Crops ›› 2023, Vol. 39 ›› Issue (4): 77-84.doi: 10.16035/j.issn.1001-7283.2023.04.012

Previous Articles     Next Articles

Bioinformatics and Expression Analysis of GzCIPK7-5B Gene in Wheat

Zhao Pengpeng(), Li Luhua, Ren Mingjian, An Chang, Hong Dingli, Li Xin, Xu Ruhong()   

  1. College of Agriculture,Guizhou University/Guizhou Branch of National Wheat Improvement Center, Guiyang 550025,Guizhou, China
  • Received:2022-03-04 Revised:2022-05-06 Online:2023-08-15 Published:2023-08-15

RichHTML

6

PDF (PC)

92

Abstract:

CIPK is a kind of serine/threonine protein kinase specifically targeting calcineurin B similar protein in plant calcium receptors,which plays an important role in calcium ion signal transduction.To explore the function of GzCIPK7-5B gene in wheat,the GzCIPK7-5B gene from wheat variety‘Guizimai 1’was cloned by RT-PCR and analyzed by bioinformatics.RT-PCR was used to detect the expression of GzCIPK7-5B gene in different tissues (roots, stems, leaves and grains) of ‘Guizimai 1’.qRT-PCR was used to detect the expression levels of GzCIPK7-5B at three stages (10, 25, 35days post anthesis) of grain.The results showed that the length of GzCIPK7-5B open reading frame was 1296bp,encoding 431 amino acids. The protein contained a conserved domain of serine-threonine protein kinase family and had the characteristics of CIPKs family genes. The encoded protein contained 29 phosphorylation sites and had no transmembrane structure. It was an unstable hydrophilic nuclear protein without signal peptide. The sequence similarity of GzCIPK7-5B gene and TdCIPK7-5B of wild two-grain wheat was the highest, and the protein sequence homology was 100%.GzCIPK7-5B gene was expressed in roots, stems,leaves and grains. In the three important periods of anthocyanin synthesis of ‘Guizimai 1’,the expression levels of 25 and 35days post anthesis were significantly higher than that of 10days after flowering.

Key words: Wheat, GzCIPK7-5B gene, RT-PCR cloning, Bioinformatics, Gene expression

Table 1

Primers used in the experiment"

引物名称Name of primer 引物序列Sequence of primer 用途Usage
CIPK7-5B-F CCTCTAGAATGGCCGTCGCCAAGAGCA 基因克隆
CIPK7-5B-R CCCCCGGGTCACAATTCCTCGCATCCATGCCAC 基因克隆
CIPK7-5B-F CGTCTTCCTCCAGCTCGTCTCC qRT-PCR和RT-PCR
CIPK7-5B-R GATGAGGACGTTCTGCGGCTTG qRT-PCR和RT-PCR
Actin-F CCAAGGCGGAGTACGATGAGTCT qRT-PCR和RT-PCR
Actin-R TTCATACAGCAGGCAAGCACCAT qRT-PCR和RT-PCR

Table2

Softwares and usage of biological information analysis"

在线软件Online software 用途Usage
http://web.expasy.org/prot param/ 蛋白的理化性质
https://www.cbs.dtu.dk/services/SignalP-3.0/ 信号肽预测
https://web.expasy.org/protscale/ 亲疏水性预测
https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi 保守结构域预测
http://www.cbs.dtu.dk/services/ 磷酸化位点预测
https://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ 亚细胞定位预测
https://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ 二级结构预测
https://swissmodel.expasy.org/ 三级结构预测
MAGE 7.0 进化树构建
DNAMAN 8.0 多重序列比对

Fig.1

PCR amplification and electrophoresis of GzCIPK7-5B gene M: DL2000; 1: PCR product"

Fig.2

Amino acid composition of GzCIPK7-5B Ala: Alanine; Arg: Arginine; Asn: Asparagine; Asp: Aspartic acid; Cys: Cysteine; Gln: Glutamicacid; Glu: Glutamic acid; Gly: Glycine; His: Histidine; Ile: Isoleucine; Leu: Leucine; Lys: Lysine; Met: Methionine; Phe: Phenylalanine; Pro: Proline; Ser: Serine; Thr: Threonine; Trp: Tryp-tophan; Tyr: Tyrosine; Val: Valine"

