欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2018, Vol. 44 ›› Issue (8): 1159-1168.doi: 10.3724/SP.J.1006.2018.01159

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆半胱氨酸蛋白酶抑制剂基因GmCYS2的功能鉴定

柯丹霞(),彭昆鹏,贾妍,曾硕,王英枝,张静怡   

  1. 信阳师范学院生命科学学院 / 大别山农业生物资源保护与利用研究院, 河南信阳 464000
  • 收稿日期:2017-11-23 接受日期:2018-03-26 出版日期:2018-08-10 网络出版日期:2018-04-20
  • 通讯作者: 柯丹霞
  • 基金资助:
    国家自然科学基金项目(31400213);河南省科技攻关计划项目(182102110448);信阳师范学院青年骨干教师资助计划项目(2015);信阳师范学院“南湖学者奖励计划”青年项目(2016)

Functional Characterization of Soybean Cystatins Gene GmCYS2

Dan-Xia KE(),Kun-Peng PENG,Yan JIA,Shuo ZENG,Ying-Zhi WANG,Jing-Yi ZHANG   

  1. College of Life Sciences / Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains, Xinyang Normal University, Xinyang, Henan 464000, China
  • Received:2017-11-23 Accepted:2018-03-26 Published:2018-08-10 Published online:2018-04-20
  • Contact: Dan-Xia KE
  • Supported by:
    the National Natural Science Foundation of China(31400213);Henan Province Science and Technology Research Projects(182102110448);the Funding Scheme for Young Core Teachers of Xinyang Normal University(2015);Nanhu Scholars Program for Young Scholars of XYNU(2016)

RichHTML

24

PDF

3925

PDF

29

摘要：

半胱氨酸蛋白酶抑制剂(cystatin, CYS)在结瘤, 根瘤发育和衰老过程中起重要作用。本研究克隆了1个大豆CYS家族基因GmCYS2, 氨基酸序列比对及进化树分析表明, GmCYS2与木豆(Cajanus cajan) CYS相似性最高。在体外对该基因编码蛋白进行了表达和纯化, 重组蛋白GmCYS2的酶活性抑制实验显示, 该蛋白对L类和B类组织蛋白酶的抑制活性明显高于对H类组织蛋白酶, 并且在30 d根瘤提取物中的抑制活性高于在60 d根瘤提取物中。此外, 构建GmCYS2的植物表达载体, 通过百脉根毛根转化法获得转GmCYS2基因的超表达复合体植株。GmCYS2的超量表达增加了百脉根的结瘤数目, 并且上调共生标记基因的表达。结果说明GmCYS2蛋白具有一定的酶活性抑制作用, 并且正调控百脉根共生结瘤过程。

关键词: 大豆, 半胱氨酸蛋白酶抑制剂, 半胱氨酸蛋白酶, 共生结瘤, 百脉根

Abstract:

CYS (cystatin) plays an important role in nodulation, nodule development and senescence. In this study, we cloned a soybean CYS family gene GmCYS2, which had the highest similarity with pigeonpea (Cajanus cajan) CYS, shown by amino acid sequence alignment and phylogenetic tree analysis. The gene encoding protein was expressed and purified in vitro. Enzyme activity inhibition experiment of recombinant protein GmCYS2 showed that the cathepsin inhibitory activity of GmCYS2 protein on L and B classes was higher than that on H class, and cathepsin inhibitory activity in nodule extract from nodule at 30 d was generally higher than that at the 60 d. In addition, the plant overexpression vector was constructed and the GmCYS2-overexpression composite plants were obtained by Lotus japonicus hairy root transformation method. Overexpression of GmCYS2 increased the number of nodules in Lotus japonicus plants and up regulated the expression levels of symbiotic marker genes. These results suggest that GmCYS2 protein can inhibit the enzyme activity and positively regulate the symbiotic nodulation of Lotus japonicus.

Key words: Glycine max, cystatin, cysteine protease, symbiotic nodulation, Lotus japonicus

表1

本研究中所使用的引物"

引物名称
Primer name		 引物序列
Sequence of primer (5′-3′)
F-GmCYS2-pro. TACGGGGGATTGGTC
R-GmCYS2-pro. TCACTGCGTGGAAGGAG
F-OX CGggatccATGGCGGCGTTGATAAG
R-OX GGggtaccTCACTGCGTGGAAGGAG
F-NIN-rt AACTCACTGGAAACAGGTGCTTTC
R-NIN-rt CTATTGCGGAATGTATTAGCTAGA
F-ENOD40-1-rt GGAGGTATGCTCAAACATTC
R-ENOD40-1-rt GTAACTTCTCAAGAGAAGACC
F-ENOD40-2-rt CAAAACTCGTTATGTTGCGG
R-ENOD40-2-rt CACCTCAAAGGAAGAAGAACA
F-GmCYS2-rt CAACAAGTGGTGTCTG
R-GmCYS2-rt TCACTGCGTGGAAGGAG
F-Polyubiquitin TTCACCTTGTGCTCCGTCTTC
R-Polyubiquitin AACAACAGCACACACAGACAATC

