HU Hui-min,CAI Wan-han,MU Ke-bin,et al.Study on the Heterologous Expression of GMCDPK SK5 Gene in Soybean ABA Signaling Pathway[J].Soybean Science,2021,40(06):737-747.[doi:10.11861/j.issn.1000-9841.2021.06.0737]
大豆ABA信号途径GmCDPK SK5基因异源表达探究
- Title:
- Study on the Heterologous Expression of GMCDPK SK5 Gene in Soybean ABA Signaling Pathway
- Keywords:
- Soybean; GmCDPK SK5; Promoter; GUS staining; ABA; Ca2+; Stomatal movement
- 文献标志码:
- A
- 摘要:
- 为探究大豆CDPKI家族成员GmCDPK SK5参与防御逆境胁迫的途径,本研究分析外源ABA作用不同时间对大豆中GmCDPK SK5基因表达量的影响,克隆基因上游启动子序列并分析其顺式作用元件,构建GmCDPK SK5pro∷GUS融合植物表达载体,转化烟草和拟南芥,并通过GUS组织化学染色和定量表达分析方法研究GmCDPK SK5启动子对外源施加的Ca2+和ABA的响应。结果显示:施加外源ABA能够诱导GmCDPK SK5基因下调表达。启动子顺式作用元件分析表明该启动子含有3个TATA盒和23个CAAT盒基本转录元件和多种与胁迫相关的顺式作用元件。烟草瞬时表达系统中,Ca2+浓度增加,GUS酶活性增加,在50 mmol?L-1 Ca2+条件下,处理组中GUS酶活性是对照的2.1倍;ABA浓度增加,GUS酶活性减弱,在0.1 μmol?L-1 ABA条件下,对照组中的GUS酶活性是处理的5倍;ABA能诱导GmCDPK SK5基因下调表达。在拟南芥中,与WT和cpk4-1突变体株系相比,在外源施加ABA的条件下,GmCDPK SK5过表达株系在种子萌发、幼苗生长和气孔运动等方面表现得最敏感。3个株系中与ABA相关的基因ABF2、ABF4、ABI5、RD29A、RAB18和KIN1差异表达。结果说明GmCDPK SK5启动子对外源施加的Ca2+和ABA有响应,且GmCDPK SK5通过影响与响应ABA相关基因的表达来增强植株对ABA的敏感性。
- Abstract:
- In order to explore the ways of CDPKI family member GmCDPK SK5 participating in the defense against adversity stress, in this study, the promoter sequence of GmCDPK SK5 gene was cloned, and the GmCDPK SK5pro∷GUS fusion plant expression vector was constructed to transform tobacco and Arabidopsis. The results showed that the application of exogenous ABA induced down-regulation of GmCDPK SK5 gene expression. Analysis of promoter cis-acting elements showed that the promoter contains 3 TATA boxes and 23 CAAT boxes basic transcription elements and a variety of cis-acting elements related to stress. GUS histochemical staining and quantitative expression analysis were used to study the response of GmCDPK SK5 promoter to exogenous Ca2+ and ABA. The results showed that the activity of GUS enzyme increased with the increase of Ca2+ concentration. Under the condition of 50 mmol?L-1 Ca2+, the activity of GUS enzyme in the treatment group was 2.1 times of the control group. While the GUS activity decreased with the increase of ABA concentration. Under 0.01 μmol?L-1 ABA, the GUS activity in the control group was 5 times of the treatment group. Therefore ABA can induce the down-regulation of GmCDPK SK5. In Arabidopsis, compared with WT and cpk4-1 mutant lines, GmCDPK SK5 overexpression lines were the most sensitive to exogenous ABA in seed germination, seedling growth and stomatal movement. The ABA-related genes (ABF2, ABF4, ABI5, RD29A, RAB18 and KIN1) in the three lines were differentially expressed. The results showed that GmCDPK SK5 promoter responds to exogenous Ca2+ and ABA, and GmCDPK SK5 enhances the sensitivity of plants to ABA by affecting the expression of ABA-related genes.
