大豆GmGSTa基因启动子克隆及表达调控功能分析
- Title:
- Cloning and Expression Regulatory Analysis of GmGSTa Gene Promoter in Soybean
- 摘要:
- GmGSTa是与种子高活力膜联蛋白GmANN和钙依赖蛋白激酶GmCDPK SK5均存在相互作用的蛋白，为探究大豆GmGSTa启动子的逆境响应机制及调控功能，采用CTAB法提取大豆叶片DNA，并利用PCR技术克隆GmGSTa启动子序列。对获得的启动子序列进行顺式作用元件分析，鉴定其中的核心元件及胁迫与激素响应元件。构建融合载体GMGSTAPRO::GUS，采用农杆菌介导法分别转入烟草叶片和拟南芥。在此基础上，开展非生物胁迫及ABA诱导表达试验，观察该启动子在拟南芥中的组织特异性和发育阶段依赖性表达情况。通过GUS组织化学染色及GUS酶活性测定，对启动子驱动下的基因表达情况进行定性与定量分析。结果显示：分离得到2 316 bp的GmGSTa启动子序列，其含有TATA-box、CAAT-box等核心元件，以及DRE1、MYB、ABRE等多种胁迫与激素响应元件。GmGSTa启动子在烟草中受150 mmol·L-1 NaCl、250 mmol·L-1甘露醇和高温高湿（40 ℃，相对湿度95%）胁迫显著诱导，GUS染色深度随胁迫程度增强而加深。在拟南芥中，其活性呈现组织特异性与发育阶段依赖性，在根、叶、雄蕊及成熟种子中显著表达。50~150 mmol·L-1 NaCl、150~250 mmol·L-1甘露醇及8~10 μmol·L-1 ABA 处理可显著增强启动子活性，其中250 mmol·L-1甘露醇诱导下GUS酶活性较对照提升幅度最大。研究结果表明，GmGSTa启动子是一个功能性的调控元件，能够响应多种非生物胁迫及ABA信号诱导，其活性表现出浓度依赖性和组织发育特异性。该启动子可作为潜在的诱导型高效启动子靶点，为作物抗逆基因工程育种提供新的调控元件。
相似文献/References:
[1]刘章雄,李卫东,孙石,等.1983～2010年北京大豆育成品种的亲本地理来源及其遗传贡献[J].大豆科学,2013,32(01):1.[doi:10.3969/j.issn.1000-9841.2013.01.002]
LIU Zhang-xiong,LI Wei-dong,SUN Shi,et al.Geographical Sources of Germplasm and Their Nuclear Contribution to Soybean Cultivars Released during 1983 to 2010 in Beijing[J].Soybean Science,2013,32(04):1.[doi:10.3969/j.issn.1000-9841.2013.01.002]
[2]李彩云,余永亮,杨红旗,等.大豆脂质转运蛋白基因GmLTP3的特征分析[J].大豆科学,2013,32(01):8.[doi:10.3969/j.issn.1000-9841.2013.01.003]
LI Cai-yun,YU Yong-liang,YANG Hong-qi,et al.Characteristics of a Lipid-transfer Protein Gene GmLTP3 in Glycine max[J].Soybean Science,2013,32(04):8.[doi:10.3969/j.issn.1000-9841.2013.01.003]
[3]王明霞,崔晓霞,薛晨晨,等.大豆耐盐基因GmHAL3a的克隆及RNAi载体的构建[J].大豆科学,2013,32(01):12.[doi:10.3969/j.issn.1000-9841.2013.01.004]
WANG Ming-xia,CUI Xiao-xia,XUE Chen-chen,et al.Cloning of Halotolerance 3 Gene and Construction of Its RNAi Vector in Soybean (Glycine max)[J].Soybean Science,2013,32(04):12.[doi:10.3969/j.issn.1000-9841.2013.01.004]
[4]张春宝,李玉秋,彭宝,等.线粒体ISSR与SCAR标记鉴定大豆细胞质雄性不育系与保持系[J].大豆科学,2013,32(01):19.[doi:10.3969/j.issn.1000-9841.2013.01.005]
ZHANG Chun-bao,LI Yu-qiu,PENG Bao,et al.Identification of Soybean Cytoplasmic Male Sterile Line and Maintainer Line with Mitochondrial ISSR and SCAR Markers[J].Soybean Science,2013,32(04):19.[doi:10.3969/j.issn.1000-9841.2013.01.005]
[5]卢清瑶,赵琳,李冬梅,等.RAV基因对拟南芥和大豆不定芽再生的影响[J].大豆科学,2013,32(01):23.[doi:10.3969/j.issn.1000-9841.2013.01.006]
LU Qing-yao,ZHAO Lin,LI Dong-mei,et al.Effects of RAV gene on Shoot Regeneration of Arabidopsis and Soybean[J].Soybean Science,2013,32(04):23.[doi:10.3969/j.issn.1000-9841.2013.01.006]
[6]杜景红,刘丽君.大豆fad3c基因沉默载体的构建[J].大豆科学,2013,32(01):28.[doi:10.3969/j.issn.1000-9841.2013.01.007]
DU Jing-hong,LIU Li-jun.Construction of fad3c Gene Silencing Vector in Soybean[J].Soybean Science,2013,32(04):28.[doi:10.3969/j.issn.1000-9841.2013.01.007]
[7]张力伟,樊颖伦,牛腾飞,等.大豆“冀黄13”突变体筛选及突变体库的建立[J].大豆科学,2013,32(01):33.[doi:10.3969/j.issn.1000-9841.2013.01.008]
ZHANG Li-wei,FAN Ying-lun,NIU Teng-fei?,et al.Screening of Mutants and Construction of Mutant Population for Soybean Cultivar "Jihuang13”[J].Soybean Science,2013,32(04):33.[doi:10.3969/j.issn.1000-9841.2013.01.008]
[8]盖江南,张彬彬,吴瑶,等.大豆不定胚悬浮培养基因型筛选及基因枪遗传转化的研究[J].大豆科学,2013,32(01):38.[doi:10.3969/j.issn.1000-9841.2013.01.009]
GAI Jiang-nan,ZHANG Bin-bin,WU Yao,et al.Screening of Soybean Genotypes Suitable for Suspension Culture with Adventitious Embryos and Genetic Transformation by Particle Bombardment[J].Soybean Science,2013,32(04):38.[doi:10.3969/j.issn.1000-9841.2013.01.009]
[9]王鹏飞,刘丽君,唐晓飞,等.适于体细胞胚发生的大豆基因型筛选[J].大豆科学,2013,32(01):43.[doi:10.3969/j.issn.1000-9841.2013.01.010]
WANG Peng-fei,LIU Li-jun,TANG Xiao-fei,et al.Screening of Soybean Genotypes Suitable for Somatic Embryogenesis[J].Soybean Science,2013,32(04):43.[doi:10.3969/j.issn.1000-9841.2013.01.010]
[10]刘德兴,年海,杨存义,等.耐酸铝大豆品种资源的筛选与鉴定[J].大豆科学,2013,32(01):46.[doi:10.3969/j.issn.1000-9841.2013.01.011]
LIU De-xing,NIAN Hai,YANG Cun-yi,et al.Screening and Identifying Soybean Germplasm Tolerant to Acid Aluminum[J].Soybean Science,2013,32(04):46.[doi:10.3969/j.issn.1000-9841.2013.01.011]