山葡萄CBF基因表达载体构建及对拟南芥根生长的影响

doi:10.13560/j.cnki.biotech.bull.1985.2016.01.019

生物技术通报 ›› 2016, Vol. 32 ›› Issue (1): 109-114.doi: 10.13560/j.cnki.biotech.bull.1985.2016.01.019

山葡萄CBF基因表达载体构建及对拟南芥根生长的影响

王法微¹, 董金晔², 李晓薇¹, 刘洋², 吴学彦², 王南¹, 董园园¹, 陈欢², 李海燕^{1, 2}

1. 吉林农业大学生物反应器与药物开发教育部工程研究中心, 长春 130118;
2. 吉林农业大学生命科学学院, 长春 130118

收稿日期:2015-02-28 出版日期:2016-01-09 发布日期:2016-01-22
作者简介:王法微, 男, 助理研究员, 研究方向：植物分子生物学;E-mail：fw-1980@163.com
基金资助:
国家自然科学基金项目(31201144, 31271746), 吉林省发改委项目(JF2012C002-04), 吉林农业大学国家大学生创新创业训练计划项目(201410193036)

The Construction of Expression Vector of Gene CBF from Vitis amurensis and the Effects of It on the Root Growth of Arabidopsis

WANG Fa-wei¹, DONG Jin-ye², LI Xiao-wei¹, LIU Yang², WU Xue-yan², WANG Nan¹, DONG Yuan-yuan¹, CHEN Huan², LI Hai-yan^{1, 2}

1. Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118;
2. College of Life Sciences, Jilin Agricultural University, Changchun 130118

Received:2015-02-28 Published:2016-01-09 Online:2016-01-22

摘要/Abstract

摘要： 为了研究山葡萄CBF基因调节植物对盐胁迫的应答机理, 分别构建了山葡萄VaCBF1、VaCBF2和VaCBF3的植物过表达载体。经酶切及琼脂糖电泳检测证实3个基因均插入到pBASTA中, 表明表达载体构建成功。然后, 分别将3个植物过表达载体转入农杆菌EHA105中, 并通过浸花法浸染拟南芥。利用除草剂筛选获得3个基因的拟南芥过表达株系。最后, 对野生型拟南芥与转基因拟南芥进行盐胁迫处理, 发现OE-CBF2转基因植株的主根伸长长度显著长于其它植株, 3个转基因株系的侧根长度也明显长于野生型植株。上述结果表明山葡萄CBF基因可能在植物盐胁迫中对根部生长发育起到非常重要的调控作用。

关键词: 山葡萄, CBF基因, 表达载体, 根生长

Abstract: In order to investigate the response mechanism of CBF gene in the regulation of plant to the salt stress, the plant over-expression vectors of VaCBF1, VaCBF2 and VaCBF3 from Vitis amurensis were constructed. The results by restriction enzyme digestion and agarose gel electrophoresis showed that 3 genes were correctly inserted into pBASTA, indicating that the expression vectors were successfully constructed. Then, 3 over-expressed vectors were transformed into Agrobacterimu tumefaciens EHA105, and Arabidopsis thaliana was leached using floral dip method. The transgenic plants of A. thaliana with over-expression of 3 genes were screened by herbicide Basta. Furthermore, wild and transgenic plants were treated with salt stress. The results revealed that the elongation length of primary root of OE-CBF2 transgenic plant was longer than others, and the lateral roots in 3 genes transgenic plants were longer than wild ones. These results indicate that the CBF genes of V. amurensis play the critical regulation role in the root development during salt stress.

Key words: Vitis amurensis, CBF gene, expression vector, root growth

王法微, 董金晔, 李晓薇, 刘洋, 吴学彦, 王南, 董园园, 陈欢, 李海燕. 山葡萄CBF基因表达载体构建及对拟南芥根生长的影响[J]. 生物技术通报, 2016, 32(1): 109-114.

WANG Fa-wei, DONG Jin-ye, LI Xiao-wei, LIU Yang, WU Xue-yan, WANG Nan, DONG Yuan-yuan, CHEN Huan, LI Hai-yan. The Construction of Expression Vector of Gene CBF from Vitis amurensis and the Effects of It on the Root Growth of Arabidopsis[J]. Biotechnology Bulletin, 2016, 32(1): 109-114.

