启动子探针载体的构建及橡胶树白粉菌启动子筛选鉴定
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摘要
旨在构建适于快速检测橡胶树白粉病菌(Oidium heveae)HO-73启动子的探针载体。以卡那霉素抗性基因Kan作为报告基因,在pUC19骨架载体,构建获得启动子探针载体pUC19-K,连入CaMV35S启动子做启动子探针载体功能验证;利用启动子探针载体pUC19-K对预测的启动子片段LY1、LY2、LY3、LY4进行筛选鉴定。将CaMV35S启动子连入到启动子探针载体中,得到可检测启动子活性的启动子探针载体pUC19-K;应用生物信息学软件对部分橡胶树白粉菌HO-73全基因组数据预测,得到4个理论上具有活性的启动子序列LY1、LY2、LY3、LY4,利用构建的启动子活性探针载体进行活性比较,卡那耐受性实验检测发现含LY2和LY3的菌株随卡那霉素浓度的升高耐受性更强,最终得到2个活性较强的启动子LY2和LY3。以上结果表明,构建的启动子活性探针载体可以有效、灵敏地用于HO-73强启动子的筛选和启动子活性检测。
This study aims to construct a probe vector suitable for rapidly detecting the promoter of Oidium heveae HO-73. Thekanamycin-resistance gene was used as a reporter gene to construct the promoter probe vector pUC19-K in the pUC19 backbone vector,and itwas ligated with the CaMV35 S promoter to verify the function of the promoter probe vector. The promoter probe vector pUC19-K was employed toscreen and identify the predicted promoter fragments LY1,LY2,LY3,and LY4. The CaMV35 S promoter was ligated into the promoter probevector to obtain the promoter probe vector pUC19-K that can detect the promoter activity. Using bioinformatics software to predict the wholegenome data of Oidium heveae HO-73,4 theoretically active promoter sequences,LY1,LY2,LY3,and LY4,were obtained,and theiractivities were compared using the constructed promoter activity probe vector. The Kana tolerance test showed that strains containing LY2 andLY3 were more tolerant with increasing concentrations of kanamycin,resulting in two more active promoters,LY2 and LY3.The above resultsindicate that the constructed promoter activity probe vector can be effectively and sensitively used for screening the strong promoter of HO-73 and detecting promoter activity.
This study aims to construct a probe vector suitable for rapidly detecting the promoter of Oidium heveae HO-73. Thekanamycin-resistance gene was used as a reporter gene to construct the promoter probe vector pUC19-K in the pUC19 backbone vector,and itwas ligated with the CaMV35 S promoter to verify the function of the promoter probe vector. The promoter probe vector pUC19-K was employed toscreen and identify the predicted promoter fragments LY1,LY2,LY3,and LY4. The CaMV35 S promoter was ligated into the promoter probevector to obtain the promoter probe vector pUC19-K that can detect the promoter activity. Using bioinformatics software to predict the wholegenome data of Oidium heveae HO-73,4 theoretically active promoter sequences,LY1,LY2,LY3,and LY4,were obtained,and theiractivities were compared using the constructed promoter activity probe vector. The Kana tolerance test showed that strains containing LY2 andLY3 were more tolerant with increasing concentrations of kanamycin,resulting in two more active promoters,LY2 and LY3.The above resultsindicate that the constructed promoter activity probe vector can be effectively and sensitively used for screening the strong promoter of HO-73 and detecting promoter activity.
引文
[1]陈明文.我国天然橡胶产业发展形势与因应策略[J].农业经济问题, 2016, 10:91-94, 112.
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[3]万三连,梁鹏,缪卫国,等.橡胶树与白粉病菌Oidium heveae亲和互作组织细胞学研究[J].植物保护, 2014, 40(3):26-36.
[4]Peramuna A, Bae H, Rasmussen EK, et al. Evaluation of syntheticpromoters in Physcomitrella patens[J]. Biochemical andBiophysical Research Communications, 2018, 500:418-422.
[5]Song Y, Nikoloff JM, Fu G, et al. Promoter screening from Bacillus subtilis in various conditions hunting for synthetic biology andindustrial applications[J]. PLoS One, 2016, 11(7):e0158447.
[6]Tao YB, He ll, Niu LJ, et al. Isolation and characterization of anubiquitin extension protein gene(JcUEP)promoter from Jatropha curcas[J]. Planta, 2015, 241(4):823-836.