图1

大豆GmCYS2与其他植物中同源蛋白的序列比对(A)及进化树分析(B) 图A中用黄色、红色、绿色和蓝色方框标出半胱氨酸蛋白酶抑制剂的主要保守结构域。图B中标尺代表遗传相似性, 指不同植物间同源蛋白进化关系的远近。"

图2

His-GST-GmCYS2融合蛋白的序列分析 氨基酸第5~10位: His标签; 第14~233位: GST标签; 第244~250位: TEV(烟草蚀纹病毒)切割位点; 251~355位: GmCYS2蛋白序列(去掉N端26个氨基酸编码的信号肽)。"

图3

GmCYS2蛋白的体外表达、抗体检测和蛋白纯化 A: SDS-PAGE分析蛋白在BL21(DE3)菌株中的表达情况; B: Western blot分析, 抗-His抗体检测; C: 纯化蛋白的SDS-PAGE分析。M1: 蛋白marker; M2: Western marker; M3: 蛋白预染marker; PC1: BSA (1 μg); PC2: BSA (2 μg); NC: 未诱导的全细胞裂解液。1: 15℃诱导16 h的全细胞裂解液; 2: 37℃诱导4 h的全细胞裂解液; NC1: 未诱导的细胞裂解液上清液; NC2: 未诱导的细胞裂解液沉淀; 3: 15℃诱导16 h的细胞裂解液上清液; 4: 15℃诱导16 h的细胞裂解液沉淀; 5: 37℃诱导4 h的细胞裂解液上清液; 6: 37℃诱导4 h的细胞裂解液沉淀; 7: 从37℃诱导4 h的细胞裂解液上清液中纯化的蛋白。"

图4

重组蛋白GmCYS2在不同时期根瘤提取物中的抑制活性分析 样品分别为30 d和60 d根瘤提取物。反应液中加入纯化的GmCYS2蛋白, 另外分别加入组织蛋白酶L类、B类和H类3种不同底物, 试验重复3次。* P < 0.05。"

图5

GmCYS2基因的过量表达对百脉根共生结瘤的影响 A: 复合体植株接种MAFF303099根瘤菌30 d后的结瘤表型。毛状根表达pMUb:GmCYS2 (GmCYS2-OX)或空载体pU1301 (CK), Bar = 10 mm; B: 单株复合体百脉根植株的平均结瘤数目; C: 根据单株根瘤数划分的不同结瘤种类所占的相对百分比; D: 阳性毛根的GUS鉴定; E: RT-PCR检测阳性毛根中GmCYS2基因的表达。M: DL2000 DNA marker; 1: 阳性质粒对照; 2: 空载体对照; 3~5: 复合体植株。"

表2

过表达GmCYS2复合体百脉根植株的结瘤数目统计"

试验
Experiment		 对照植株
CK		 超表达植株
GmCYS2-OX		 P值
P-value
1 5.42±1.53 (n=30) 10.33±2.35 (n=25) < 0.01
2 6.18±2.05 (n=23) 9.87±2.48 (n=20) < 0.01

图6

转基因毛根中GmCYS2以及共生基因的转录水平检测 荧光定量PCR检测GmCYS2以及共生基因 NIN、ENOD40-1和ENOD40-2 在超表达植株(GmCYS2-OX)和对照植株(CK)阳性毛根中的转录水平。* P<0.05, ** P<0.01。"