参考文献

［1］ Boyer JS. Plant productivity and environment［J］. Science, 1982, 218(4571)：443-448.
［2］ Lobell DB, Schlenker W, Costa-Roberts J. Climate trends and global crop production since 1980［J］. Science, 2011, 333(6042)：616-620.
［3］张俊环, 王玉柱, 孙浩元, 等. 外源水杨酸对低温下杏花抗氧化酶和CBF转录因子表达的影响［J］. 植物生理学报, 2014, 50(2)：171-177.
［4］ Cramer GR, Urano K, Delrot S, et al. Effects of abiotic stress on plants：a systems biology perspective［J］. BMC Plant Biol, 2011, 11：163.
［5］ Yamaguchi-Shinozaki K, Shinozaki K. Transcriptional regulatory networks in cellular response and the tolerance to dehydration and cold stresses［J］. Annu Rev Plant Biol, 2006, 57：781-803.
［6］ Stockinger EJ, Gilmour SJ, Thomashow MF. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit［J］. Proc Natl Acad Sci USA, 1997, 94：1035-1040.
［7］ Thomashow MF. Plant cold acclimation：Freezing tolerance genes and regulatory mechanisms［J］. Annual Review of Plant Physiology and Plant Molecular Biology, 1999, 50：571-599.
［8］林元震, 郭海, 刘纯鑫, 等. 赤桉抗寒转录因子ICE1基因的分子克隆与表达分析［J］. 植物生理学报, 2011, 47：488-494.
［9］ Chinnusamy V, Ohta M, Kanrar S, et al. ICE1, a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis［J］. Gene Dev, 2003, 17(8)：1043-1054.
［10］ Wang Y, Jiang CJ, Li YY, et al. CsICE1 and CsCBF1：two transcription factors involved in cold responses in Camellia sinensis［J］. Plant Cell Rep, 2012, 31(1)：27-34.
［11］ Liu Q, Kasuga M, Sakuma Y, et al. Two transcription factors, DREB1 and DREB2 with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis［J］. Plant Cell, 1998, 10(8)：1391-1406.
［12］ Satoko M, Mizoi J, Yoshida T, et al. Comprehensive analysis of rice DREB2-type genes that encode transcription factors involved in the expression of abiotic stress-responsive genes［J］. Mol Genet Genomics, 2010, 283(2)：185-196.
［13］ Qin F, Kakimoto M, Sakuma Y, et al. Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L.［J］ Plant J, 2007, 50(1)：54-69.
［14］ Zhou ML, Tang YX, Wu YM. Genome-wide analysis of AP2/ERF transcription factor family in Zea mays［J］. Curr Bioinform, 2012, 7(3)：324-332.
［15］ Sazegari S, Niazi A. Isolation and molecular characterization of wheat(Triticum aestivum)dehydration responsive element binding factor(DREB)isoforms［J］. Aust J Crop Sci, 2012, 6(6)：1037-1044.
［16］ Shen YG, He SJ, Zhang WK, et al. An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold, dehydration and ABA stress［J］. Theor Appl Genet, 2003, 106(5)：923-930.
［17］ Jing T, Chang Q, Li W, et al. Stress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfa［J］. Plant Cell Tiss Org, 2010, 100(2)：219-227.
［18］ Mizoi J, Ohori T, Moriwaki T, et al. GmDREB2A;2, a canonical dehydration-responsive element-binding protein2-type transcription factor in soybean, is posttranslationally regulated and mediates dehydration-responsive element-dependent gene expression［J］. Plant Physiol, 2013, 161(1)：346-361.
［19］ Xiao H, Siddigua M, Braybrook S, et al. Three grape CBF/DREB1 genes respond to low temperature, drought and abscisic acid［J］. Plant Cell Environ, 2006, 29(7)：1410-1421.
［20］ Gilmour SJ, Fowler SG, Thomashow MF. Arabidopsis transcriptional activators CBF1, CBF2, and CBF3 have matching functional activities［J］. Plant Mol Biol, 2004, 54(5)：767-781.
［21］ Novillo F, Alonso JM, Ecker JR, et al. CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis［J］. Proc Natl Acad Sci USA, 2004, 101(11)：3985-3990.
［22］ Skinner JS, Zitzewitz J, Szucs P, et al. Structural, functional, and physogenetic characterization of a large CBF gene family in barley［J］. Plant Mol Biol, 2005, 59(4)：533-551.
［23］ Zhang L, Li Z, Li J, et al. Ectopic overexpression of SsCBF1, a CRT/DRE-binding factor from the nightshade plant Solanum lycopersicoides, confers freezing and salt tolerance in transgenic Arabidopsis［J］. PLoS One, 2013, 8(6)：e61810.
［24］王法微, 刘洋, 吴学彦, 等. 山葡萄VaCBF1转录因子基因的克隆与表达分析［J］. 西北农林科技大学学报：自然科学版, 2013, 12(41)：86-92.
［25］ Xu MY, Li LH, Fan YL, et al. ZmCBF3 overexpression improves tolerance to abiotic stress in transgenic rice(Oryza sativa)without yield penalty［J］. Plant Cell Rep, 2013, 30(10)：1949-1957.
［26］ Li ZJ, Zhang LL, Li JF, et al. Isolation and functional characterization of the ShCBF1 gene encoding a CTR/DRE-binding factor from the wild tomato species Solanum habrochaites［J］. Plant Physiol Bioch, 2014, 74：294-303.
［27］ Duan LN, Dietrich D, Ng CH, et al. Endodermal ABA signaling promotes lateral root quiescence during salt stress in Arabidopsis seedlings［J］. Plant Cell, 2013, 25(1)：324-341.

