玉米钼辅助因子硫酸化酶基因启动子的克隆与功能验证

doi:10.3724/SP.J.1005.2014.0584

遗传 ›› 2014, Vol. 36 ›› Issue (6): 584-591.doi: 10.3724/SP.J.1005.2014.0584

玉米钼辅助因子硫酸化酶基因启动子的克隆与功能验证

高学焕, 付凤玲, 牟巍, 周树峰, 张素芝, 李晚忱

四川农业大学玉米研究所, 成都 611130

收稿日期:2013-09-30 修回日期:2014-01-02 出版日期:2014-06-20 发布日期:2014-05-28
通讯作者: 李晚忱,教授,博士生导师,研究方向：玉米遗传育种与生物技术。E-mail:aumdyms@sicau.edu.cn E-mail:gaoxh2011@qq.com
作者简介:高学焕,硕士研究生,专业方向：植物分子生物学。E-mail:gaoxh2011@qq.com
基金资助:
国家转基因重大科技专项(编号：2013ZX08003-005)资助

Cloning and functional validation of promoter of mo-molybdopterin cofactor sulfurase gene in maize

Xuehuan Gao, Fengling Fu, Wei Mu, Shufeng Zhou, Suzhi Zhang, Wanchen Li

Maize Research Institute, Sichuan Agricultural University, Chengdu 611130, China

Received:2013-09-30 Revised:2014-01-02 Online:2014-06-20 Published:2014-05-28

摘要/Abstract

摘要：

为克服组成型启动子启动外源基因过量表达引起的诸多问题,同源克隆(Mo-molybdopterin cofactor sulfurase)基因（ABA3）的启动子(ABA3s)序列,并用PlantCARE软件分析其非生物逆境应答元件, 实时定量PCR检测ABA3基因在非生物逆境诱导下的差异表达后。然后,用该启动子构建启动GUS（β-glucuronidase）基因的表达载体, 基因枪法转化玉米愈伤组织。经组织化学染色法检测其表达后, 在高渗、高盐、低温胁迫处理及ABA诱导下检测GUS酶荧光值与荧光素酶(内参)发光值的比值(GUS/LUC), 以此评价ABA3s启动子在非生物逆境胁迫下的启动活性。结果表明, ABA3基因在模拟干旱、低温、高温、高盐胁迫及ABA、乙稀诱导下差异表达, 说明该基因的启动子(ABA3s)具有非生物逆境诱导活性。序列分析表明, ABA3s启动子全长777 bp, 含有ARE、HSE、MBS、TGA、Circadian等多种非生物逆境胁迫应答元件。用ABA3s启动GUS基因构建的表达载体转化的玉米愈伤组织, 响应干旱、低温、高温、高盐胁迫等多种非生物逆境胁迫, 及ABA和乙稀诱导, GUS检测呈阳性。在8%甘露醇高渗条件下, GUS/LUC比值比空白对照高6倍。上述结果表明, ABA3s启动子具有非生物逆境诱导特性, 经进一步验证其功能后, 可用于玉米抗逆转基因研究。

关键词: 玉米, 非生物胁迫, 钼辅助因子硫酸化酶, 启动子

Abstract:

To overcome the problems caused by the over-expression of exogenous genes under the control of constitutive promoters, the promoter (ABA3s) sequence of maize (Zea mays) mo-molybdopterin cofactor sulfurase gene (ABA3) was cloned homologously, analyzed for its abiotic stress-responsive elements by the PlantCARE software, and detected for differential expression of the ABA3 gene under the abiotic stresses by real-time quantitative PCR. Then, this promoter was used to construct expression vector to start GUS (β-glucuronidase) gene, and transform maize calli by biolistics. After identification by histochemcal staining, the ratio of the GUS activity relative to the luciferase activity (internal control) (GUS/LUC) was measured under the stresses of hypertonic, high salt, low temperature, and the induction of ABA, and used to evaluate the activity of the ABA3s promoter in response to abiotic stresses. The results showed that the ABA3 gene was differentially expressed under the stress of simulative drought, low temperature, high temperature, high salt, and the induction of ABA and ethylene, indicating that the promoter (ABA3s) of this gene is induced by abtiotic stress. The sequence analysis showed that the ABA3s promoter is 777 bp long, and contains abiotic stress-responsive elements ARE, HSE, MBS, TGA and circadian. The transformed calli by the expression vector of the GUS gene under the control of the ABA3s promoter showed positive in GUS detection in response to the abiotic stresses of drought, low temperature, high temperature, high salt, and the induction of ABA and ethylene. The GUS/LUC ratio was six folds higher than the blank control under the hypertonic stress of 8% mannitol. It is concluded that the promoter ABA3s is inducible in response to abiotic stresses, and might be applied to transgenic research of maize for abiotic tolerance after further functional evaluation.

