz E.Two genes encoding the soybean translation elongation factor eE F-1αare transcribed in seedling leaves[J].Plant Molecular Biology,1991,17(3):351-360
[21]李子银,陈受宜.环境因子胁迫下水稻翻译延伸因子1A基因的诱导表达[J].植物学报,1999,41(8):800-806
[22]陈燕,王伟倩,张红莉,夏群芳,孙恒吉,李瑞沙.拟南芥翻译延伸因子EF1A的亚细胞定位研究[J].河南师范大学学报,2015,43(1):106-115
[23]阳永学,李振波,童超波,黄军艳,于景印,董彩华,刘胜毅.甘蓝型油菜半胱氨酸蛋白酶基因BnCP51及其启动子的克隆与表达[J].中国油料作物学报,2016,38(4):406-414
[24]董乐,姚丽梅,戴聪杰,张乐,黄苹苹,王建颖.大黄鱼β-actin抗体的制备与组织表达谱分析[J].海洋科学,2014,38(12):22-28
[25]贾伟,李春娟,闫彩霞,赵小波,王娟,孔青,孙全喜,单世华.花生AhNAC53基因的克隆及干旱胁迫下的表达分析[J].核农学报,2018,32(10):1898-1907
[26]Bradford M M.A rapid and sensitive method of or the quantitation of microgram quantitative of protein using the principle of protein-dye binding[J].Analytical Biochemistry,1976,72:248-254
[27]李振波,王琢玉,刘学群,覃瑞,黄军艳,董彩华,刘胜毅.甘蓝型油菜Bn19070的表达与同源基因At2g19070的amiRNAi研究[J].中国油料作物学报,2011,33(3):197-202
[28]Berberich T,Sugawara K,Harada M,Kusano T.Molecular cloning,characterization and expression of an elongation factor 1αgene in maize[J].Plant Molecular Biology,1995,29(3):611-615
[29]Tanaka S,Ikeda K,Ono M,Miyasaka H.Isolation of several antistress genes from a mangrove plant Avicennia marina[J].World Journal of Microbiology&Biotechnology,2002,18(8):801-804
[30]Vijaykumar D,Ramachander T V N,Mahishi L H,Kaul R,Pyati P,Paul B,Rawal S K.Molecular cloning,characterization and tissuespecific expression of an elongation factor 1A gene in Saccharum officinarum L.[J].Plant Science,2002,162(2):315-321
[31]孙伟,李燕,赵彦修,张慧.盐地碱蓬延伸因子(SsE F-1α)的克隆与表达分析[J].西北植物学报,2004,24(9):1657-1661
[32]潘刚,韩舒睿,沈智超,邸柄荣,孙银苹,赵卫国,楼程富,潘一乐.桑树橡胶延伸因子基因的克隆与表达分析[J].蚕业科学,2009,35(4):718-721
[33]Costa A,Giacomo M D,MassarelliⅠ,Palma M D,Leone A,Grillo M S.Isolation,characterization and expression of an elongation factor 1αgene in potato(Solanum tuberosum)cell cultures[J].Giornale Botanico Italiano,2010,144(3):618-625
[34]张玉芳,赵丽娟,曾幼玲.基因表达研究中内参基因的选择与应用[J].植物生理学报,2014,50(8):1119-1125
[35]Gue'nin S,Mauriat M,Pelloux J,Van Wuytswinkel O,Bellini C,Gutierrez L.Normalization of qRT-PCR data:the necessityof adopting a systematic,experimental conditions-specific,validation of references[J].Journal of Experimental Botany,2009,60(2):487-493
[2]Kozera B,Rapacz M.Reference genes in real-time PCR[J].Journal of Applied Genetics,2013,54(4):391-406
[3]Janska A,Hodek J,Svoboda P,Zamecnik J,Prasil I T,Vlasakova E,Milella L,Ovesna J.The choice of reference gene set for assessing gene expression in barley(Hordeum vulgare L.)under low temperature and drought stress[J].Molecular Genetics&Genomics Mgg,2013,288(11):639-649
[4]Gilda J E,Ghosh R,Cheah J X,West T M,Bodine S C,Gomes AV.Western blotting inaccuracies with unverified antibodies:need for a Western blotting minimal reporting standard(WBMRS)[J].PLoSOne,2015,10(8):e0135392
[5]Nguyen D Q,Eamens A L,Grof C P L.Reference geneidentification for reliable normalisation of quantitative RT-PCR data in Setaria viridis[J].Plant Methods,2018,14(1):24-35
[6]赵亚运,张玉,曹明月,黄红丽,李娟,薛良义.大黄鱼microRNA荧光定量PCR中内参基因的选择与评价[J].核农学报,2017,31(7):1299-1309
[7]Joseph J T,Poolakkalody N J,Shah J M.Plant reference genes for development and stress response studies[J].Journal of Biosciences,2018,43(1):173-187
[8]Lilly S T,Drummond R S,Pearson M N,MacD iarmid R M.Identification and validation of reference genes for normalization of transcripts from virus infected Arabidopsis thaliana[J].Molecular Plant Microbe Interactions.2011,24(3):294-304
[9]覃迎姿,黄先益,叶兴枝,王爱勤,李杨瑞.植物延伸因子eE F1A研究进展[J].广西农业科学,2009,40(5):472-477
[10]蒋素华,王默霏,宋彩霞,梁芳,李艳辉,崔波.萼脊兰EF1α基因片段的克隆及序列分析[J].贵州农业科学,2016,44(5):5-8
[11]Pittman Y R,Kandl K,Lewis M,Valente L,Kinzy T G.Coordination of eukaryotic translation elongation factors 1A(eE F1A)function in action organization and translation elongation by the guanine nucleotide exchange factor eE F1Bα[J].Journal of Biological Chemistry,2009,284(7):4739-4747
