Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2015, Vol. 41 ›› Issue (07): 1056-1063.doi: 10.3724/SP.J.1006.2015.01056

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Screening of AhCaM-Interactive Proteins in Peanuts Using Yeast Two Hybrid System

YANG Sha1,LI Yan1,GUO Feng1,ZHANG Jia-Lei1,MENG Jing-Jing1,LI Meng2,WAN Shu-Bo1,*,LI Xin-Guo1,*   

  1. 1 Biotechnology Research Center of Shandong Academy of Agricultural Sciences / Shandong Provincial key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Ji’nan 250100, China; 2 Human Resources Department of Shandong Academy of Agricultural Sciences Ji’nan 250100, China
  • Received:2014-11-06 Revised:2015-05-04 Online:2015-07-12 Published:2015-05-15
  • Contact: 万书波, E-mail: wansb@saas.ac.cn; 李新国, E-mail: lixinguo@tom.com E-mail:yangsha0904@126.com

Abstract:

Calmodulin is the most important known receptor of intracellular Ca2+, which is involved in regulation of many physiological activities. To further study the mechanism of CaM, we isolated and identified proteins that interact with AhCaM by yeast two-hybrid system. The pGBKT7-AhCaM bait vector without toxicity or auto-activation was firstly constructed with the peanut CaM gene. Then, the total RNA of peanut leaf was extracted and the mRNA was isolated, purified to be used as the templates to synthesize ds-cDNA by SMART technology. Ds-cDNA was amplified by long distance PCR. At last, the CaM interaction proteins were screened through co-transformation with bait vector and cDNA prey library. In the peanut cDNA library, five proteins interacting with AhCaM were identified. Among them, NAD kinase was a well-known protein to interact with CaM protein, and ubiquitin could interact with AhCaM, playing a role in development and stress resistance. The significant functional correlation between AhCaM and its interacting proteins peanut will help to elucidate the possible mechanisms of AhCaM in improving the tolerance of transgenic plants.

Key words: cDNA library of peanut leaf, AhCaM, Ubiquitin, Yeast two hybrid system, Protein interaction

[1]Hepler P K. Calcium: A central regulator of plant growth and development. Plant Cell, 2005, 17: 2142–2155



[2]张海平. 钙调控花生(Arachis hypogaea L.)生长发育的细胞生理机制研究. 福建农业大学硕士论文, 福建福州, 2003



Zhang H P. Study on Physiology and Eytology Mechanism of Peanut (Arachis hypogaea L.) Growth and Development Regulated by Calcium. MS Thesis of Fujian Agriculture and Forestry University, Fuzhou, China, 2003 (in Chinese with English abstract)



[3]Snedden W A, Fromm H. Calmpdulin as a versatile calcium signal transducer in plants. New Phytol, 2001, 151: 35–66



[4]Krebs J, Heizmann C W. Calcium-binding proteins and the EF-hand principle. New Compr Biochem, 2007, 41: 51–93



[5]Vetter S W, Leclerc E. Novel aspects of calmodulin target recognition and activation. Eur J Biochem, 2003, 270: 404–414



[6]Shimoda Y, Han L, Yamazaki T. Rhizobial and fungal symbioses show different requirements for calmodulin binding to calcium calmodulin-dependent protein kinase in lotus japonicus. Plant Cell, 2012, 24: 304–321



[7]韦慧彦, 郭振清, 崔素娟. 钙不依赖性钙调素结合蛋白的研究进展. 生物化学与生物物理进展, 2007, 34: 124–131



Wei H Y, Guo Z Q, Cui S J. Aro calmodulin and Ca-independent calmodulin-binding proteins. Prog Biochem Biophys, 2007, 34: 124–131 (in Chinese with English abstract)



[8]Ranty B, Aldon D, Galaud J P. Plant calmodulins and calmodulin-related proteins. Plant Signal Behav, 2006, 1: 96–104