Fig.3

Prediction of amino acid conservative domain in GzCIPK7-5B"

Fig.4

Prediction of signal peptide of GzCIPK7-5B protein"

Fig.5

Prediction of affinity and hydrophobicity of GzCIPK7-5B protein"

Fig.6

Prediction of transmembrane structure of GzCIPK7-5B protein"

Fig.7

Prediction of phosphorylation site of GzCIPK7-5B protein"

Fig.8

Prediction of secondary structure of GzCIPK7-5B protein"

Fig.9

Prediction of tertiary structure ofGzCIPK7-5B protein"

Fig.10Phy

logenetic tree analysis of GzCIPK7-5B gene"

Fig.11

Multiple sequence alignment of amino acids GzCIPK7-5B"

Fig.12

Expression profile analysis of GzCIPK7-5B"

Fig.13

Relative expression levels of GzCIPK7-5B in the critical period of anthocyanin synthesis in GZ1 grains “**”indicates the significant difference at the level of 0.01"

[1] 马瑞, 李世贵, 刘维刚, 等. 植物CBL-CIPK信号系统的功能及其响应非生物胁迫作用机制研究进展. 植物生理学报, 2021, 57(3):521-530.
[2] Wang Y, Li T, John S J, et al. A CBL-interacting protein kinase TaCIPK27 confers drought tolerance and exogenous ABA sensitivity in transgenic Arabidopsis. Plant Physiology Biochemistry, 2018, 123:103-113.
doi: 10.1016/j.plaphy.2017.11.019
[3] 许静, 高景阳, 李程成, 等. 过表达ZmCIPKHT基因增强植物耐热性. 作物学报, 2022, 48(4):851-859.
doi: 10.3724/SP.J.1006.2022.13013
[4] Wang R K, Li L L, Cao Z H, et al. Molecular clonging and fuctional characterization of a novel apple MdCIPK6L gene reveals its involvement in multiple abiotic stress tolerance in transgenic plants. Plant Molecular Biology, 2012, 79(1/2):123-135.
doi: 10.1007/s11103-012-9899-9
[5] Zhang Y M, Ling H J J, Dong W, et al. SiCBL4) interacts with SiCIPK24, modulates plant salt stress tolerance. Plant Molecular Reporter, 2017, 35(6):634-646.
[6] Song S J, Feng Q N, Li Chun L, et al. A Tonoplast-associated calcium-signaliling module dampens ABA signaling during stomatal movement. Plant Physiology, 2018, 177(4):1666-1678.
doi: 10.1104/pp.18.00377
[7] Sanya S K, Kanwar P, Yadav A K, et al. Arabidopsis CBL interacting protein kinase 3 interavts with ABR1, an APETALA2 domain transcription factor, to regulate ABA responses. Plant Science, 2017, 254:48-59.
doi: 10.1016/j.plantsci.2016.11.004
[8] Zhao J F, Sun Z F, Zheng J, et al. Cloning and characterization of a novel CBL-interacting protein kinase from maize. Plant Molecular Biology, 2009, 69(6):661-674.
doi: 10.1007/s11103-008-9445-y pmid: 19105030
[9] Mahajaan S, Sopory S K, Tuteja N. Cloning and characterization of CBL-CIPK signaling components fromalegume(Pisum sativum). FEBS Journal, 2006, 27, 3(5):907-925.
[10] Huang C L, Ding S, Zhang H, et al. CIPK 7 is involved in cold response by interacting with CBL1 in Arabidopsis thaliana. Plant Science, 2011, 181(1):57-64.
doi: 10.1016/j.plantsci.2011.03.011
[11] Qiu Q S, Gou Y, Detrich M, et al. Regulation of SOS1, a plasma membrane Na+/H+ exchanger in Arabidopsis thaliana, by SOS2 and SOS3. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(12):8436-8441.
[12] Xing Y, Huang Y, Xiong L Z. Characterization of stress-responsive CIPK genes in rice for stress toleranceimprovement. Plant Physiology, 2007, 144(3):1416-1428.
