Identification of LcCDPKs and analysis of their expression patterns in response to downy mildew stresses in lychee

Abstract:【Objective】Lychee (Litchi chinensis Sonn.) is a delicious and nutritional fruit widely accepted by consumers. However, the fruits are highly susceptible to various diseases. Lychee downy blight is one of the major diseases in lychee. The calcium dependent protein kinases (CDPKs) play vital roles in regulating plant growth, development and response to abiotic or biotic stresses. However, the potential role of CDPK gene family in lychee (LcCDPK) have not been reported. Here, we aimed to identify and analyze the CDPK gene family from the lychee genome with bioinformatic technology, and investigate the phylogeny, protein structure, expression patterns and response of CDPK gene in lychee under lychee downy mildew stress. This study would provide a basis foundation for further functional characterization of the lychee CDPK genes.【Methods】The lychee genome was used to identify and analyze theLcCDPK genes by bioinformatics using a local BLASTP search of TBtool software. All putative candidates were manually verified with the InterProScan program to confirm the presence of the protein kinase domain and the CaM domain. The length of sequences, isoelectric point (pI), molecular weights (MW) and predication subcellular location of the LcCDPKs were estimated by ExPASy Compute pI/ Mw tool. The multiple alignments of amino acid sequences were performed using MEGA-X program. The neighbor-joining phylogenetic tree was constructed via applying the MEGA6 program, with bootstrapping set at 1000 replications. The gene structure of the LcCDPKs were analyzed using the Gene Structure Display Server (GSDS) program with default settings. The LcCDPK genes were mapped to lychee chromosomes based on physical location from the database of the lychee genome using MapChart. The cis- acting regulatory elements located in a region upstream 2 kb to the start codon of LcCDPK genes were identified using the PlantCARE database, and visualized with TBtools. The germplasm used in this study contained 276 lychee landraces from different geographic areas. This population was selected for the present study because the collection was representative of the diverse genetic variation in lychee. These 276 germplasm materials were provided by Institute of Fruit Trees, Guangdong Academy of Agricultural Sciences and were planted in National Fruit Tree Germplasm, Guangzhou Lychee Nursery. The pathogen of P. litchii was provided by South China Agricultural University. P. litchi were cultured in juice agar (CJA) medium at 27 ℃. The spore suspension was filtrated using double sterile layer and adjusted to 104 spores mL-1 for inoculation. For transcription analysis, the leaves of Yurong 1 (YR1) lychee were inoculated with 5 μL sporangia suspension of P. litchii (104 spores·mL-1 ) or a mock suspension (sterile water), and then the leaves were collected at the indicated time points. All tissues were immediately placed in liquid nitrogen and stored at -80 ℃. The sequencing data of lychee were available from NCBI Sequence Read Archive database under the accession number PRJNA747875. The expression levels of the LcCDPK genes were analyzed in various tissues, including female flowers ovary, root, pericarp, aril, embryo, epicarp, male flowers anther, leaf and seed. The heat map with hierarchical clustering of the LcCDPK genes were constructed using MeV4.9 software by average linkage with Euclidean distance method, to visualize the expression levels in nine tissues based on the log10 (FPKM+1) values of the LcCDPK genes. Based on the transcriptome data, the expression of the CDPK was analyzed and verified by qRT- PCR.【Results】This study identified and comprehensively analyzed the CDPK family genes based on the whole genome data of lychee. The sequence characteristics, gene structure, promoter cis-acting elements, chromosome localization, evolutionary relationships and expression patterns in different tissues under the stress of lychee downy mildew were analyzed. A total of 19 CDPK family members were identified from lychee genome, which were distributed on 11 chromosomes. According to conserved domain and phylogenetic analysis, the 19 CDPK family members were divided into four subfamilies. The gene structure analysis indicated that the number of exon-intron ranged in the LcCDPKs from 7 to 19. The protein structure domain analysis showed that all LcCDPK proteins had 1- 4 EF-hand domains. The promoter cis-acting element analysis exhibited that LcCDPKs had a large number of biological and abiotic stress response elements. Tissue-specific expression analysis showed that many LcCDPKs genes could be detected in all tissues, while a few of LcCDPKs genes were tissue specific. The differential gene expression analysis of the LcCDPK genes in response to lychee downy mildew stresses helped us identify the LcCDPK5, LcCDPK17 and LcCDPK19 as the candidate genes for disease resistance in lychee, whose relative transcript abundance rapidly increased after lychee downy mildew infection.【Conclusion】A total of 19 CDPK family genes were identified in lychee genome. The expression of the LcCDPK genes in different tissues and their expression patterns in the response tolychee downy mildew stresses elucidated that the LcCDPK5, LcCDPK17, LcCDPK19, LcCDPK3 and LcCDPK8 might play an important role in the resistances of lychee to lychee downy mildew. Our study would provide critical foundation for further functional characterization of the lychee CDPK gene family.