ThMYB3和ThDof2对ThVHAc1基因表达的调控

杨桂燕; 赵震; 赵玉琳; 张凤娇; 高彩球

doi:10.13332/j.1000-1522.20140486

ThMYB3和ThDof2对ThVHAc1基因表达的调控

1.
1 东北林业大学林木遗传育种国家重点实验室
2.
2 西北农林科技大学林学院

基金项目:

国家自然科学基金项目(31370676)、教育部“新世纪优秀人才支持计划”项目(NCET--13--0709)。

详细信息

作者简介:
杨桂燕,博士,讲师。主要研究方向:林木抗逆分子遗传育种。Email:yangguiyan@nwsuaf.edu.cn 地址:712100 陕西省杨凌邰城路3号西北农林科技大学林学院。
责任作者: 高彩球,副教授。主要研究方向:林木抗性育种。Email:gaocaiqiu@nefu.edu.cn 地址:150040 黑龙江省哈尔滨市香坊区和兴路26号东北林业大学林木遗传育种国家重点实验室。

杨桂燕,博士,讲师。主要研究方向:林木抗逆分子遗传育种。Email:yangguiyan@nwsuaf.edu.cn 地址:712100 陕西省杨凌邰城路3号西北农林科技大学林学院。
责任作者: 高彩球,副教授。主要研究方向:林木抗性育种。Email:gaocaiqiu@nefu.edu.cn 地址:150040 黑龙江省哈尔滨市香坊区和兴路26号东北林业大学林木遗传育种国家重点实验室。

计量
- 文章访问数: 2016
- HTML全文浏览量: 187
- PDF下载量: 110
出版历程
- 收稿日期: 2015-01-04
- 发布日期: 2015-12-30

Upstream regulators of ThVHAc1.

1.
1 State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, 150040, Harbin, Heilongjiang, P. R. China;
2.
2 Laboratory of Walnut Research Center, College of Forestry, Northwest A & F University, 712100, Yangling, Shaanxi, P. R. China.

摘要

摘要: ThVHAc1基因能响应干旱、盐、重金属等胁迫诱导,该基因能有效提高转ThVHAc1基因酵母的多种抗逆能力。为进一步研究ThVHAc1基因的抗逆调控机理,本研究利用酵母单杂交技术钓取ThVHAc1可能的上游调控因子,并利用基因枪瞬时共转化技术进行初步验证。酵母单杂交结果显示,ThMYB3基因能识别ThVHAc1基因上游MYB顺式作用元件和含有MYB元件的启动子片段。ThDof2基因能识别ThVHAc1基因上游DOF顺式作用元件和含有DOF元件的启动子片段。但ThMYB3和ThDof2均不能识别突变后的元件及含对应突变元件的启动子片段。将ThMYB3和ThDof2分别构建成效应载体,各元件、突变元件、启动子片段及突变的启动子片段分别构建报告载体进行共表达验证。结果发现:效应载体ThMYB3和含MYB元件及含MYB元件的ThVHAc1启动子片段的报告载体,ThDof2和含DOF元件及含DOF元件的ThVHAc1启动子片段的报告载体瞬时共表达烟草叶片能观察到GUS染色,且GUS活性较高;而与相应突变元件及片段的共转化则观察不到烟草叶片的GUS染色,且GUS活性很低。说明只有非突变的元件和启动子片段才能与效应载体进行互作识别,激活GUS基因的表达,验证了酵母单杂交获得的上游调控基因的识别特异性。表明ThMYB3和ThDof2可能通过与相应启动子元件的结合调控ThVHAc1基因。
- 刚毛柽柳 /
- 酵母单杂交 /
- VHAc基因 /
- 启动子 /
- 调控因子
Abstract: ThVHAc1 gene was reported to be responsive to drought, salt and heavy metal stresses, and overexpressed ThVHAc1 yeast could improve effectively multi-stress tolerance. To further understand the stress regulation mechanism of ThVHAc1, we screened the upstream regulators that could specifically recognize the cis-elements DOF and MYB in the ThVHAc1 promoter by yeast one-hybrid assay. The results showed that the ThMYB3 gene could specifically recognize the MYB motif and the promoter segment that contains MYB motif, and the ThDof2 gene could specifically recognize the DOF motif and the promoter segment that includes DOF motif in the ThVHAc1 promoter. However, ThMYB3 and ThDof2 could not recognize the mutated motifs and promoter segments containing these mutated motifs. Then ThMYB3 and ThDof2 were independently constructed as effecter vector, and MYB and DOF motifs as well as their mutates and promoters were independently constructed as reporter vectors for co-expression assays. And the results of co-transient expression of the effecters and reporters confirmed that ThDof2 and ThMYB3 can specifically bind to the ThVHAc1 promoter. ThMYB3 and reporters containing MYB motif and ThVHAc1 promoter segments including MYB motif, ThDof2 and reporters containing DOF motif and the ThVHAc1 promoter segments including DOF motif could stain the GUS expression in tobacco leaves and showed high GUS activities. However, other co-expression referred to mutated motifs could not activate the GUS expression, indicating that only normal cis-elements and promoter segments could be recognized by effecters to activate the GUS expression. Our study suggests the effectiveness of ThMYB3 and ThDof2 as ThVHAc1 upstream regulators.
- Tamarix hispida /
- yeast one hybrid /
- VHAc gene /
- promoter /
- upstream regulator

