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

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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
  • Contact: Xu Ruhong E-mail:920848894@qq.com;xrhgz@163.com

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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"

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