[7]Li X, Bi ZH, Di R, et al. Identification of powdery mildew responsivegenes in Hevea brasiliensis through mRNA differential display[J].Int J Mol Sci, 2016, 17(2):181.
[8]贾娇.灰葡萄孢钙调磷酸酶基因β亚基及其启动子的克隆与序列分析[D].保定:河北农业大学, 2008.
[9]萨姆布鲁克J,拉塞尔DW.分子克隆实验指南[M].第3版.黄培堂,译.北京:科学出版社, 2008.
[10]Gao Y, Zan XL, Wu XF, et al. Identification of fungus-responsivecis-acting element in the promoter of Brassica juncea chitinasegene, BjCHI1[J]. Plant Science, 2014, 215-216(215-216C):190-198.
[11]Haapalainen M, Karp M, Metzler MC. Isolation of strong promotersfrom Clavibacter xyli subsp cynodontis using a promoter probeplasmid[J]. Biochimica Et Biophysica Acta, 1996, 1305(3):130-134.
[12]Prestridge DS. Predicting Pol II promoter sequences usingtranscription factor binding sites[J]. J Mol Biol, 1995, 249(5):923-932.
[13]Sandelin A, Carninci P, Lenhard B, et al. Mammalian RNApolymerase II core promoters:insights from genome-widestudies[J]. Nat Rev Genet, 2007, 8(6):424-436.
[14]杨乌日吐,林昊.基于多特征尺度的大肠杆菌启动子预测[J].计算机工程与科学, 2010, 32(9):148-151.
[15]Zhang X, Gao T, Peng Q, et al. A strong promoter of a noncry, gene directs expression of the cry1Ac, gene in Bacillus thuringiensis[J]. Applied Microbiology&Biotechnology, 2018,102(8):1-13.
[16]赵静.利用启动子探针载体分离与筛选Arthrobacter sp. A3冷诱导启动子[D].兰州:兰州大学, 2010.
[17]Wang Q, Yi Z, Lin S, et al. Transgenic Bt cotton driven by the greentissue-specific promoter shows strong toxicity to lepidopteran pestsand lower Bt toxin accumulation in seeds[J]. Science China LifeSciences, 2016, 59(2):172-182.
[18]Woodfint RM, Chen PR, Ahn J, et al. Identification of the MUC2promoter as a strong promoter for intestinal gene expression throughgeneration of transgenic quail expressing GFP in gut epithelialcells[J]. Int J Mol Sci, 2017, 18(1):196.
[19]Jong AD, Pietersma H, Cordes M, et al. PePPER:a webserver forprediction of prokaryote promoter elements and regulons[J].BMC Genom, 2012, 13(1):1-10.
[20]万三连,梁鹏,缪卫国,等.橡胶树白粉病菌分生孢子在不同介质的萌发过程及其内容物的变化[J].植物病理学报,2014, 44(6):595-602.
[21]Saidi Y, Schaefer DG, Goloubinoff P, et al. The CaMV 35S promoterhas a weak expression activity in dark grown tissues of moss[J].Plant Signaling&Behavior, 2009, 4(5):457-459.
[22]Torre FDl, Rodríguez R, Jorge G, et al. Genetic transformation ofEucalyptus globulus, using the vas cular-specific EgCCR, as analternative to the constitutive CaMV35S, promoter[J]. Plant CellTissue&Organ Culture, 2014, 117(1):77-84.
[23]Martínez F, Arif A, Nebauer SG, et al. A fungal transcription factorgene is expressed in plants from its own promoter and improvesdrought tolerance[J]. Planta, 2015, 242(1):1-14.
[24]徐茂军.新型食品添加剂卡那霉素抗性基因编码蛋白的安全性评价[J].食品科学, 2001, 22:96-98.
[25]白泽涛,柴国华,石磊,等.油菜毛状体发育相关基因GLABRA2启动子的克隆和功能验证[J].农业生物技术学报,2010, 18(2):210-217.
[2]金华斌,田维敏,史敏晶.我国天然橡胶产业发展概况及现状分析[J].热带农业科学, 2017, 5:98-104.
[3]万三连,梁鹏,缪卫国,等.橡胶树与白粉病菌Oidium heveae亲和互作组织细胞学研究[J].植物保护, 2014, 40(3):26-36.
[4]Peramuna A, Bae H, Rasmussen EK, et al. Evaluation of syntheticpromoters in Physcomitrella patens[J]. Biochemical andBiophysical Research Communications, 2018, 500:418-422.
