LIU Su-shuang,LI Xin-yu,WANG Ze-bo,et al.Functional Analysis of Soybean GmPM31 Gene and Transformation into Arabidopsis thaliana[J].Soybean Science,2022,41(05):526-535.[doi:10.11861/j.issn.1000-9841.2022.05.0526]
大豆GmPM31基因的功能分析及拟南芥转化
- Title:
- Functional Analysis of Soybean GmPM31 Gene and Transformation into Arabidopsis thaliana
- Keywords:
- soybean; GmPM31 gene; protein interaction; promoter analysis; overexpression
- 文献标志码:
- A
- 摘要:
- 大豆GmPM31基因编码蛋白属于植物小分子热激蛋白sHSP-CI(Class I)亚家族的成员，为研究其在大豆抗逆过程中的生物学功能，对GmPM31蛋白进行亚细胞定位分析；采用酵母双杂交和双分子荧光互补方法验证Gm2-MMP与GmPM31蛋白在体内的互作关系；构建GmPM31启动子5’端不同缺失片段的植物表达载体，通过烟草叶片瞬时表达系统分析其驱动GUS基因的表达水平；利用农杆菌侵染法将GmPM31基因转化拟南芥。结果表明：大豆基质金属蛋白酶Gm2-MMP与小分子热激蛋白GmPM31存在体内互作关系。经40 ℃高温高湿胁迫处理，与对照组相比，P2062与P1800启动子驱动表达的GUS活性显著增加，说明-1 800~-1 535 bp区段对GmPM31基因响应高温高湿胁迫信号起关键作用。繁种过表达GmPM31转基因拟南芥植株，筛选得到5株T2代拟南芥阳性植株。
- Abstract:
- Soybean GmPM31 gene encoding protein is a member of sHSP-CI (Class I) subfamily of plant small molecule heat shock protein. In order to study the biological function of GmPM31 protein in soybean stress tolerance， subcellular localization analysis of GmPM31 protein was performed. Yeast two-hybrid and Bimolecular Fluorescent Complimentary methods were used to verify the interaction between Gm2-MMP and GmPM31 protein in vivo. Plant expression vectors with different missing fragments at the 5′ end of GmPM31 promoter were constructed， and the expression level of drived GUS gene was analyzed by transient expression system in tobacco leaves. GmPM31 gene was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens infection. The results showed that soybean matrix metalloproteinase Gm2-MMP interacts with small molecule heat shock protein GmPM31 in vivo. Compared with the control group， the activity of GUS driven by the P2062 and P1800 promoters was significantly increased under high temperature and high humidity stress at 40 ℃， indicating that -1 800~-1 535 bp region plays a key role in the response of the GmPM31 gene to high temperature and high humidity stress signals. Transgenic Arabidopsis plants overexpressing GmPM31 were propagated， and five T2-generation Arabidopsis positive plants were screened.
参考文献/References:
［1］王曙明， 孟凡凡， 郑宇宏， 等. 大豆高产育种研究进展[J]. 中国农学通报， 2010， 26(9): 162-166.（WANG S M， MENG F F， ZHENG Y H， et al. Progress of soybean breeding for high yield[J]. Chinese Agricultural Science Bulletin， 2010， 26(9): 162-166.）[2]舒英杰， 陶源， 王爽， 等. 高等植物种子活力的生物学研究进展[J]. 西北植物学报， 2013， 33(8): 1709-1716.（SHU Y J， TAO Y， WANG S， et al. Research progress on seed vigor biology of higher plant[J]. Acta Botanica Boreali-Occidentalia Sinica， 2013， 33(8): 1709-1716.）[3]CHEN W L， ZHOU Q， LI L， et al. Comparing ultraweak bio-chemiluminescence emission in wounded green and etiolated soybean cotyledons[C]. Imaging， Manipulation， and Analysis of Biomolecules， Cells， and Tissues IV. International Society for Optics and Photonics， 2006.[4]陈志英， 胡健， 王长进， 等. 利用转录组测序对不同贮藏温度处理玉米种子的苗期差异表达基因分析[J]. 玉米科学， 2021， 29(6): 35-40，49.（CHEN Z Y， HU J， WANG C J， et al. Analysis of differentially expressed genes in seedling stage of maize seeds treated with different storage temperatures by transcriptome sequencing[J]. Journal of Maize Sciences， 2021， 29(6): 35-40，49.）[5]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.）[6]REN C， BILYEU K D， BEUSELINK P R. Composition， vigor， and proteome of mature soybean seeds developed under high temperature[J]. Proteomics Center Publications (MU)， 2009，49(3):1010-1022.[7]SAHA R R， SULTANA W. Influence of seed ageing on growth and yield of soybean[J]. Bangladesh Journal of Botany， 2008， 37(1): 21-26.[8]KEBEDE H， SMITH J R， RAY J D. A new gene that controls seed coat wrinkling in soybean[J]. Euphytica， 2013， 189(2): 309-320.[9]王依隆， 赵海红， 沈英姿， 等. 