doi: 10.1104/pp.107.101295
[13] 晋霞.小麦盐胁迫响应基因TaCIPK25的功能研究. 武汉:华中科技大学, 2017.
[14] Tian Q Y, Zhang X X, Yang A, et al. CIPK 23 is involved in iron acquisition of Arabidopsis by affecting ferric chelate reductase activity. Plant Science, 2016, 246:70-79.
doi: S0168-9452(16)30010-3 pmid: 26993237
[15] Zhang X, Li Z C, Li X J, et al. CBL3 and CIPK 18 are required for the function of NHX5 and NHX6 in mediating Li+ homeostasis in Arabidopsis. Journal of Plant Physiology, 2020, 255:153295.
doi: 10.1016/j.jplph.2020.153295
[16] Peng H, Yang T, Whitaker BD, et al. Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP glucosyl- transferase involved in fruit anthocyanin biosynthesis. BMC Plant Biology, 2016, 16(1):197.
doi: 10.1186/s12870-016-0888-z pmid: 27609111
[17] Zong Y, Xi X, Li S, et al. Allelic variation and transcriptional isoforms of wheat TaMYC1gene regulating anthocyanin synthesis in pericarp. Plant Science, 2017, 8:1645.
[18] Yu Y H, Xia X L, Yin W L, et al. Comparative genomic analysis of CIPK gene family in Arbidopsis and Populus. Plant Growth Regulation, 2007, 52(2):101-110.
doi: 10.1007/s10725-007-9165-3
[19] Li L B, Zhang Y R, Liu K C, et al. Identtification and bioinfprmatics analysis of SnRK2 and CIPKfamily genes in Sorghum. Agricultural Sciences in China, 2010, 9(1):19-30.
doi: 10.1016/S1671-2927(09)60063-8
[20] Cui X Y, Du Y T, Fu T F, et al. Wheat CBL-interacting protein kinase 23 positively regulates drought stress and ABA responses. British Medical Council Plant Biology, 2018, 18(1):93.
[21] 余爱丽, 赵晋锋, 王高鸿, 等. 两个谷子CIPK基因在非生物逆境胁迫下的表达分析. 作物学报, 2016, 42(2):295-302.
[22] 冯志娟, 徐盛春, 刘娜, 等. CIPK基因对逆境胁迫及激素的响应特征. 植物遗传资源学报, 2017, 18(6):1168-1178.
doi: 10.13430/j.cnki.jpgr.2017.06.019
[23] 李亚坤, 陈乃钰, 杨晓雪, 等. 紫花苜蓿MsCIPK8基因的克隆与表达分析. 植物遗传资源学报, 2020, 21(2):491-499.
[24] Xu J, Li H D, Chen L Q, et al. A prontein kinase, interacting with two calcineurin B-like proteins, regulates K+transporter AKT1 in Arabidopsisi. Cell, 2006, 125:1347-1360.
doi: 10.1016/j.cell.2006.06.011
[25] Lee S C, Lan W Z, Kim B G, et al. A protein phosphorylation/dephosphorylation network regulates a plant potassium channel. Proceedings of the National Academy of Sciences, 2007, 104(40):15959-15964.
[26] Ho C H, Lin S H, Hu H C, et al. CHL1functions as a nitrate sensor in plants. Cell, 2009, 138:1184-1194.
doi: 10.1016/j.cell.2009.07.004
[27] 毕惠惠, 贺亚伟, 毛伟伟, 等. 小麦TaCIPK8基因的表达分析及其与TaCBLs的互作. 植物遗传资源学报, 2018, 19(2):296-304.
doi: DOI:10.13430/j.cnki.jpgr.2018.02.013
[28] 时丕彪, 洪立洲, 王军, 等. 藜麦CqCIPK7基因的克隆与表达分析. 江苏农业学报, 2020, 36(4):1068-1072.
[1] Zhang Mingwei, Ding Jinfeng, Zhu Xinkai, Guo Wenshan. Analysis of High-Yielding Planting Density and Nitrogen Application in Super-Late Sowing Wheat Following Rice [J]. Crops, 2023, 39(4): 126-135.
[2] Song Xiao, Zhang Keke, Yue Ke, Huang Chenchen, Huang Shaomin, Sun Jianguo, Guo Tengfei, Guo Doudou, Zhang Shuiqing, Pei Minnan. Differences of Enzyme Activities and Bacterial Communities in Rhizosphere Soil of Wheat Varieties with Different Nitrogen Efficiency [J]. Crops, 2023, 39(4): 188-194.
