Cloning,Subcellular Localization and Expression Analysis of GhCRK26 Gene in Cotton

doi:10.7668/hbnxb.20195920

Abstract

Abstract:

As an important subfamily of receptor kinases,cysteine-rich receptor kinases play a key role in plant growth and development.The aim of this study was to analyze the relationship between the GhCRK26 gene and the development of cotton fiber,and to provide a theoretical basis for the improvement of cotton fiber quality.This study selected upland cotton Line 9 and sea island cotton Xinhai 16,and collected samples at different periods of fiber development and root,stem and leaf tissues.The expression pattern of GhCRK26 was analyzed by qRT-PCR;the gene was cloned by PCR;a phylogenetic tree was constructed with the help of bioinformatics tools to predict the physicochemical properties of the protein,its transmembrane structure and signal peptide;the promoter cis-acting elements were analyzed by the PlantCare database;and the localization of the protein was clarified by the subcellular localization assay.The results showed that the expression of GhCRK26 gene showed highly significant differences at 10,20 and 30 days after flowering in two varieties;the highest expression levels were found in the leaves of Line 9,while in Xinhai 16,the expression of stem and leaves did not show significant differences.A 2 049 bp CDS sequence encoding 682 amino acids was successfully cloned.The evolutionary tree showed that GhCRK26 protein was the most distantly related to Hibiscus trionum,and the closest to Gossypium tomentosum and Gossypium barbadense;the protein was hydrophilic and unstable,containing a transmembrane structure and a signal peptide;methyl jasmonate and abscisic acid response elements were identified in the promoter region;and the subcellular localization confirmed that it was localized in the cell membrane.The above studies provide a basis for further in-depth analysis of the function of GhCRK26 gene in fiber development.

Key words: Cotton, Fiber, GhCRK26, Gene cloning, Expression analysis, Subcellular localization

CLC Number:

LI Jing, PANG Bo, ZHANG Ru, ZHAERJIAMALI Abudubieke, WANG Zhengrui, SHI Shunyu, FAN Zhenzhen, MA Jingjing, CHEN Jialin, SONG Wu, LI Shengmei, GAO Wenwei. Cloning,Subcellular Localization and Expression Analysis of GhCRK26 Gene in Cotton[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(6): 62-70. doi: 10.7668/hbnxb.20195920.

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Figures/Tables 13

Tab.1 Bioinformatics prediction websites

数据库/软件 Databases/softwares	预测功能 Predictive functions	网址 Website
Expasy	蛋白质理化性质预测	https://web.expasy.org/protparam/
ProtScale	氨基酸亲疏水性分析	https://prosite.expasy.org/
TMHMM 2.0	跨膜结构域预测	https://services.healthtech.dtu.dk/services/TMHMM-2.0/
SignalP-5	信号肽序列预测及剪切位点分析	https://services.healthtech.dtu.dk/services/SignalP-5.0/
NetPhos 3.1	磷酸化位点预测	https://services.healthtech.dtu.dk/services/NetPhos-3.1/
SOPMA	蛋白质二级结构预测	https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html
SWISS-MODEL	三级结构	https://www.swissmodel.expasy.org/
NCBI CD Search	保守结构域注释	https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
NCBI BLAST	同源序列比对与检索	https://blast.ncbi.nlm.nih.gov/Blast.cgi
CLUSTALW	多序列比对分析	https://www.genome.jp/tools-bin/clustalw^[23]
ESPript 3.0	多序列比对结果可视化美化	https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi
PlantCare	启动子顺式作用元件预测	https://bioinformatics.psb.ugent.be/webtools/plantcare/html/
EvolView	系统发育树可视化与美化	https://evolgenius.info//evolview-v2/#login
MEGA 11	进化树构建	—
TBtools	启动子序列提取	—

Fig.1 Expression of GhCRK26 in parents Different letters show significant difference at P<0.01 level.The same as Fig.2.

Fig.2 Expression of GhCRK26 in root,stem and leaf tissues of Line 9 and Xinhai 16

Fig.3 Cloning of the GhCRK26 gene M.DL2000 Marker;1.PCR amplification fragment of GhCRK26 gene.

Fig.4 Sequence comparison of GhCRK26 protein in different species

Fig.5 Phylogenetic tree analysis of GhCRK26 protein sequences from different species

Fig.6 Protein characteristics encoded by GhCRK26 gene A.Prediction of amino acid affinity and hydrophobicity of the GhCRK26-encoded protein; B.Prediction of the transmembrane region of the GhCRK26-encoded protein.

Fig.7 Signal peptide prediction and phosphorylation site prediction of the protein encoded by the GhCRK26 gene A.Signal peptide prediction of GhCRK26;B.Phosphorylation site prediction of GhCRK26.

Fig.8 Secondary structure and tertiary structure of the protein encoded by the GhCRK26 gene A.Secondary structure of GhCRK26;B.Tertiary structure of GhCRK26.

Fig.9 Analysis of conserved domains in GhCRK26

Fig.10 Cis-acting element analysis of the promoter region of the GhCRK26 gene

Fig.11 PHG∷GhCRK26 bacteriophage PCR detection A.PCR detection of PHG∷GhCRK26 bacterial solution by cloning primers:M.DL2000 Marker,1.PCR results of PHG∷GhCRK26 bacterial solution;B.PCR detection of specific primers for GhCRK26 bacterial solution:M.DL2000 Marker,1.PCR results of PHG∷GhCRK26 bacterial solution.

Fig.12 Subcellular localization of GhCRK26 protein The first line is the transformed tobacco cells by empty load and the second line is the cells transformed by the fusion vector.GFP corresponds to the fluorescence field of view,Bright field represents the bright field of view,and Merge shows the superimposed fusion field of fluorescence and bright field.

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