大豆GmPHD3基因的克隆及逆境表达分析[J]. 大豆科学， 2020， 39(4): 500-508.（WANG Y L， ZHAO H H， SHEN Y Z， et al. Cloning and abiotic expression analysis of GmPHD3 gene in soybean[J]. Soybean Science， 2020， 39(4): 500-508.）[10]朱雅婧， 周亚丽， 刘骕骦， 等.GmHMADP参与高温高湿下大豆种子活力形成及铜镉胁迫响应的研究[J]. 中国农业科学， 2018， 51(14): 2642-2654.（ZHU Y J， ZHOU Y L， LIU S S， et al. GmHMADP involved in seed vigor formation of soybean under high temperature and humidity stress and its study responsive to copper and cadmium stress[J]. Scientia Agricultura Sinica， 2018， 51(14): 2642-2654.）[11]LUCIANO A E， ANDREIA C， ALBERTO T， et al. Soybean seed vigor: Uniformity and growth as key factors to improve yield[J]. Agronomy， 2020， 10(4): 545.[12]ZHOU Q Q， FU Z Y， LIU H J， et al. Mining novel kernel size related genes by pQTL mapping and multi-omics integrative analysis in developing maize kernels[J]. Plant Biotechnology Journal， 2021， 19(8): 1489-1491.[13]HENRIET C， BALLIAU T， AIME D， et al. Proteomics of developing pea seeds reveals a complex antioxidant network underlying the response to sulfur deficiency and water stress[J]. Journal of Experimental Botany， 2021， 72(7): 2611-2626.[14]万会娜， 于月华， 王怡， 等. 大豆GmNAC131基因的生物信息学及表达分析[J]. 大豆科学， 2021， 40(2): 186-194.（WAN H N， YU Y H， WANG Y， et al. Bioinformatics and expression analysis of GmNAC131 gene in soybean[J]. Soybean Science， 2021， 40(2): 186-194.）[15]柏锡， 陈云， 杨雪， 等.GmSTK12基因转化大豆及对转化体生物量影响的研究[J]. 东北农业大学学报， 2021， 52(9): 10-18，46.（BAI X， CHEN Y， YANG X， et al. Research on biomass production by overexpression of a serine/threonine protein kinase， GmSTK12 in soybean[J]. Journal of Northeast Agricultural University， 2021， 52(9): 10-18，46.）[16]刘骕骦. 大豆基质金属蛋白酶基因Gm1-MMP和Gm2-MMP的分离以及响应高温高湿胁迫的功能分析[D]. 南京: 南京农业大学， 2017.（LIU S S. Isolation and functional analysis of soybean matrix metalloproteinases Gm1-MMP and Gm2-MMP involved in high temperature and high humidity stress response[D]. Nanjing: Nanjing Agricultural University， 2017.）[17]LIU S S， JIA Y H， ZHU Y J， et al. Soybean matrix metalloproteinase Gm2-MMP relates to growth and development and confers enhanced tolerance to high temperature and humidity stress in transgenic Arabidopsis[J]. Plant Molecular Biology Reporter， 2018， 36(1): 94-106.[18]WATERS E R. The evolution， function， structure， and expression of the plant sHSPs[J]. Journal of Experimental Botany， 2013， 64(2):391-403.[19]杨芳， 杨仕梅， 罗雪， 等. 百脉根Hsp70s基因家族的生物信息学分析[J]. 山地农业生物学报， 2020， 39(5): 1-8.（YANG F， YANG S M， LUO X， et al. Bioinformatics analysis of Hsp70s gene family in Lotus japonicus[J]. Journal of Mountain Agriculture and Biology， 2020， 39(5): 1-8.）[20]ZHANG N， SHI J W， ZHAO H Y， et al. Activation of small heat shock protein (SlHSP17.7) gene by cell wall invertase inhibitor (SlCIF1) gene involved in sugar metabolism in tomato[J]. Gene， 2018， 679: 90-99.[21]栗振义， 龙瑞才， 张铁军， 等. 植物热激蛋白研究进展[J]. 生物技术通报， 2016， 32(2): 7-13.（LI Z Y， LONG R C， ZHANG T J， et al. Research progress on plant heat shock protein[J]. Biotechnology Bulletin， 2016， 32(2): 7-13.)[22]KEY J L， LIN C Y， CHEN Y M. Heat shock proteins of higher plants[J]. Proceedings of the National Academy of Sciences of the United States of America， 1981， 78(6): 3526-3530.[23]JI X R， YU Y H， NI P Y， et al. Genome-wide identification of small heat-shock protein (HSP20) gene family in grape and expression profile during berry development[J]. BMC Plant Biology， 2019， 19(1): 433.[24]ZHAO P， WANG D D， WANG R Q， et al. Genome-wide analysis of the potato Hsp20 gene family: Identification， genomic organization and expression profiles in response to heat stress[J]. BMC genomics， 2018， 19(1): 61.[25]张宁， 姜晶， 史洁玮. 番茄HSP20基因家族的全基因组鉴定、系统进化及表达分析[J]. 沈阳农业大学学报， 2017， 48(2): 137-144.