[3] Fu Xiaoyi, Wang Hongguang, Liu Zhilian, Li Dongxiao, He Mingqi, Li Ruiqi. Effects of Water Stress on Growth of Different Wheat Varieties at Seedling Stage and Selection of Drought Resistant Varieties [J]. Crops, 2023, 39(4): 224-229.
[4] Chen Yuanyuan, Li Guangsheng, Liu Yang, He Yuqi, Zhou Meiliang, Fang Zhengwu. Molecular Cloning and Functional Identification of Resistance Gene FtTIR of Tartary Buckwheat to Blight [J]. Crops, 2023, 39(4): 44-51.
[5] Liu Ying, Gu Yunyi, Zhang Weiyang, Yang Jianchang. Research Advances in the Effects of Water and Nitrogen and Their Interaction on the Grain Yield, Water and Nitrogen Use Efficiencies of Wheat [J]. Crops, 2023, 39(4): 7-15.
[6] Li Hongsheng, Li Shaoxiang, Yang Zhonghui, Yang Jiali, Liu Kun, Xiong Shian, Li Fuqian, Guo Hui, Yang Mujun. Comparison ofPhenotype and Marker Detection in Seed Purity of Thermo-Photo Sensitive Two-Line WheatHybrids [J]. Crops, 2023, 39(4): 71-76.
[7] Li Haoran, Li Ruiqi, Li Yanming. Review of the Changes of Wheat Row Spacing Forms and the Affecting Factors in Haihe Plain [J]. Crops, 2023, 39(3): 12-19.
[8] Li Junzhi, Chang Xuhong, Wang Demei, Wang Yanjie, Yang Yushuang, Zhao Guangcai. Effects of Nitrogen Application Levels on Yield and Quality of Different Strong Gluten Wheat Varieties [J]. Crops, 2023, 39(3): 148-153.
[9] Luo Siwei, Shi Xiunan, Jia Yonghong, Zhang Jinshan, Wang Kai, Li Dandan, Wang Runqi, Dong Yanxue, Shi Shubing. Effects of Drip Irrigation Capillary Spacing and Drop Spacing on Photosynthesis, Dry matter Accumulation, and Yield Formation of Uniformly Sown Winter Wheat [J]. Crops, 2023, 39(3): 230-237.
[10] Qiu Kaihua, Fang Shumei, Liang Xilong. Functional Analysis of SRRM1-Like Transcription Factor of Magnaporthe grisea [J]. Crops, 2023, 39(3): 246-253.
[11] Bai Kaihong, Abie Xiaobing, Xu Xiaoli, Jiang Na, Li Jianqiang, Luo Laixin. Analysis of Fungal Diversity in Seeds of Tartary Buckwheat from Liangshan, Sichuan Province [J]. Crops, 2023, 39(3): 260-266.
[12] Zhang Haibin, Wu Xiaohua, Yu Meiling, Wang Xiaobing, Ye Jun, Cui Siyu, Li Yuanqing, Wang Zhanxian, Zhang Hongxu, Xue Wei, Li Yan, Cui Guohui, Zhao Xuanwei, Liu Juan. AMMI Model Analysis of Grain Yield of Wheat Varieties (Lines) in Inner Mongolia Regional Trials [J]. Crops, 2023, 39(3): 27-34.
[13] Li Guangsheng, Lu Xiang, Lai Dili, Zhang Kaixuan, Wang Haihua, Zhou Meiliang. Molecular Cloning and Functional Analysis of Resistance Gene FtABCG12 of Tartary Buckwheat to Blight [J]. Crops, 2023, 39(3): 43-50.
[14] Li Jing, Li Pengcheng, He Yongbin, Xing Yaling, Meng Fanhua, Zhou Qian, Nan Ming. Multivariate Analysis and Comprehensive Evaluation of Main Characteristics of 16 Russian Winter Wheat Varieties [J]. Crops, 2023, 39(3): 58-65.
[15] Zhang Yufen, Qi Jingkai, Wang Guiling, Zhao Baoping, Zhou Lei. Study on Geographical Origin of Buckwheat Based on Mineral Element Fingerprint [J]. Crops, 2023, 39(3): 66-74.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!