[1] Martinez M, Diaz-Mendoza M, Carrillo L, Diaz I . Carboxy terminal extended phytocystatins are bifunctional inhibitors of papain and legumain cysteine proteinases. FEBS Lett, 2007,581:2914-2918
doi: 10.1016/j.febslet.2007.05.042
[2] Stubbs M T, Laber B, Bode W, Huber R, Jerala R, Lenarcic B, Turk V . The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction. EMBO J, 1990,9:1939-1947
[3] Margis R, Reis E M, Villeret V . Structural and phylogenetic relationships among plant and animal cystatins. Arch Biochem Biophys, 1998,359:24-30
doi: 10.1006/abbi.1998.0875 pmid: 9799556
[4] Martinez M, Cambra I, Carrillo L, Diaz-Mendoza M, Diaz I . Characterization of the entire cystatin gene family in barley and their target cathepsin L-like cysteine-proteases, partners in the hordein mobilization during seed germination. Plant Physiol, 2009,151:1531-1545
doi: 10.1104/pp.109.146019
[5] Benchabane M, Schlüter U, Vorster J, Goulet M C, Michaud D . Plant cystatins. Biochimie, 2010,92:1657-1666
doi: 10.1016/j.biochi.2010.06.006
[6] Arai S, Matsumoto I, Emori Y, Abe K . Plant seed cystatins and their target enzymes of endogenous and exogenous origin. J Agric Food Chem, 2002,50:6612-6617
doi: 10.1021/jf0201935
[7] Belenghi B, Acconcia F, Trovato M, Perazzolli M, Bocedi A, Polticelli F, Ascenzi P, Delledonne M . AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death. Eur J Biochem, 2003,270:2593-2604
[8] Hwang J E, Hong J K, Lim C J, Chen H, Je J, Yang K A, Kim D Y, Choi Y J, Lee S Y, Lim C O . Distinct expression patterns of two Arabidopsis phytocystatin genes, AtCYS1 and AtCYS2, during development and abiotic stresses. Plant Cell Rep, 2010,29:905-915
[9] Carrillo L, Martinez M, Ramessar K, Cambra I, Castañera P, Ortego F, Díaz I . Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases. Plant Cell Rep, 2011,30:101-112
doi: 10.1007/s00299-010-0948-z
[10] Popovic M, Andjelkovic U, Burazer L, Lindner B, Petersen A, Gavrovic-Jankulovic M . Biochemical and immunological characterization of a recombinantly-produced antifungal cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa). Phytochemistry, 2013,94:53-59
[11] Tan Y, Wang S, Liang D, Li M, Ma F . Genome-wide identification and expression profiling of the cystatin gene family in apple (Malus × domestica Borkh.). Plant Physio Biochem, 2014,79:88-97
[12] Tan Y X, Li M J, Ma F W . Overexpression of MpCYS2, a phytocystatin gene from Malus prunifolia(Willd.) Borkh., confers drought tolerance and protects against oxidative stress in Arabidopsis. Plant Cell Tiss Org, 2015,123:15-27
[13] Tan Y X, Li M J, Yang Y L, Sun X, Wang N, Liang B W, Ma F W . Overexpression of MpCYS4, a phytocystatin gene from Malus prunifolia(Willd.) Borkh., enhances stomatal closure to confer drought tolerance in transgenic Arabidopsis and apple. Front Plant Sci, 2017,8:33
[14] Tan Y X, Yang Y L, Li C, Liang B W, Li M J, Ma F W . Overexpression of MpCYS4, a phytocystatin gene from Malus prunifolia(Willd.) Borkh., delays natural and stress-induced leaf senescence in apple. Plant Physiol Biochem, 2017,115:219-228
[15] Tan Y X, Wei XY, Wang P, Sun X, Li M J, Ma F W . A phytocystatin gene from Malus prunifolia(Willd.) Borkh., MpCYS5, confers salt stress tolerance and functions in endoplasmic reticulum stress response in Arabidopsis. Plant Mol Biol Rep, 2016,34:62-75
