Bioinformatics Analysis and Functional Verification of GhPKE1 inUpland Cotton

doi:10.13304/j.nykjdb.2020.1054

Abstract

Abstract:

To study the function of GhPKE1 in cotton， the structural characteristics of DNA sequence， physicochemical properties， hydrophobicity and secondary structure of GhPKE1， a heavy metal domain gene， were analyzed using bioinformatics method. And the virus-induced gene silencing （VIGS） carrier was constructed to infect cotton. The results showed that the length of GhPKE1 gene sequence was 1 048 bp， and its coden protein relative molecular weight was 27.54 kD， the isoelectric point was 9.3 and the instability coefficient was 35.56. After treated with Na₂SO₄ and CdCl₂， the relative expression levels of GhPKE1 in silencing plant were significant lower than those in CK， and the resistances of silencing plant were significant decreased comparing with CK. The above results indicated that GhPKE1 gene played an important role in the response of cotton to stress， which laid a foundation for further research on the stress resistance mechanism of cotton.

Key words: Gossypium hirsutum L．, GhPKE1, bioinformatics analysis, VIGS

摘要：

为研究棉花重金属结构域基因GhPKE1在棉花中的作用，采用生物信息学方法对GhPKE1 DNA序列的结构特性及其编码蛋白质的理化性质、疏水性和蛋白二级结构等进行分析，并根据GhPKE1的结构特点构建了GhPKE1的病毒诱导基因沉默（VIGS）载体侵染棉花。结果表明，GhPKE1基因序列全长1 048 bp，蛋白质相对分子质量27.54 kD，等电点9.3，不稳定系数35.56。分别用硫酸钠（Na₂SO₄）和氯化镉（CdCl₂）胁迫处理沉默GhPKE1的三叶期棉苗，处理后植株表现出明显的表型差异。qRT-PCR表达模式分析表明，用pYL156：GhPKE1侵染过的棉花中GhPKE1转录水平与对照相比显著降低；与对照相比，沉默GhPKE1棉花幼苗的抗Na₂SO₄和抗CdCl₂性显著减弱。综上所述，GhPKE1基因在棉花响应逆境胁迫过程中发挥重要作用，为进一步研究棉花的抗逆机制奠定基础。

关键词: 陆地棉, GhPKE1, 生物信息学分析, VIGS

CLC Number:

S562

Qinqin WANG, Xiugui CHEN, Xuke LU, Shuai WANG, Yuexin ZHANG, Yapeng FAN, Quanjia CHEN, Wuwei YE. Bioinformatics Analysis and Functional Verification of GhPKE1 inUpland Cotton[J]. Journal of Agricultural Science and Technology, 2022, 24(1): 38-45.

王琴琴, 陈修贵, 陆许可, 王帅, 张悦新, 范亚朋, 陈全家, 叶武威. 陆地棉GhPKE1的生物信息学分析及功能验证[J]. 中国农业科技导报, 2022, 24(1): 38-45.

Figures/Tables 6

Table 1 Primer sequence of GhPKE1

引物名称 Primer name	引物序列 Primer sequence （5’-3’）
KL-GhPKE1	F：GAGAACACGGGGGACTCTAGAATGGCTGAAAAGGTGACGATAATGG
KL-GhPKE1	R：CGATCGGGGAAATTCGAGCTCTTACATGATCGAGCAACCTTGTGGG
V-GhPKE1	F：AGAAGGCCTCCATGGGGATCCATGGCTGAAAAGGTGACGATAATGG
V-GhPKE1	R：GAGACGCGTGAGCTCGGTACCTCTTCTCACCGTCTTTGGGCTTTTC
q-GhPKE1	F：ACAAGTTATGTTACAAAGGTGGTGG
q-GhPKE1	R：TCTCAGCTTCTTTGGGTTTTTCCGG
GhHistone3 （AF024716）	F：TCAAGACTGATTTGCGTTTCCA
GhHistone3 （AF024716）	R：GCGCAAAGGTTGGTGTCTTC

Fig.1 Hydrophilicity/hydrophilicity analysis of GhPKE1protein

Fig.2 Prediction of transmembrane domain and signal peptide in GhPKE1 proteinA： Transmembrane domain analysis of GhPKE1 protein； B： Signal peptide prediction of GhPKE1 protein

Fig.3 GhPKE1 bioinformatics analysisA： Secondary structure of GhPKE1 protein； B： Prediction of GhPKE1 protein globulin region and disorder region； C： Secondary structure of GhPKE1 protein； D： Interaction network of GhPKE1 protein in Arabidopsis thaliana

Fig.4 Prediction of GhPKE1 phosphorylation sites and functional areaA： NetPhos 3.1 Server predicts GhPKE1 phosphorylation sites； B： PROSITE predicts GhPKE1 functional area

Fig.5 Cotton phenotype and the relative expression level of GhPKE1 after VIGS infectionA： Cotton phenotype under Na2SO4 stress for 12 h； B： Cotton phenotype under CdCl2 stress for 12 h； C： Relative expression level of GhPKE1 after Na2SO4 stress； D： Relative expression level of GhPKE1 after CdCl2 stress

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