[9]Liu H T, Li G L, Chang H, Sun D Y, Zhou R G, Li B. Calmodulin-binding protein phosphatase PP7 is involved in thermo-tolerance in Arabidopsis. Plant Cell Environ, 2007, 30: 156–164



[10]Bouche N, Yellin A, Snedden W A, Fromm H. Plant-special calmodulin-binding preteins. Annu Rev Plant Biol, 2005, 56: 435–466



[11]Yoo J H, Park C Y, Kim J C. Direct interaction of a divergent CaM isoform and the transcription factor, MYB2, enhances salt tolerance in Arabidopsis. J Biol Chem, 2005, 280: 435–466



[12]Yang T B, Chaudhuri S, Yang L H, Du L Q, Poovaiah B W. A calcium/calmodulin regulated member of the receptor-like kinase family confers cold tolerance in plants. J Biol Chem, 2010, 285: 7119–7126



[13]Yang T B, Shad Ali G, Yang L H, Du L Q, Reddy A S N, Poovaiah B W. Calcium/ calmodulin regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants. Plant Signal Behav, 2010, 5: 991–994



[14]Yang S, Wang F, Guo F, Meng J J, Li X G, Dong S T, Wan S B. Exogenous calcium alleviates photoinhibition of PSII by improving the xanthophyll cycle in peanut (Arachis Hypogaea) leaves during heat stress under high irradiance. PLOS One, 2013, 8: e71214



[15]郭佳岩, 宋婀莉, 马素参, 高友鹤. 快速LacZ检测法提高酵母双杂交试验的敏感度. 生物技术, 2004, 11(4): 31–33



Guo J Y, Song E L, Ma S S, Gao Y H. Rapid LacZ screening method increases the sensitivity of yeast two-hybrid screening. Biotechnology, 2004, 11(4): 31–33 (in Chinese with English abstract)



[16]孟玉环, 单世华, 李春娟, 庄伟建, 蔡宁波. 花生(Arachis hypogaea L.) CaM基因克隆与序列分析. 花生学报, 2007, 36(2): 11–15



Meng Y H, Shan S H, Li C J, Zhuang W J, Cai N B. Cloning of two genomics genes and sequence analysis encoding calmodulin from peanut (Arachis hypogaea L.). J Peanut Sci, 2007, 36(2): 11–15 (in Chinese with English abstract)



[17]韩亮, 张新军, 黄世思, 翟永功, 常智杰. 黑色素瘤相关抗原MAAT1 p15与LRP6的相互作用及其对Wnt信号通路的调控. 中国生物化学与分子生物学报. 2004, 20: 827–832



Han L, Zhang X J, Huang S S, Zhai Y G, Cahng Z J. MAAT1 p15 interacts with LRP6 and regulates Wnt signaling pathway. Chin J Biochem Mol Biol, 2004, 20: 827–832 (in Chinese with English abstract)



[18]Yin Y, Vafeados D, Tao Y. A new class of transcription factors mediates brassino steroid-regulated gene expression in Arabidopsis. Cell, 2005, 120: 249–259



[19]Roberts D M. Calcium-modulated proteins: targets of intracellular calcium signals in higher plant. Annu Rev Plant Physiol Plant Mol Biol, 1992, 43: 3753–414



[20]Jarrett H W, Brown C J, Black C C, Cormiergy M J. Evidence that calmodulin is in the chloroplast of peas and serves as regulatory role in photosynthesis. J Biol Chem, 1982, 257: 13795–13804



[21]Newmeyer D D, Ferguson-Miller S. Mitochondria: Releasing power for life and unleashing the machineries of death. Cell, 2003, 112: 481–490



[22]Lemasters J J, Qian T, Bradham C A, Brenner D A, Cascio W E, Trost L C, Nishimura Y. Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death. J Bioenerg Biomembr, 1999, 31: 305–319



