Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (13): 2442-2461.doi: 10.3864/j.issn.0578-1752.2018.13.002

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

Identification and Expression Analysis of CRK Gene Family in Upland Cotton

ZHANG ZhongQi1, WANG Jiao1,2, JIN Wei1, GE DongDong1, LIU Kang1, LÜ FenNi1, SUN Jing1   

  1. 1National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095; 2Taiyuan University of Technology, Taiyuan 030024
  • Received:2018-02-14 Online:2018-07-01 Published:2018-07-01

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Abstract: 【Objective】 The cysteine-rich receptor kinase (CRK) is one of the largest family of receptor-like kinases in plants, and plays important roles in plant growth and development, hormone signal transduction and stress tolerance. So far, few cotton CRK genes have been reported. In silico identification, bioinformatics and expression analysis of CRK family genes in upland cotton on whole genome level lays the foundation for in-depth study and utilization of CRK family genes in cotton. 【Method】 The conserved stress-antifung domain sequence downloaded from the Pfam database was used as query sequence to search the Gossypium hirsutum (cv. TM-1) genome database to identify cotton CRKs by using BLASTp program; the theoretical isoelectric point and molecular weight, signal peptide, transmembrane domain, subcellular localization of cotton CRK proteins was predicted applying Compute pI/Mw, SignalP, TMHMM Server V2.0, WoLF POSRT online program respectively; amino acid sequence alignment of CRK proteins in cotton and Arabidopsis thaliana was performed using ClustalX1.8 software. phylogenetic relationships of cotton and Arabidopsis CRK proteins were analyzed with MEGA5.0; the chromosome location, gene structure and conserved domain were visualized with TBtools. The promoter sequences of cotton CRK genes were In silico analyzed by searching PlantCARE database; phosphorylation sites were predicted with PlantPhos. RNA-Seq data were download the from the NCBI database, TPM values were calculated using transcriptome quantification tool Kallisto, the heatmap of CRK gene expression was drawn with online tools Morpheus. 【Result】 There are 70 CRK genes in upland cotton genome distributed on 14 chromosomes, 52 genes (74.3% of the total) are intensively distributed in clusters on A6/D6, A9/D9 and A10/D10 chromosomes characterized by collinear relationships between A/D chromosomes. These CRK genes encode proteins containing 302-901 amino acids, 58 proteins (82.9%) have a transmembrane domain, mainly located in the chloroplasts, plasmalemma and extracellular. Phosphorylation site prediction results showed that cotton and Arabidopsis CRK share 5 consensus phosphorylation sites, including three serine phosphorylation motifs and two threonine phosphorylation motifs. The promoter regions of 65 cotton CRK genes (account for 92.9%) contain at least one stress hormone response element, and 69 (98.6%) genes contain at least one biotic or abiotic stress response element. RNA-Seq data analyses showed that the tissue expression patterns of CRKs could be divided into three types, and that the expression of some CRK genes were altered in response to salt, drought, cold, heat stress and inoculation with Verticillium dahliae Kleb. GhCRK25 was predominantly expressed in roots, stems, leaves, and ovules, but barely accumulated in fibers. ABA, GA3, SA, PEG-6000, NaCl, and Verticillium dahliae Vd991 can stimulate rapid up-regulation of GhCRK25 expression. GhCRK25-silenced cottonby using virus induced gene silencing technology (VIGS) showed increased susceptibility to Verticillium dahliae Vd991. 【Conclusion】 There are 70 members of CRK family gene in the upland cotton genome. They have conserved gene structure and functional domain, diverse tissue expression characteristics; most of cotton CRK genes are responsive to hormone and stress stimulus.

Key words: upland cotton, cysteine-rich protein kinase, gene family, stress tolerance, GhCRK25

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