（ZHANG N， JIANG J， SHI J W， et al. Genome-wide identification， phyletic evolution and expression analysis of the HSP20 gene family in tomato[J]. Journal of Shenyang Agricultural University， 2017， 48(2): 137-144.）[26]张泽， 裴鑫， 鲁仪增， 等. 花楸树小热激蛋白23.8基因(SPHSP23.8)克隆与表达分析[J]. 植物资源与环境学报， 2020， 29(5): 9-20.（ZHANG Z， PEI X， LU Y Z， et al. Cloning and expression analysis on small heat shock protein 23.8 gene（SpHSP23.8） in Sorbus pohuashanensis[J]. Journal of Plant Resources and Environment， 2020， 29(5): 9-20.）[27]MARIE H A M， AURELIA R， MARIE P H， et al. The mitochondrial small heat shock protein HSP22 from pea is a thermosoluble chaperone prone to co-precipitate with unfolding client proteins[J]. International Journal of Molecular Sciences， 2019， 21(1): 97.[28]张驰， 刘丹丹，刘建中. 植物J蛋白的生物学功能及其作用机制[J]. 浙江大学学报(农业与生命科学版)， 2018， 44(3): 275-282.（ZHANG C， LIU D D， LIU J Z. Biological functions and action mechanisms of J-domain proteins in plants[J]. Journal of Zhejiang University(Agriculture and Life Sciences)， 2018， 44(3): 275-282.）〖LL〗[29]RASHED M A S， ABOU-DEIF M H， KHALIL K M， et al. Expression levels of heat shock proteins through western blot and real-time polymerase chain reaction in maize[J]. Jordan Journal of Biological Sciences， 2021， 14(4): 671-676.[30]刘骕骦， 邱颖胜， 刘燕敏， 等. 大豆GmPM31基因生物信息学、组织表达及高温高湿响应分析[J]. 大豆科学， 2021， 40(5): 612-619.（LIU S S， QIU Y S， LIU Y M， et al. Analysis of soybean GmPM31 bioinformatics， tissue expression and response to high temperature and high humidity[J]. Soybean Science， 2021， 40(5): 612-619.）[31]宋蒙飞， 王星， 张开京， 等. 黄瓜DnaJ基因家族鉴定及对高温胁迫的表达响应[J]. 南京农业大学学报， 2021， 44(2): 267-277.（SONG M F， WANG X， ZHANG K J， et al. Identification of DnaJ gene family in cucumber and its expression response to high temperature stress[J]. Journal of Nanjing Agricultural University， 2021， 44(2): 267-277.）[32]CHEN G， HU J， LIAN J， et al. Functional characterization of OsHAK1 promoter in response to osmotic/drought stress by deletion analysis in transgenic rice[J]. Plant Growth Regulation， 2019， 88(3): 241-251.[33]ZHENG J， LIN S， ZHANG Q， et al. Functional identification and regulation of the PtDrl02 gene promoter from triploid white poplar[J]. Plant Cell Reports， 2010， 29(5): 449-460.[34]DIVYA K， KISHOR P B K， BHATNAGAR M P， et al. Isolation and functional characterization of three abiotic stress-inducible (Apx， Dhn and Hsc70) promoters from pearl millet (Pennisetum glaucum L.)[J]. Molecular Biology Reports， 2019， 46(6): 6039-6052.[35]申威. 山葡萄CBL-CIPKs调控网络中两个关键基因响应逆境胁迫的功能分析[D]. 宁夏: 宁夏大学， 2019.（SHEN W. Functional analysis of two key stress-related genes in CBL-CIPKs regulatory network in Vitis Amurensis[D]. Ningxia: Ningxia University， 2019.）[36]SONG H M， FAN P X， SHI W L， et al. Expression of five AtHsp90 genes in Saccharomyces cerevisiae reveals functional differences of AtHsp90s under abiotic stresses[J]. Journal of Plant Physiology， 2010， 167(14): 1172-1178.[37]FU X M. Chaperone function and mechanism of small heat-shock proteins[J]. Acta Biochimica Et Biophysica Sinica， 2014， 46(5): 347-356.[38]HUANG L J， CHENG G X， KHAN A， et al.Ca HSP164， a small heat shock gene in pepper， is involved in heat and drought tolerance[J]. Protoplasma， 2018， 256(1): 39-51.
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(05):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(05):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(05):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(05):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(05):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(05):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(05):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(05):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(05):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(05):46.[doi:10.3969/j.issn.1000-9841.2013.01.011]
备注/Memo
收稿日期：2022-02-13