新牧1号苜蓿两种抗逆相关启动子的功能分析

doi:10.11686/cyxb2016071

草业学报 ›› 2017, Vol. 26 ›› Issue (1): 131-141.DOI: 10.11686/cyxb2016071

新牧1号苜蓿两种抗逆相关启动子的功能分析

晁朝霞¹, 任燕萍¹, 钱进², 姚正培¹, 许磊³, 张桦^1,*

1.新疆农业大学农学院,新疆乌鲁木齐 830052;
2.江苏金色农业科技发展有限公司,江苏盐城 224100;
3.南京农业大学草业学院,江苏南京 210095

收稿日期:2016-03-02 出版日期:2017-01-20 发布日期:2017-01-20
通讯作者: E-mail:hazelzhang@163.com
作者简介:晁朝霞(1989-),女,山东菏泽人,在读硕士。E-mail:xiangyin.cool@163.com
基金资助:
新疆维吾尔自治区自然科学基金(项目编号:2014211B017)资助

Functional analysis of two stress-related promoters in Medicago varia cultivar Xinmu No.1

CHAO Zhao-Xia¹, REN Yan-Ping¹, QIAN Jin², YAO Zheng-Pei¹, XU Lei³, ZHANG Hua^1,*

1.College of Agriculture, Xinjiang Agricultural University, Urumqi 830052, China;
2.Jiangsu Golden Agricultural Science and Technology Development Co., Ltd, Yancheng 224100, China;
3.Prataculture Institute, Nanjing Agricultural University, Nanjing 210095, China

Received:2016-03-02 Online:2017-01-20 Published:2017-01-20

摘要/Abstract

摘要： 前期已成功克隆了新牧1号苜蓿MvP5CS与MvNHX1基因和二者的启动子序列。在此基础上,本试验分别构建了含有Mvp5cs和Mvnhx1启动子的调控GUS报告基因的植物表达载体,并通过农杆菌介导法得到了含有Mvnhx1与Mvp5cs启动子的转基因烟草植株。PCR检测证明了两种启动子已稳定的整合到烟草基因组中;GUS组织染色证明两种启动子均具有启动基因表达的功能。通过测量GUS活性来探究两种不同的诱导型启动子在4种非生物胁迫下(干旱、盐、脱落酸、赤霉素)的表达活性。结果表明,1)Mvnhx1与Mvp5cs启动子均响应盐、脱落酸、赤霉素、干旱胁迫,与CaMV35S启动子相比,差异显著。2)Mvnhx1启动子在盐胁迫、脱落酸胁迫下所起诱导作用要优于Mvp5cs启动子。在48 h 100 mmol/L、150 mmol/L NaCl胁迫处理时,Mvnhx1启动子GUS活性分别是Mvp5cs启动子的2.03和3.23倍,差异显著。在25 μmol/L、 50 μmol/L ABA处理下,Mvnhx1启动子的活性整体高于另两种启动子,差异显著。3)Mvp5cs启动子在干旱胁迫、赤霉素胁迫所起诱导作用优于Mvnhx1启动子。干旱胁迫时,Mvp5cs启动子的GUS活性在36 h是同时间Mvnhx1启动子的2.22倍。70 μmol/L GA处理时,Mvp5cs启动子在36 h达到最大值,是同时间段Mvnhx1启动子的1.79倍。

Abstract: MvP5CS and MvNHX1 genes of the Medicago varia cultivar Xinmu No.1 and their promoter sequences have been successfully cloned in previous work. Based on this research, in this study two plant expression vectors, each containing Mvp5cs and Mvnhx1 promoters with a GUS gene as the reporter, were constructed and two tobacco transgenic plants with the promoter obtained by Agrobacterium-mediated method. PCR analysis showed that the two promoters had been stably integrated into the tobacco genome and GUS-staining proved that they have the function of activating gene expression. We explored this function by measuring GUS activity under four different abiotic stresses: drought, salinity, abscisic acid (ABA) and gibberellin (GA). The results showed that Mvnhx1 and Mvp5cs responded to the four stresses and had significant differences from the CaMV35S promoter. The Mvnhx1 promoter was superior to Mvp5cs under salt and ABA stress. After 48 h treatments of 100 and 150 mmol/L NaCl stress, the GUS activity of Mvnhx1 was 2.03 times and 3.23 times that of Mvp5cs. Under treatments of 25 and 50 μmol/L ABA stress, Mvnhx1 activity was significantly higher than the other two promoters. Mvp5cs was superior to Mvnhx1 under drought and GA stress. The GUS activity of the Mvp5cs was 2.22 times that of Mvnhx1 after 36 h treatment of drought stress. The Mvp5cs promoter reached its maximum value, which was 1.79 times that of Mvnhx1, after 36 h treatment with 70 μmol/L GA stress.