Key words: maize, abiotic stress, mo-molybdopterin cofactor sulfurase, promoter

高学焕, 付凤玲, 牟巍, 周树峰, 张素芝, 李晚忱. 玉米钼辅助因子硫酸化酶基因启动子的克隆与功能验证[J]. 遗传, 2014, 36(6): 584-591.

Xuehuan Gao, Fengling Fu, Wei Mu, Shufeng Zhou, Suzhi Zhang, Wanchen Li. Cloning and functional validation of promoter of mo-molybdopterin cofactor sulfurase gene in maize[J]. HEREDITAS(Beijing), 2014, 36(6): 584-591.

参考文献

[1]Tambo JA, Abdoulaye T. Climate change and agricultural technology adoption: the case of drought tolerant maize in rural Nigeria. Mitig Adapt Strat Glob Chan, 2012, 17(3): 277-292.
[2]Fu FL, Feng ZL, Gao SB, Zhou SF, Li WC. Evaluation and quantitative inheritance of several drought-relative traits in maize. Agri Sci Chin, 2008, 7(3): 280-290.
[3]Lu Y, Hao Z, Xie C, Crossa J, Araus JL, Gao S, Vivek BS, Magorokosho C, Mugo S, Makumbi D, Taba S, Pan G, Li X, Rong T, Zhang S, Xu Y. Large-scale screening for maize drought resistance using multiple selection criteria evaluated under water-stressed and well-watered environments. Field Crop Res, 2011, 124(1): 37-45.
[4]Neumann PM. Coping mechanisms for crop plants in drought-prone environments. Ann Bot, 2008, 101(7): 901- 907.
[5]Shuja MN, Ali W, Iqbal A, Ali I, Munir I, Ahmad D, Inamullah, Shaheenshah, Ahmad G, Khan MA, Swati ZA. Maize breeding for marginal lands: Physiological and molecular approach to decipher response and selection of maize recombinant inbred lines (RILs) under water deficit at early growth stage. Afr J Biotechnol, 2011, 10(18): 3521-3527.
[6]Tao D, Mu Y, Fu FL, Li WC. Transformation of maize with trehalose synthase gene cloned from. Saccharomyces cerevisiae Biotechnology, 2008, 7(2): 258-265.
[7]Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol, 2000, 124(4): 1854-1865.
[8]Karim S, Aronsson H, Ericson H, Pirhonen M, Leyman B, Welin B, Mantyla E, Palva ET, Van Dijck P, Holmstrom KO. Improved drought tolerance without undesired side effects in transgenic plants producing trehalose. Plant Mol Biol, 2007, 64(4): 371-386.
[9]Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol, 1999, 17(3): 287-291.
[10]Yamaguchi-Shinozaki K, Mino M, Mundy J, Chua NH. Analysis of an ABA-responsive rice gene promoter in transgenic tobacco. Plant Mol Biol, 1990, 15(6): 905-912.
[11]Ono A, Izawa T, Chua NH, Shimamoto K. The rab16B promoter of rice contains two distinct abscisic acid-respon-sive elements. Plant Physiol, 1996, 112(2): 483-491.
[12]Rai M, He C, Wu R. Comparative functional analysis of three abiotic stress-inducible promoters in transgenic rice. Transgenic Res, 2009, 18(5): 787-799.
[13]Buchanan CD, Klein PE, Mullet JE. Phylogenetic analysis of 5’-noncoding regions from the ABA-responsive rab16/17 gene family of sorghum, maize and rice provides insight into the composition, organization and function of cis-re-gulatory modules. Genetics, 2004, 168(3): 1639-1654.
[14]Cao X, Costa LM, Biderre-Petit C, Kbhaya B, Dey N, Perez P, McCarty DR, Gutierrez-Marcos JF, Becraft PW. Abscisic acid and stress signals induce Viviparous1 expression in seed and vegetative tissues of maize. Plant Physiol, 2007, 143(2): 720-731.
[15]Wu S, Yu Z, Wang F, Li W, Ye C, Li J, Tang J, Ding J, Zhao J, Wang B. Cloning, characterization, and transformation of the phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) in maize (Zea mays L.). Mol Biotechnol, 2007, 36(2): 102-112.
[16]Theologis A, Ecker JR, Palm CJ, Federspiel NA, Kaul S, White O, Alonso J, Altafi H, Araujo R, Bowman CL, Brooks SY, Buehler E, Chan A, Chao Q, Chen H, Cheuk RF, Chin CW, Chung MK, Conn L, Conway AB, Conway AR, Creasy TH, Dewar K, Dunn P, Etgu P