[12]Bhadula S K,Ristic Z.Heat-stress induced synthesis of chloroplast protein synthesis elongation factor(EF-Tu)in a heat-tolerant maize line[J].Planta,2001,212(3):359-366
[13]Momcilovic I,Ristic Z.Expression of chloroplast protein synthesis elongation factor,EF-Tu,in two lines of maize with contrasting tolerance to heat stress during early stages of plant development[J].Journal of Plant Physiology,2007,164(1):90-99
[14]Infante C,Asensio E,Ca1avate J P,Manchado M.Molecular characterization and expression analysis of five different elongation factor 1 alpha genes in the flatfish Senegalese sole(Solea senegalensis Kaup):differential gene expression and thyroid hormones dependence during metamorphosis[J].BMC Molecular Biology,2008,9(1):19
[15]Wan H,Zhao Z,Qian C,Sui Y,Malik A A,Chen J.Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber[J].Analytical Biochemistry,2010,399(2):257-261
[16]Mallona I,Lischewski S,Weiss J,Hause B,Egeacortines M.Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida[J].BMC Molecular Biology,2010,10(4):4-14
[17]Qi J,Yu S,Zhang F,Shen X,Zhao X,Yu Y,Zhang D.Reference gene selection for real-time quantitative polymerase chain reaction of mRNA transcript levels in Chinese cabbage(Brassica rapa ssp.pekinensis)[J].Plant Molecular Biology Reporter,2010,28(4):597-604
[18]王志敏,牛义,许俊强,孙梓健,丁泽琴,杨修勤,汤青林,宋明.结球甘蓝花粉蛋白延伸因子基因的克隆及其序列分析[J].园艺学报,2013,40(10):1990-1998
[19]彭亚兰,王友绍.红树植物桐花树EF1A基因的克隆与表达分析[J].生态科学,2014,33(4):704-712
[20]Aguilar F,Montandon P E,Stutz E.Two genes encoding the soybean translation elongation factor eE F-1αare transcribed in seedling leaves[J].Plant Molecular Biology,1991,17(3):351-360
[21]李子银,陈受宜.环境因子胁迫下水稻翻译延伸因子1A基因的诱导表达[J].植物学报,1999,41(8):800-806
[22]陈燕,王伟倩,张红莉,夏群芳,孙恒吉,李瑞沙.拟南芥翻译延伸因子EF1A的亚细胞定位研究[J].河南师范大学学报,2015,43(1):106-115
[23]阳永学,李振波,童超波,黄军艳,于景印,董彩华,刘胜毅.甘蓝型油菜半胱氨酸蛋白酶基因BnCP51及其启动子的克隆与表达[J].中国油料作物学报,2016,38(4):406-414
[24]董乐,姚丽梅,戴聪杰,张乐,黄苹苹,王建颖.大黄鱼β-actin抗体的制备与组织表达谱分析[J].海洋科学,2014,38(12):22-28
[25]贾伟,李春娟,闫彩霞,赵小波,王娟,孔青,孙全喜,单世华.花生AhNAC53基因的克隆及干旱胁迫下的表达分析[J].核农学报,2018,32(10):1898-1907
[26]Bradford M M.A rapid and sensitive method of or the quantitation of microgram quantitative of protein using the principle of protein-dye binding[J].Analytical Biochemistry,1976,72:248-254
[27]李振波,王琢玉,刘学群,覃瑞,黄军艳,董彩华,刘胜毅.甘蓝型油菜Bn19070的表达与同源基因At2g19070的amiRNAi研究[J].中国油料作物学报,2011,33(3):197-202
[28]Berberich T,Sugawara K,Harada M,Kusano T.Molecular cloning,characterization and expression of an elongation factor 1αgene in maize[J].Plant Molecular Biology,1995,29(3):611-615
[29]Tanaka S,Ikeda K,Ono M,Miyasaka H.Isolation of several antistress genes from a mangrove plant Avicennia marina[J].World Journal of Microbiology&Biotechnology,2002,18(8):801-804
[30]Vijaykumar D,Ramachander T V N,Mahishi L H,Kaul R,Pyati P,Paul B,Rawal S K.Molecular cloning,characterization and tissuespecific expression of an elongation factor 1A gene in Saccharum officinarum L.[J].Plant Science,2002,162(2):315-321
[31]孙伟,李燕,赵彦修,张慧.盐地碱蓬延伸因子(SsE F-1α)的克隆与表达分析[J].西北植物学报,2004,24(9):1657-1661
[32]潘刚,韩舒睿,沈智超,邸柄荣,孙银苹,赵卫国,楼程富,潘一乐.桑树橡胶延伸因子基因的克隆与表达分析[J].蚕业科学,2009,35(4):718-721
[33]Costa A,Giacomo M D,MassarelliⅠ,Palma M D,Leone A,Grillo M S.Isolation,characterization and expression of an elongation factor 1αgene in potato(Solanum tuberosum)cell cultures[J].Giornale Botanico Italiano,2010,144(3):618-625
[34]张玉芳,赵丽娟,曾幼玲.基因表达研究中内参基因的选择与应用[J].植物生理学报,2014,50(8):1119-1125
[35]Gue'nin S,Mauriat M,Pelloux J,Van Wuytswinkel O,Bellini C,Gutierrez L.Normalization of qRT-PCR data:the necessityof adopting a systematic,experimental conditions-specific,validation of references[J].Journal of Experimental Botany,2009,60(2):487-493