[16] Song C, Kim T, Chung W S, Lim C O . The Arabidopsis phytocystatin AtCYS5 enhances seed germination and seedling growth under heat stress conditions. Mol Cells, 2017,40:577-586
[17] Christoff A P, Passaia G, Salvati C, Alves-Ferreira M, Margis- Pinheiro M, Margis R . Rice bifunctional phytocystatin is a dual modulator of legumain and papain-like proteases. Plant Mol Biol, 2016,92:193-207
doi: 10.1007/s11103-016-0504-5 pmid: 27325119
[18] Sun X, Yang S, Sun M, Wang S, Ding X, Zhu D, Ji W, Cai H, Zhao C, Wang X, Zhu Y . A novel Glycine soja cysteine proteinase inhibitor GsCPI14, interacting with the calcium/calmodulin- binding receptor-like kinase GsCBRLK, regulated plant tolerance to alkali stress. Plant Mol Biol, 2014,85:33-48
[19] Labudda M, Różańska E, Szewińska J, Sobczak M, Dzik J M . Protease activity and phytocystatin expression in Arabidopsis thaliana upon heterodera schachtii infection. Plant Physiol Biochem, 2016,109:416-429
[20] Yu Y, Zhang G, Li Z, Cheng Y, Gao C, Zeng L, Chen J, Yan L, Sun X, Guo L, Yan Z . Molecular cloning, recombinant expression and antifungal activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.). Genes(Basel), 2017,8:265
[21] Schmutz J, Cannon S B, Schlueter J, Ma J, Mitros T, Nelson W, Hyten D L, Song Q, Thelen J J, Cheng J, Xu D, Hellsten U, May G D, Yu Y, Sakurai T, Umezawa T, Bhattacharyya M K, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du J, Tian Z, Zhu L, Gill N, Joshi T, Libault M, Sethuraman A, Zhang X C, Shinozaki K, Nguyen H T, Wing R A, Cregan P, Specht J, Grimwood J, Rokhsar D, Stacey G, Shoemaker R C, Jackson S A . Genome sequence of the palaeopolyploid soybean. Nature, 2010,463:178-183
doi: 10.1038/nature08670 pmid: 20075913
[22] Severin A J, Woody J L, Bolon Y T, Joseph B, Diers B W, Farmer A D, Muehlbauer G J, Nelson R T, Grant D, Specht J E, Graham M A, Cannon S B, May G D, Vance C P, Shoemaker R C . RNA-Seq atlas of Glycine max: a guide to the soybean transcriptome. BMC Plant Biol, 2010,10:160
doi: 10.1186/1471-2229-10-160 pmid: 3017786
[23] Van Wyk S G, Du Plessis M, Cullis C A, Kunert K J, Vorster B J . Cysteine protease and cystatin expression and activity during soybean nodule development and senescence. BMC Plant Biol, 2014,14:294
doi: 10.1186/s12870-014-0294-3
[24] Yuan S, Li R, Wang L, Chen H, Zhang C, Chen L, Hao Q, Shan Z, Zhang X, Chen S, Yang Z, Qiu D, Zhou X . Search for nodulation and nodule development-related cystatin genes in the genome of soybean (Glycine max). Front Plant Sci, 2016,7:1595
[25] Ke D X, Li X Y, Han Y P, Cheng L, Yuan H Y, Wang L . ROP6 is involved in root hair deformation induced by Nod factors in Lotus japonicus. Plant Physiol Biochem, 2016,108:488-498
doi: 10.1016/j.plaphy.2016.08.015 pmid: 27592173
[26] 柯丹霞, 李祥永, 彭昆鹏, 韩雅彭 . 百脉根Rac1基因启动子的克隆与表达分析. 信阳师范学院学报(自然科学版), 2018,31:46-51
Ke D X, Li X Y, Peng K P, Han Y P . Cloning and expression analysis of the promoter region of Rac1 gene of Lotus japonicus. J Xinyang Normal Univ (Nat Sci Edn), 2018,31:46-51 (in Chinese with English abstract)
[27] Li Y, Zhou L, Li Y, Chen D, Tan X, Lei L, Zhou J . A nodule-specific plant cysteine proteinase, AsNODF32, is involved in nodule senescence and nitrogen fixation activity of the green manure legume Astragalus sinicus. New Phytol, 2008,180:185-192