[1]	吴坤坤, 徐行, 季策, 任建峰, 李伟明, 张庆华. 斑马鱼notch3基因真核表达载体的构建及其表达分析[J]. 生物技术通报, 2022, 38(1): 179-186.
[2]	巩元勇, 赵丽华, 闫飞, 郭书巧, 束红梅, 倪万潮. 琉璃苣BoD6D载体的构建及转化[J]. 生物技术通报, 2021, 37(3): 227-232.
[3]	胡晓, 王宝宝, 窦少华, 姜南, 付常振, 金行, 高凤山. 烟台黑猪SLA-2基因真核表达载体的构建及表达[J]. 生物技术通报, 2021, 37(10): 143-151.
[4]	王衍莉, 杨义明, 范书田, 赵滢, 许培磊, 路文鹏, 李昌禹. 基于SSR分子标记的73份山葡萄及杂交后代的遗传多样性分析[J]. 生物技术通报, 2021, 37(1): 189-197.
[5]	孟利, 杜彩萍. 大鼠His-Akt1重组蛋白的真核表达、蛋白纯化及活性鉴定[J]. 生物技术通报, 2020, 36(12): 98-103.
[6]	陈和锋, 朱晁谊, 李爽. 产瓦伦西亚烯酿酒酵母的表达载体适配及发酵碳氮源优化[J]. 生物技术通报, 2020, 36(1): 209-219.
[7]	翟晓鑫, 高花, 姜平, 许崇波, 张宗辉, 李文哲, 高凤山. 荷包猪SLA-2-HB01真核表达载体的构建及在PK15细胞中的表达[J]. 生物技术通报, 2018, 34(12): 132-139.
[8]	赵佳福, 许厚强, 宋书弦, 段志强, 陈祥. BLM解旋酶基因的克隆、表达载体构建及表达研究[J]. 生物技术通报, 2018, 34(11): 152-159.
[9]	赵忠娟,魏艳丽,李纪顺,王贻莲,杨合同. 一种根癌农杆菌介导的耐盐椒样薄荷含芽茎段转化系统[J]. 生物技术通报, 2017, 33(7): 126-132.
[10]	肖自华,高飞,孙会改,周宜君. 蒙古沙冬青AmMYB4-like基因的克隆与表达分析[J]. 生物技术通报, 2017, 33(5): 108-116.
[11]	张晶晶, 金小宝, 李小波, 汪洁, 马艳. 过表达Glypican-3对高转移性肝癌细胞HCCLM3生物学行为的影响[J]. 生物技术通报, 2017, 33(12): 176-184.
[12]	束红梅, 郭书巧, 巩元勇, 倪万潮. 油菜素内酯基因BAS1根系表达载体的构建及烟草的遗传转化[J]. 生物技术通报, 2015, 31(6): 106-110.
[13]	侯莹, 孙西魁, 唐明青. 从功能部件看小干扰RNA表达载体的发展[J]. 生物技术通报, 2015, 31(3): 64-69.
[14]	孙璐, 刘志文, 邹丹, 李丹, 潘博, 丛丽娜. 海参溶菌酶枯草芽孢杆菌基因工程菌构建[J]. 生物技术通报, 2014, 0(6): 150-154.
[15]	江伟华, 刘益丽, 江明锋. 大肠杆菌外源蛋白表达载体稳定性的研究进展[J]. 生物技术通报, 2014, 0(5): 25-31.

山葡萄CBF基因表达载体构建及对拟南芥根生长的影响

The Construction of Expression Vector of Gene CBF from Vitis amurensis and the Effects of It on the Root Growth of Arabidopsis

PDF (PC)

可视化

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

本文评价