参考文献/References:
[1]Mcainsh M R, Pittman J K. Shaping the calcium signature[J]. New Phytologist, 2009, 181(2): 275-294.[2]Liese A, Romeis T. Biochemical regulation of in vivo function of plant calcium-dependent protein kinases (CDPK)[J]. Biochimica et Biophysica Acta(BBA)-Molecular Cell Research, 2013, 1833(7): 1582-1589.[3]Xie K, Chen J, Wang Q, et al. Direct phosphorylation and activation of a mitogen-activated protein kinase by a calcium-dependent protein kinase in rice[J]. Plant Cell, 2014, 26(7): 3077-3089.[4]Mori I C, Yoshiyuki M, Yang Y, et al. CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-Type anion- and Ca2+-permeable channels and stomatal closure[J]. PLoS Biology, 2006, 4(10): e327.[5]Brandt B, Brodsky D E, Xue S, et al. Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branchedABI1 PP2C phosphatase action[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(26): 10593-10598.[6]Zhu S Y, Yu X C, Wang X J, et al. Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction inArabidopsis[J]. Plant Cell, 2007, 19(10): 3019-3036.[7]Choi H I, Park H J, Ji H P, et al. Arabidopsis calcium-dependent protein kinase AtCPK32 interacts with ABF4, a transcriptional regulator of abscisic acid-responsive gene expression, and modulates its activity[J]. Plant Physiology, 2005, 139(4): 1750-1761.[8]Kobayashi M, Ohura I, Kawakita K, et al. Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato nadph oxidase [J]. Plant Cell, 2007, 19(3): 1065-1080.[9]Asano T, Hakata M, Nakamura H, et al. Functional characteri-sation of OsCPK21, a calciumdependent protein kinase that confers salt tolerance in rice[J]. Plant Molecular Biology, 2011, 75(1-2): 179-191.[10]Liu F, Yoo B C, Lee J Y, et al. Calcium-regulated phosphorylation of soybean serine acetyltransferase in response to oxidative stress[J]. Journal of Biological Chemistry, 2006, 281(37): 27405-27415.[11]Choi H I , Hong J H , Ha J O , et al. ABFs, a family of ABA-responsive element binding factors[J]. Journal of Biological Chemistry, 2000, 275(3): 1723-1730.[12]Finkelstein R R, Lynch T J. The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor[J]. Plant Cell, 2000, 12(4): 599-609.[13]Lang V, Palva E T. The expression of a rab-related gene, rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana(L.) Heynh[J]. Plant Molecular Biology, 1992, 21(3): 581-582.[14]Kurkela S, Borg-Franck M. Structure and expression of KIN2, one of two cold- and ABA-induced genes of Arabidopsis thaliana[J]. Plant Molecular Biology, 1992, 19(4): 689-692.[15]Yamaguchi-Shinozakiaib K, Shinozaki K. A nove1 cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low temperature, or high-salt stress[J]. The Plant Cell, 1994, 6: 251-264.[16]牛娟. 大豆CDPK蛋白基因CDPK-SK5的分离、表达分析与亚细胞定位[D]. 南京: 南京农业大学, 2012. (Nu J. Separate, expression and subcellular location of CDPK protein gene, CDPK-SK5 in soybean[D]. Nanjing: Nanjing Agricultural University, 2012.)[17]宋利茹, 王爽, 牛娟, 等. 春大豆种子田间劣变性和劣变抗性的差异蛋白质组学研究[J]. 中国农业科学, 2015, 48(1): 23-32. (Song L R, Wang S, Niu J, et al. Differentially proteomics analysis of pre-harvest seed deterioration and deterioration resistance in spring soybean[J]. Scientia Agricultura Sinica, 2015, 48(1): 23-32.)[18]王爽. 高温高湿下大豆钙依赖蛋白激酶基因在种子活力中的功能分析[D]. 南京: 南京农业大学, 2016. (Wang S. Function analysis of soybean [Glycine max (L.) Merr.] CDPK genes on seed vigor under high temperature and humidity[D]. Nanjing: Nanjing Agricultural University, 2016.)[19]陈明. GmCOL4、GmZTL1与顺式元件HSE响应高温高湿以及调控GmSBH1的研究[D]. 南京: 南京农业大学, 2019. (Chen M. Study on the function of GmZTL1, GmCOL4 and cis-element and the co-regulation to GmSBH1[D]. Nanjing: Nanjing Agricultural University, 2019.)
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(06):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(06):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(06):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(06):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(06):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(06):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(06):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(06):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(06):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(06):46.[doi:10.3969/j.issn.1000-9841.2013.01.011]
备注/Memo
收稿日期:2021-04-25