[5]Song Y, Nikoloff JM, Fu G, et al. Promoter screening from Bacillus subtilis in various conditions hunting for synthetic biology andindustrial applications[J]. PLoS One, 2016, 11(7):e0158447.
[6]Tao YB, He ll, Niu LJ, et al. Isolation and characterization of anubiquitin extension protein gene(JcUEP)promoter from Jatropha curcas[J]. Planta, 2015, 241(4):823-836.
[7]Li X, Bi ZH, Di R, et al. Identification of powdery mildew responsivegenes in Hevea brasiliensis through mRNA differential display[J].Int J Mol Sci, 2016, 17(2):181.
[8]贾娇.灰葡萄孢钙调磷酸酶基因β亚基及其启动子的克隆与序列分析[D].保定:河北农业大学, 2008.
[9]萨姆布鲁克J,拉塞尔DW.分子克隆实验指南[M].第3版.黄培堂,译.北京:科学出版社, 2008.
[10]Gao Y, Zan XL, Wu XF, et al. Identification of fungus-responsivecis-acting element in the promoter of Brassica juncea chitinasegene, BjCHI1[J]. Plant Science, 2014, 215-216(215-216C):190-198.
[11]Haapalainen M, Karp M, Metzler MC. Isolation of strong promotersfrom Clavibacter xyli subsp cynodontis using a promoter probeplasmid[J]. Biochimica Et Biophysica Acta, 1996, 1305(3):130-134.
[12]Prestridge DS. Predicting Pol II promoter sequences usingtranscription factor binding sites[J]. J Mol Biol, 1995, 249(5):923-932.
[13]Sandelin A, Carninci P, Lenhard B, et al. Mammalian RNApolymerase II core promoters:insights from genome-widestudies[J]. Nat Rev Genet, 2007, 8(6):424-436.
[14]杨乌日吐,林昊.基于多特征尺度的大肠杆菌启动子预测[J].计算机工程与科学, 2010, 32(9):148-151.
[15]Zhang X, Gao T, Peng Q, et al. A strong promoter of a noncry, gene directs expression of the cry1Ac, gene in Bacillus thuringiensis[J]. Applied Microbiology&Biotechnology, 2018,102(8):1-13.
[16]赵静.利用启动子探针载体分离与筛选Arthrobacter sp. A3冷诱导启动子[D].兰州:兰州大学, 2010.
[17]Wang Q, Yi Z, Lin S, et al. Transgenic Bt cotton driven by the greentissue-specific promoter shows strong toxicity to lepidopteran pestsand lower Bt toxin accumulation in seeds[J]. Science China LifeSciences, 2016, 59(2):172-182.
[18]Woodfint RM, Chen PR, Ahn J, et al. Identification of the MUC2promoter as a strong promoter for intestinal gene expression throughgeneration of transgenic quail expressing GFP in gut epithelialcells[J]. Int J Mol Sci, 2017, 18(1):196.
[19]Jong AD, Pietersma H, Cordes M, et al. PePPER:a webserver forprediction of prokaryote promoter elements and regulons[J].BMC Genom, 2012, 13(1):1-10.
[20]万三连,梁鹏,缪卫国,等.橡胶树白粉病菌分生孢子在不同介质的萌发过程及其内容物的变化[J].植物病理学报,2014, 44(6):595-602.
[21]Saidi Y, Schaefer DG, Goloubinoff P, et al. The CaMV 35S promoterhas a weak expression activity in dark grown tissues of moss[J].Plant Signaling&Behavior, 2009, 4(5):457-459.
[22]Torre FDl, Rodríguez R, Jorge G, et al. Genetic transformation ofEucalyptus globulus, using the vas cular-specific EgCCR, as analternative to the constitutive CaMV35S, promoter[J]. Plant CellTissue&Organ Culture, 2014, 117(1):77-84.
[23]Martínez F, Arif A, Nebauer SG, et al. A fungal transcription factorgene is expressed in plants from its own promoter and improvesdrought tolerance[J]. Planta, 2015, 242(1):1-14.
[24]徐茂军.新型食品添加剂卡那霉素抗性基因编码蛋白的安全性评价[J].食品科学, 2001, 22:96-98.
[25]白泽涛,柴国华,石磊,等.油菜毛状体发育相关基因GLABRA2启动子的克隆和功能验证[J].农业生物技术学报,2010, 18(2):210-217.
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