HTML全文

参考文献(17)

[1]	DIETZ K J, TAVAKOLI N, KLUGE C, et al. Significance of the V-type Atpase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level[J] . Journal of Experimental Botany, 2001, 52: 1969-1980.
[1]	HAO Y L, ZHU B Z, LUAN C G, et al. Cloning and identification of sunflower seed-specific promoter Ha ds10 G1 [J] . Journal of Agricultural Biotechnology, 2006, 14(6): 922-925.
[2]	KANE P, KUEHN M, HOWALD-STEVENSON I, et al. Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H+-ATPase[J] . Journal of Biological Chemistry, 1992, 267(1): 447-454.
[3]	UMEMOTO N, YOSHIHISA T, HIRATA R,et al. Roles of the Vma3 gene product, subunit c of the vacuolar membrane H+-ATPase on vacuolar acidification and protein transport-a study with Vma3-disrupted mutants of Saccharomyces cerevisiae[J] . Journal of Biological Chemistry, 1990, 265(30): 18447-18453.
[4]	TYAGI W, RAJAGOPAL D, SINGLA-PAREEK S L, et al. Cloning and regulation of a stress-regulated Pennisetum glaucum vacuolar ATPase c gene and characterization of its promoter that is expressed in shoot hairs and floral Organs[J] . Plant and Cell Physiology, 2005, 46(8): 1411-1422.
[5]	NITZ I, BERKEFELD H, PUZIO P S,et al. Pyk10, a seedling and root specific gene and promoter from Arabidopsis thaliana[J] . Plant Science, 2001, 161(2): 337-346.
[6]	郝彦玲, 朱本忠, 栾春光, 等. 向日葵种子特异性启动子Ha ds10 G1的克隆及其功能验证[J] . 农业生物技术学报, 2006, 14(6): 922-925.
[7]	ZAREI A, KRBES A P, YOUNESSI P,et al. Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis[J] . Plant Molecular Biology, 2011, 75(4-5): 321-331.
[8]	ZHENG L, LIU G, MENG X,et al. A WRKY gene from Tamarix hispida, ThWRKY4, mediates abiotic stress responses by modulating reactive oxygen species and expression of stress-responsive genes[J] . Plant Molecular Biology, 2013, 82(4-5): 303-320.
[9]	GAO C, WANG Y, JIANG B,et al. A novel vacuolar membrane H+-ATPase c subunit gene(ThVHAc1) from Tamarix hispida confers tolerance to several abiotic stresses in Saccharomyces cerevisiae[J] . Molecular Biology Reports, 2011, 38(2): 957-963.
[10]	WANG L, XU C, WANG C,et al. Characterization of a eukaryotic translation initiation factor 5A homolog from Tamarix androssowii involved in plant abiotic stress tolerance[J] . BMC Plant Biology, 2012, 12(1): 118.
[11]	GAO C, WANG Y, LIU G,et al. Expression profiling of salinity-alkali stress responses by large-scale expressed sequence tag analysis in Tamarix hispida[J] . Plant Molecular Biology, 2008, 66(3): 245-258.
[12]	YANAGISAWA S, SCHMIDT R J. Diversity andsimilarity among recognition sequences of Dof transcription factors[J] . The Plant Journal, 1999, 17(2): 209-214.
[13]	JI X, LIU G, LIU Y,et al. The bZIP protein from Tamarix hispida, ThbZIP1, Is ACGT elements binding factor that enhances abiotic stress signaling in transgenic Arabidopsis[J] . BMC Plant Biology, 2013, 13(1): 151,
[14]	HOSODA K, IMAMURA A, KATOH E,et al. Molecular structure of the GARP family of plant Myb-related DNA binding motifs of the Arabidopsis response regulators[J] . The Plant Cell Online, 2002, 14(9): 2015-2029,
[15]	WANG L, QIN L, LIU W, et al. A novel ethylene-responsive factor from Tamarix hispida, ThERF1, is a GCC-box and DRE-motif binding protein that negatively modulates abiotic sress tolerance in Arabidopsis[J] . Physiologia Plantarum, 2014, 152(1): 84-87
[16]	SUN C, PALMQVIST S, OLSSON H,et al. A novel WRKY transcription factor, Susiba2, participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1 promoter[J] . The Plant Cell Online, 2003, 15(9): 2076-2092.

施引文献

资源附件(0)

计量

文章访问数: 2016
HTML全文浏览量: 187
PDF下载量: 110
被引次数: 0

ThMYB3和ThDof2对ThVHAc1基因表达的调控

计量

出版历程

Upstream regulators of ThVHAc1.

计量

出版历程

目录