Identification of CDPK family genes in Cili (Rosa roxburghii Tratt.) and its expression in response to calcium levels

Abstract：【Objective】Calcium-dependent protein kinases (CDPKs/CPKs) are a class of serine/threonine-type protein kinases, which are widespread Ca²⁺ sensors in plants. This work was aimed at identifying and analyzing the RrCDPK gene family in Cili (Rosa roxburghii Tratt.) and exploring its expression response to different calcium levels.【Methods】The cutting nursery trees of R. roxburghii Tratt.‘Guinong 5’with basically the same growth were selected as the experimental materials in April 2021 in the R. roxburghii Tratt. Resource Garden of the Agricultural College of Guizhou University. After literature review and a series of pre-experiment screening, this work set up 3 Ca2+ concentration gradients: 0, 0.5, 2 mmol· L^{- 1} and 3 sampling time points: 0, 1, 7 d to explore the response of RrCDPKs to no calcium, low concentration and high concentration calcium treatments. Additionally, based on R. roxburghii Tratt. genome, the RrCDPK gene family was identified and analyzed by bioinformatics methods. The Arabidopsis thaliana CDPKs (AtCDPKs) protein sequences downloaded from Tair database (https:// www.arabidopsis.org/) were used as queries to search against R. roxburghii Tratt. genome data. The pu-tative genes were identified based on a local BLASTP search in TBtools software (E-value＜1×10^-5 , bit score＞100). Combined with SMART and Pfam database for further screening, the genes with the protein kinase domain (Pfam database ID: PF00069) and the EF-hand domain (PF13499) were recognized as the final CDPK family members. With the help of ExPASy, WoLF PSORT, MEME, NCBI- CDD, iTOL and plantCARE online website, and MEGA7 and TBtools software, the physicochemical properties of the encoded proteins, the chromosome location, subcellular localization, gene structures, conserved motifs and conserved domains, phylogeny, promoter cis-acting elements were obtained. Finally, transcriptome sequencing and qRT- PCR were used to analyze the tissue expression specificity the RrCDPKs and their expression response under the different calcium levels.【Results】A total of 16 CDPK genes with serine/threonine protein kinase and EF- hand domains were identified from the R. roxburghii genome. They were randomly distributed on the other 6 chromosomes except for chromosome 4 and were named as the RrCDPK1-16 according to their chromosome location from the top to bottom. The structural analysis displayed that the gene length ranged from 1496 bp (RrCDPK6) to 5524 bp (RrCDPK14), the lengths of the proteins from 393 aa (RrCDPK6) to 561 aa (RrCDPK16), the molecular weights form 44.02 (RrCDPK6) ku-62.98 ku (RrCDPK16); RrCDPKs were hydrophilic proteins; except for RrCDPK5/8, other members were acidic proteins; unlike RrCDPK1/4/11/12/14/15, others were stable proteins. The gene structure was quite different, the number of exons was 2-10, and all the RrCDPKs included 6 conserved motifs. The subcellular localization predicted that the RrCDPKs were localized in the nucleus and various organelles, mainly in the cytoplasm. The RrCDPKs were divided into four subfamilies by evolutionary analysis and most closely related to those of Fragaria × ananassa Duch., followed by Malus pumila Mill., and farther than A. thaliana (L.) Heynh. and Oryza sativa L.. The analysis of promoter cis- acting elements in the upstream 2000 bp sequence showed that most of them contained light response elements, various hormone response elements and stress response elements. Transcriptome data from different organs and fruit developmental stages showed that the RrCDPKs had spatiotemporal expression specificity. Among them, the high expression of the RrCDPK3/5/10/ 13 in the stems and the RrCDPK4/8/9/15 in the leaves indicated that they might be related to vegetative growth. The RrCDPK2/8/11/15 was continuously up-regulated with fruit development, so that it reached the highest level at the later stage, which was consistent with the accumulation trend of flavonoids. The expression of the RrCDPK1/10/12/14, RrCDPK7 and RrCDPK3/4/5/13/16 was the highest in the early stage, the fruit expansion stage, and the early and middle stages of fruit development, respectively. Therefore, it was speculated that these RrCDPKs were involved in the regulation of various biological activities synthesis of substances. The response of the RrCDPKs to different calcium levels showed that after calcium supply, a few RrCDPKs showed obvious up-regulation effect: the expression levels of the RrCDPK4/8 in the leaves 1 day after the treatment and the RrCDPK1/2 in the roots 7 days after the treatment were significantly increased (p＜0.05) under 0.5 mmol· L^{- 1} Ca^{2 +} ; the RrCDPK4/10/12 in the leaves and the RrCDPK9 in the roots 1 day after the treatment, and the RrCDPK9 in the leaves and the RrCDPK2 in the roots 7 days after the treatment were significantly up-regulated under 2 mmol·L^-1 Ca2+ . It was suggested that the RrCDPKs in the leaves and roots could be expressed differently in response to the different calcium levels and treatment times.【Conclusion】Totally 16 RrCDPK genes were identified in the whole genome of R. roxburghii Tratt., and all of them contained the typical serine/threonine protein kinase and EF-hand domains. The expression of the RrCDPK genes in R. roxburghii were spatiotemporally specific, indicating that they might play an important role in the different organs and developmental stages of fruits. The RrCDPK family members might play different roles in response to the dif-ferent calcium levels, especially the RrCDPK1/2/4/8/9/10/12. The results could provide relevant information for further revealing the function of R. roxburghii Tratt. CDPK gene family and its response mechanism to the external calcium environment.