晁朝霞, 任燕萍, 钱进, 姚正培, 许磊, 张桦. 新牧1号苜蓿两种抗逆相关启动子的功能分析[J]. 草业学报, 2017, 26(1): 131-141.

CHAO Zhao-Xia, REN Yan-Ping, QIAN Jin, YAO Zheng-Pei, XU Lei, ZHANG Hua. Functional analysis of two stress-related promoters in Medicago varia cultivar Xinmu No.1[J]. Acta Prataculturae Sinica, 2017, 26(1): 131-141.

参考文献

[1] Zhu Y X, Li Y. Modern Molecular Biology[M]. Beijing: Higher Education Press, 2002: 255-265.
朱玉贤, 李毅. 现代分子生物学[M]. 北京: 高等教育出版社, 2002: 255-265.
[2] Odell J T, Nagy F, Chua N H. Identification of DNA sequences required for activity of the Cauliflower mosaic virus 35S promoter. Nature, 1985, 3(13): 810-812.
[3] Cao H W. Functional Analysis of BADH Gene Promoter from Chrysanthemum lavandulifolium and the Isolation of Inducible Promoter[D]. Beijing: Beijing Forestry University, 2010.
曹华雯. 甘菊 BADH 基因启动子功能鉴定及诱导型启动子的分离[D]. 北京: 北京林业大学, 2010.
[4] Ye R, Zhou F, Lin Y. Two novel positive cis-regulatory elements involved in green tissue-specific promoter activity in rice ( Oryza sativa L ssp). Plant Cell Reports, 2012, 31(7): 1159-1172.
[5] Freitas R L, Carvalho C M, Fietto L G, et al . Distinct repressing modules on the distal region of the SBP 2 promoter contribute to its vascular tissue-specific expression in different vegetative organs. Plant Molecular Biology, 2007, 65(5): 603-614.
[6] Kasuga M, Liu Q, Miura S, et al . Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotechnology, 1999, 17(3): 287-291.
[7] Zheng H Q, Lin S Z, Zhang Q, et al . Isolation and analysis of a TIR-specific promoter from poplar. Forestry Studies in China, 2007, 9(2): 95-106.
[8] Kohli A, Griffiths S, Palacios N, et al . Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35 S promoter and confirms the predominance of micro homology mediated recombination. Plant, 1999, 17(6): 591-601.
[9] Cummins J, Ho M W, Ryan A. Hazardous CaMV 35 S promoter. Nature Biotechnology, 2000, 18(4): 361-363.
[10] Zhang Y H, Ji J, Wang G. Transgenic plants and biological safety. Crops, 2003, 6(6): 4-7.
张艳华, 季静, 王罡. 转基因植物与生物安全性. 作物杂志, 2003, 6(6): 4-7.
[11] McNeil S D. Betanies and related osmoprotectants targets for metabolic engineering of stress resistance. Plant Physiology, 1999, 120(4): 945-949.
[12] Yin X Y, Yang A F, Zhang K W, et al . Production and analysis of transgenic maize with improved salt tolerance by the introduction of AtNHX 1 gene. Acta Botanica Sinica, 2004, 46(7): 854-861.
尹小燕, 杨爱芳, 张可炜, 等. 转 AtNHX 1基因玉米的产生及其耐盐性分析. 植物学报:英文版, 2004, 46(7): 854-861.
[13] Banjara M, Zhu L, Shen G, et al . Expression of an Arabidopsis sodium /proton antiporter gene ( AtNHX 1) in peanut to improve salt tolerance. Plant Biotechnology Reports, 2012, 6(1): 59-67.
[14] Zhang Z Y, Chen J B, Song J L, et al . Cloning and expression analysis of P 5 CS gene from wild soybean ( Glycine soja ) seedling under salt stress. Journal of Plant Genetic Resources, 2014, 15(4): 844-849.
张兆元, 陈吉宝, 宋佳璘, 等. 野生大豆 P 5 CS 基因的克隆及对盐胁迫反应. 植物遗传资源学报, 2014, 15(4): 844-849.