[23]Hajnoczky G, Csordas G, Yi M. Old players in a new role: mitochondria-associate membranes, VDAC, and ryanodine receptors as contributors to calcium signal propagation from endoplasmic reticulum to the mitochondria. Cell Calcium, 2002, 32: 363–377



[24]Smalle J, Kurepa J, Yang P, Emborg T J, Babiychuk E, Kushnir S, Vierstra R D. The pleiotropic role of the 26S proteasome subunit RPN10 in Arabidopsis growth and development supports a substrate-specific function in abscisic acid signaling. Plant Cell, 2003, 15: 965–980



[25]Belknap W R, Garbarino J E. The role of ubiquitin in plant senescence and stress responses. Trends Plant Sci, 1996, 1: 331–335



[26]Patrick G N, Zukerberg L, Nikolic M, de la Monte S, Dikkes P, Tsai L H. Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature, 1999, 402: 615–622



[27]Abdel-Hamid M I, Khairou K S, Hassan R M. Kinetics and mechanism of permanganate oxidation of pectin in acid perchlorate media. Eur Polymer J, 2003, 39: 381–387



[28]Tanaka K. Proteasome: structure and biology. J Biochem, 1998, 123: 195–204



[29]Boyes D C, Nam J, Dangl J L. The Arabidopsis thaliana RPM1 disease resistance gene product is a peripheral plasma membrane protein that is degraded coincident with the hypersensitive response. Proc Natl Acad Sci USA, 1998, 95: 15849–15854



[30]Fu Q T, Li S J, Yu D Q. Identification of an Arabidopsis Nodulin-related protein in heat stress. Mol Cells, 2010, 29: 77–84