[28] Sheokand S, Dahiya P, Vincent J L, Brewin N J . Modified expression of cysteine protease affects seed germination, vegetative growth and nodule development in transgenic lines of Medicago truncatula. Plant Sci, 2005,169:966-975
doi: 10.1016/j.plantsci.2005.07.003
[29] Vincent J L, Brewin N J . Immunolocalization of a cysteine protease in vacuoles, vesicles, and symbiosomes of pea nodule cells. Plant Physiol, 2000,123:521-530
doi: 10.1104/pp.123.2.521
[30] Pfeiffer N E, Torres C M, Wagner F W . Proteolytic activity in soybean root nodules: activity in host cell cytosol and bacteroids throughout physiological development and senescence. Plant Physiol, 1983,71:797-802
[31] Zhang X, Liu S, Takano T . Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing the salt, drought, oxidation and cold tolerance. Plant Mol. Biol, 2008,68:131-143
[32] Quain M D, Makgopa M E, Cooper J W, Kunert K J, Foyer C H . Ectopic phytocystatin expression increases nodule numbers and influences the responses of soybean (Glycine max) to nitrogen deficiency. Phytochemistry, 2015,112:179-187
[33] 柯丹霞, 李祥永 . 结瘤信号途径中相关调控蛋白的研究进展. 信阳师范学院学报(自然科学版), 2015,28:621-626
doi: 10.3969/j.issn.1003-0972.2015.04.038
Ke D X, Li X Y . Research progress of key regulatory proteins in nodulation pathway. J Xinyang Normal Univ (Nat Sci Edn), 2015,28:621-626 (in Chinese with English abstract)
doi: 10.3969/j.issn.1003-0972.2015.04.038
[34] Ke D X, Fang Q, Chen C F, Zhu H, Chen T, Chang X J, Yuan S L, Kang H, Ma L, Hong Z L, Zhang Z M . Small GTPase ROP6 interacts with NFR5 and is involved in nodule formation in Lotus japonicus. Plant Physiol, 2012,159:131-143
[1] 姚术, 郭凯悦, 翟慧慧, 姚佳慧, 邓文琪, 闫玲, 黄驰, 高阳, 俞嫣然, 赵振邦, 李英慧, 王晓波, 李佳佳. 大豆苗期耐低铁综合评价及优异种质筛选[J]. 作物学报, 2026, 52(5): 1373-1387.
[2] 张晴, 杨昱, 郭茜, 岳霈尧, 殷丛丛, 牛景萍, 赵晋忠, 杜维俊, 岳爱琴. 大豆GmARA6a的克隆及响应盐胁迫的功能分析[J]. 作物学报, 2026, 52(2): 480-493.
[3] 贺红利, 张雨涵, 杨静, 程云清, 赵杨, 李星诺, 司洪亮, 张兴政, 杨向东. 大豆e1-as基因突变体的创制及生理分析[J]. 作物学报, 2025, 51(8): 2228-2239.
[4] 王克晶, 李向华. 我国珍稀的大豆属多年生烟豆和短绒野大豆物种遗传资源濒危性评估分析[J]. 作物学报, 2025, 51(8): 2009-2019.
[5] 孟然, 李赵嘉, 冯薇, 陈悦, 刘路平, 杨春燕, 鲁雪林, 王秀萍. 大豆不同生育时期耐盐性综合评价及耐盐种质筛选[J]. 作物学报, 2025, 51(8): 1991-2008.
[6] 胡蒙, 沙丹, 张晟瑞, 谷勇哲, 张世碧, 李静, 孙君明, 邱丽娟, 李斌. 大豆分枝数QTL定位及候选基因筛选[J]. 作物学报, 2025, 51(7): 1747-1756.
[7] 王琼, 邹丹霞, 陈兴运, 张威, 张红梅, 刘晓庆, 贾倩茹, 魏利斌, 崔晓艳, 陈新, 王学军, 陈华涛. 大豆开花时间和成熟期性状全基因组关联分析与候选基因预测[J]. 作物学报, 2025, 51(6): 1558-1568.
[8] 殷丛丛, 李睿琦, 岳霈尧, 李晨, 牛景萍, 赵晋忠, 杜维俊, 岳爱琴. 基于闭合哑铃介导等温扩增可视化检测大豆花叶病毒SC15方法的建立及应用[J]. 作物学报, 2025, 51(5): 1248-1260.
[9] 许睿, 何妙华, 王昊, 李卫, 任杰, 夏志强. 基于空间转录组技术解析大豆种胚对X射线辐射的响应机制[J]. 作物学报, 2025, 51(12): 3121-3132.
[10] 林洋, 史晓蕾, 陈强, 刘兵强, 杨庆, 于慧娟, 闫龙, 武小霞, 杨春燕. 大豆蛋白质脂肪及脂肪酸组分相关QTL定位[J]. 作物学报, 2025, 51(11): 2899-2910.
[11] 王浩辰, 王克晶, 韩娟, 李向华. 东南沿海短绒野大豆两种代表性生境自然种群的空间遗传结构特征:种群内取样策略研究[J]. 作物学报, 2025, 51(11): 2875-2885.
[12] 李威, 朱玉鹏, 孙宾成, 温有祥, 吴宗声, 徐一帆, 宋雯雯, 徐彩龙, 吴存祥. 转基因大豆结合免耕平作实现东北地区大豆生产轻简化[J]. 作物学报, 2025, 51(10): 2738-2749.
[13] 陈敏, 贾蓉, 张金传, 张辰煜, 褚俊聪, 姚伟, 葛军勇, 王星宇, 杨亚东, 曾昭海, 臧华栋. 半干旱区燕麦与豆科作物带状复合种植的产量优势及氮素利用特征研究[J]. 作物学报, 2025, 51(10): 2727-2737.
[14] 钱玉平, 宿兵兵, 高吉星, 阮粉花, 李亚伟, 茅林春. 玉米大豆间作对喀斯特区土壤理化性质及微生物碳代谢特征的影响[J]. 作物学报, 2025, 51(1): 273-284.
[15] 丁树启, 程彤, 王弼琨, 于德彬, 饶德民, 孟凡钢, 赵胤凯, 王晓慧, 张伟. 密植对不同年代大豆品种群体光合生产和产量形成的影响[J]. 作物学报, 2025, 51(1): 161-173.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2