[15] Igarashi Y. Characterization of the gene for Δ1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa . Plant Molecular Biology, 1997, 33(5): 857-865.
[16] Zhang J L, Li H R, Guo S Y, et al . Research advances in higher plant adaptation to salt stress. Acta Prataculturae Sinica, 2015, 24(12): 220-236.
张金林, 李惠茹, 郭姝媛, 等. 高等植物适应盐逆境研究进展. 草业学报, 2015, 24(12): 220-236.
[17] Zhang H. The Cloning and Functional Analysis of Stress-related Gene in Medicago varia Xinmu No.1[D]. Urumqi: Xinjiang Agricultural University, 2011.
张桦. 新牧1号杂花苜蓿抗逆相关基因的克隆和功能分析[D]. 乌鲁木齐: 新疆农业大学, 2011.
[18] Yang Y Y. Enhancing the Drought Resistance of Cotton Through Transformation of MvP 5 CS and MvNHX 1[D]. Urumqi: Xinjiang Agricultural University, 2012.
杨云尧. 转 MvP 5 CS 和 MvNHX 1基因提高棉花抗旱性的研究[D]. 乌鲁木齐: 新疆农业大学, 2012.
[19] Zhao Y W, Wang Y J, Bu H Y, et al . Studies on the conversion of chicory and salt tolerance of AtNHX 1 gene. Acta Prataculturae Sinica, 2009, 18(3): 103-109.
赵宇玮, 王英娟, 步怀宇, 等. AtNHX 1基因对菊苣的转化和耐盐性研究. 草业学报, 2009, 18(3): 103-109.
[20] Cui D Q. Expression Analysis of PtSEP 2, PtSEP 3 - 1, PtAP 1 - 2 and PtMCP Promoters in Nicotiana tabacum ang Populus tomentosa [D]. Beijing: Beijing Forestry University, 2011.
崔东清. PtSEP 2、 PtSEP 3 - 1、 PtAP 1 - 2和 PtMCP 基因启动子在烟草和毛白杨中的表达特性分析[D]. 北京: 北京林业大学, 2011.
[21] Bai Y N. Molecular Cloning and Activity Analysis of a Rice Gene Promoter OsBTF 3 p [D]. Beijing: Chinese Academy of Agricultural Sciences, 2008.
柏亚男. 水稻基因启动子 OsBTF 3 p 的克隆及其启动活性分析[D]. 北京: 中国农业科学院, 2008.
[22] Jefferson R A. Assaying chemeric genes in plants: the GUS gene fusion system. Plant Molecular Biology Reporter, 1987, 5(4): 387-405.
[23] Bai Y N, Wu M S, He C Y. Molecular cloning and activity analysis of a rice gene promoter OsBTF 3 p . Agricultural Sciences in China, 2009, 42(3): 862-868.
柏亚男, 吴茂森, 何晨阳. 水稻基因启动子 OsBTF 3 p 的克隆和启动活性分析. 中国农业科学, 2009, 42(3): 862-868.
[24] You Z, Chao Z X, Yao Z P, et al . Comparative analysis of drought and salt resistance and breeding value in transgenic MvNHX 1 and MvP 5 CS cotton. Cotton Science, 2015, 27(3): 198-207.
游朝, 晁朝霞, 姚正培, 等. 转 MvNHX 1和 MvP 5 CS 基因棉花耐盐抗旱性比较与育种价值分析. 棉花学报, 2015, 27(3): 198-207.
[25] Yang Y Y, Ren Y P, Su Y M, et al . The cloning and analysis of stress-related promoters in Medicago varia Xinmu No.1. Pratacultural Science, 2012, 29(12): 1887-1893.
杨云尧, 任燕萍, 苏豫梅, 等. 新牧1号苜蓿两个抗逆相关基因启动子的克隆及分析. 草业科学, 2012, 29(12): 1887-1893.
[26] Xu L M, Zhang Z B, Liang X L, et al . Advances in research on plant drought resistance gene engineering. Acta Prataculturae Sinica, 2014, 23(6): 293-303.
徐立明, 张振葆, 梁晓玲, 等. 植物抗旱基因工程研究进展. 草业学报, 2014, 23(6): 293-303.
[27] Zhang Z L, Xie Z, Zou X, et al . A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells. Plant Physiology, 2004, 134: 1500-1513.

新牧1号苜蓿两种抗逆相关启动子的功能分析

Functional analysis of two stress-related promoters in Medicago varia cultivar Xinmu No.1

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