[1] Cui Zhi-Yuan, Qin Chen-Zhan, Liu Xing-Yu, Zhang Hai, Zeng Kang, Huang Guo-Qiang, Xu Jing-Sheng. Interaction between the sugarcane tetraspanin-like protein ScTSPAN18 and 6K2 in response to SCMV infection [J]. Acta Agronomica Sinica, 2026, 52(6): 1618-1630.
[2] LIU Xiao-Ning, ZHANG Ying, CAI Man-Lei, MA Hao, MIAO Zhi-Bo, CAO Ning, LIAN Rong-Fang, XU Quan-Le. Regulation of ODAP levels in Lathyrus sativus L. via interaction between LsSAT2 and LsAAE3 [J]. Acta Agronomica Sinica, 2025, 51(8): 2220-2227.
[3] LI Fu-Yuan, YANG Yi, MA Ji-Qiong, XU Ming-Hui, LIN Liang-Bin, SUN Yi-Ding. Cloning, hormone-induced expression analysis, and interaction protein screening of OsPUB4 in rice [J]. Acta Agronomica Sinica, 2025, 51(6): 1690-1700.
[4] WANG Qing, WANG Yi-Xiu, LI Yue-Nan, LYU Yong-Hui, ZHANG Hai-Bo, LIU Na, CHENG Hong-Yan. Differences in transcriptomic responses to cadmium stress in high/low-Cd- accumulation wheat [J]. Acta Agronomica Sinica, 2025, 51(5): 1230-1247.
[5] ZHOU En-Qiang, MIAO Ya-Mei, ZHOU Yao, YAO Meng-Nan, ZHAO Na, WANG Yong-Qiang, ZHU Yu-Xiang, XUE Dong, LI Zong-Di, SHI Yu-Xin, LI Bo, WANG Kai-Hua, GU Chun-Yan, WANG Xue-Jun, WEI Li-Bin. Analysis of bZIP gene family and identification of seed development candidate genes in pea based on seed development transcriptome [J]. Acta Agronomica Sinica, 2025, 51(4): 914-931.
[6] YU Quan-Xin, YANG Zong-Tao, ZHANG Hai, CHENG Guang-Yuan, JIAO Wen-Di, ZENG Kang, LUO Ting-Xu, HUANG Guo-Qiang, WANG Lu, XU Jing-Sheng. Interaction between calmodulin-like ScCML13 of sugarcane and SCMV movement protein P3N-PIPO [J]. Acta Agronomica Sinica, 2024, 50(7): 1855-1866.
[7] WANG Lian-Nan, LI Yuan-Chao, YU Nai-Tong, MAI Wei-Tao, LI Ya-Jun, CHEN Xin. Functional identification of MeTCP3a transcription factor in cassava leaf development [J]. Acta Agronomica Sinica, 2024, 50(11): 2720-2730.
[8] YU Quan-Xin, YANG Zong-Tao, ZHANG Hai, CHENG Guang-Yuan, ZHOU Ying-Shuan, JIAO Wen-Di, ZENG Kang, LUO Ting-Xu, HUANG Guo-Qiang, ZHANG Mu-Qing, XU Jing-Sheng. Interaction of sugarcane VAMP associated protein ScPVA12 with SCMV P3N-PIPO [J]. Acta Agronomica Sinica, 2023, 49(9): 2472-2484.
[9] BAI Cheng-Cheng, YAO Xiao-Yao, WANG Yu-Lu, WANG Sai-Yu, LI Jin-Ying, JIANG You-Wei, JIN Shu-Rong, CHEN Chun-Jie, LIU Yu, WEI Xing-Yue, XU Xin-Fu, LI Jia-Na, NI Yu. Cloning of genes involved in cuticular very-long-chain alkane synthesis and its interaction with BnCER1-2 in Brassica napus [J]. Acta Agronomica Sinica, 2023, 49(4): 1016-1027.
[10] DU Juan, PENG Xiao-Jun, HOU Juan, LIU Teng-Fei, LIU Zeng, SONG Bo-Tao. Identification of potato amylase StBAM9 interacting protein and analysis of the interaction mechanism [J]. Acta Agronomica Sinica, 2023, 49(10): 2643-2653.
[11] YANG Zong-Tao, JIAO Wen-Di, ZHANG Hai, ZHANG Ke-Ming, CHENG Guang-Yuan, LUO Ting-Xu, ZENG Kang, ZHOU Ying-Shuan, XU Jing-Sheng. Interaction of sugarcane glutathione S-transferase ScGSTF1 with P3N-PIPO in response to SCMV infection [J]. Acta Agronomica Sinica, 2023, 49(10): 2665-2676.
[12] YANG Zong-Tao, LIU Shu-Xian, CHENG Guang-Yuan, ZHANG Hai, ZHOU Ying-Shuan, SHANG He-Yang, HUANG Guo-Qiang, XU Jing-Sheng. Sugarcane ubiquitin-like protein UBL5 responses to SCMV infection and interacts with SCMV-6K2 [J]. Acta Agronomica Sinica, 2022, 48(2): 332-341.
[13] LIU Shu-Xian, YANG Zong-Tao, CHENG Guang-Yuan, ZHANG Hai, ZHOU Ying-Shuan, SHANG He-Yang, HUANG Guo-Qiang, XU Jing-Sheng. Interaction of sugarcane main facilitator superfamily member ScZIFL1 with 6K2 in response to Sugarcane mosaic virus infection [J]. Acta Agronomica Sinica, 2022, 48(12): 3080-3090.
[14] XU Bin, CAO Shao-Yu, SU Tian, PENG Meng-Ling, LYU Xia, LI Zhen-Lin, ZHANG Guo-Ping, XU Jun-Qiang. Interactions between CMLs and NPG1 and related proteins in pollen germination of Brassica oleracea L. var. capitata [J]. Acta Agronomica Sinica, 2022, 48(11): 2934-2944.
[15] ZHANG Hai, CHENG Guang-Yuan, YANG Zong-Tao, LIU Shu-Xian, SHANG He-Yang, HUANG Guo-Qiang, XU Jing-Sheng. Sugarcane PsbR subunit response to SCMV infection and its interaction with SCMV-6K2 [J]. Acta Agronomica Sinica, 2021, 47(8): 1522-1530.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!