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

玉米钼辅助因子硫酸化酶基因启动子的克隆与功能验证

Cloning and functional validation of promoter of mo-molybdopterin cofactor sulfurase gene in maize

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	时子文, 何青, 赵卓凡, 刘孝伟, 张鹏, 曹墨菊. 玉米雄性不育资源的发掘与利用[J]. 遗传, 2022, 44(2): 134-152.
[2]	陈欲, 陈笑芸, 彭城, 徐俊锋, 沈洁, 李玥莹, 汪小福. 转基因玉米双抗12-5荧光RPA现场可视化检测方法的建立[J]. 遗传, 2021, 43(8): 802-812.
[3]	单婷玉, 施雯, 王翌婷, 曹孜怡, 汪保华, 方辉. 玉米盐胁迫相关性状全基因组关联分析及候选基因预测[J]. 遗传, 2021, 43(12): 1159-1169.
[4]	毛洋, 丁寄葳, 陈淑敏, 岑山, 李晓宇. SLFN14抗LINE-1分子机制研究[J]. 遗传, 2020, 42(7): 669-679.
[5]	秦中勇, 石晓, 曹平平, 褚鹰, 管蔚, 杨楠, 程禾, 孙玉洁. 细胞凋亡反应中NOXA基因启动子发挥增强子功能调节BCL2基因表达[J]. 遗传, 2020, 42(11): 1110-1121.
[6]	赵剑超, 柴壮, 郭诗萌, 刘忠华. 猪早期胚胎发育中SOX2基因启动子活性分析[J]. 遗传, 2019, 41(10): 950-961.
[7]	汪德州,莫晓婷,张霞,徐妙云,赵军,王磊. 玉米逆境响应相关转录因子ZmC2H2-1基因克隆及功能验证[J]. 遗传, 2018, 40(9): 767-778.
[8]	赵卓凡, 黄玲, 刘永明, 张鹏, 魏桂, 曹墨菊. 玉米CMS-C同质异核不育系育性恢复的遗传研究[J]. 遗传, 2018, 40(5): 402-414.
[9]	王星,张纪龙,冯秀秀,李洪杰,张根发. 植物质膜水通道蛋白转运及逆境胁迫响应的分子调控机制[J]. 遗传, 2017, 39(4): 293-301.
[10]	朱帅旗,龚一富,章丽,俞凯,王何瑜,严小军. 绿色杜氏藻不同β-胡萝卜素羟化酶基因家族胁迫应答模式研究[J]. 遗传, 2017, 39(2): 156-165.
[11]	程敏, 张文建, 邢天宇, 闫晓红, 李玉茂, 李辉, 王宁. 鸡miR-17-92基因簇上游调控区功能分析[J]. 遗传, 2016, 38(8): 724-735.
[12]	尹朝华, 李岩, 张春庆, 杨翠翠, 吴承来, 刘翔攀, 王明明. 玉米脱氧麦根酸分泌通道蛋白基因YS3启动子的克隆与启动活性分析[J]. 遗传, 2016, 38(6): 560-568.
[13]	马建辉, 仝豆豆, 张文利, 张黛静, 邵云, 杨云, 姜丽娜. 乌拉尔图小麦NAC转录因子的筛选与分析[J]. 遗传, 2016, 38(3): 243-253.
[14]	刘娟, 孙艳, 姜强, 杨春红, 黄金明, 李建斌, 侯明海, 仲跻峰, 王长法, 刘保申. INCENP基因功能性单倍型可调控启动子活性并影响公牛精液品质[J]. 遗传, 2016, 38(1): 62-71.
[15]	刘静茹, 孟莎莎, 周卫辉. 儿童孤独症相关基因NRXN1β启动子功能分析[J]. 遗传, 2015, 37(8): 801-810.