Identification of CDPK family genes and their response to abiotic stresses in Actinidia valvata

Abstract:【Objective】The experiment was conducted to identify the CDPK family genes in Actinidia valvata and analyze their expression patterns in different tissues and responses to salt and waterlogging stress【. Methods】Based on the full-length transcriptome sequencing data of third-generation RNA-seq, the CDPK family genes of Actinidia valvata were analyzed and identified by various bioinformatics methods. qRT-PCR was used to analyze the expression of these genes in stem, leaf, petiole, pedicel, se- pal and petal, as well as their expressions under different abiotic stresses. One year old KR5 kiwifruit plantlets (16 cm × 16 cm) under waterlogging stress were placed in a blue plastic turnover box (45 cm × 35 cm × 16 cm) filled with water. The water level was kept at 2 cm above the soil surface. Four time points of 0, 3, 7 and 11 d were set as sampling times. The root samples after waterlogging stress were harvested as experimental materials. KR5 potted plantlets (16 cm × 16 cm) were cultured in a plastic container (39 cm × 29 cm × 12 cm) filled with Hoagland nutrient solution. Oxygen was supplied by an oxygenerator. The concentration of salt treatment was 0.6% NaCl. The sampling time points were set as 0, 0.5, 1, 3, 5 and 7 d【. Results】A total of 63 CDPK genes, named as AvCDPK1-AvCDPK63, were iden- tified from the full-length transcriptome sequencing data of kiwifruit genotype KR5. They all have typi- cal characteristic domains: variable domain, catalytic domain (activator domain), junction domain (auto- inhibitory domain) and regulatory domain (calmodulin-like domain / CAM-LD). Furthermore, the CDS sequence length, relative molecular weight, isoelectric point, EF-hand structure, palmitoylation and my- ristoylation sites of AvCDPK family genes were analyzed. The CDS sequences of 63 AvCDPK genes family members ranged from 1068 bp to 1893 bp, with amino acid length ranging from 355 (AvCD- PK54 and 60) to 630 aa (AvCDPK18 and 19), molecular weight ranging from 40.10 to 70.97, and iso- electric point ranging from 5.10 to 9.13. Through statistical analysis, 53 AvCDPKs have four EF-hand domains, and 10 AvCDPKs have three EF-hand domains (AvCDPK3, 14, 23, 25, 28, 51, 59, 61, 62 and63). EF-hand domain is the site of recognition and binding of Ca2 +, which indicates that different mem- bers of AvCDPK family genes may play a different rolein change of Ca2 + concentrations. According to the prediction of 63 amino acid modification sites of AvCDPK proteins, 37 members have palmi- toylation site, 11 members have myristoylation site, and 15 members have both palmitoylation and my- ristoylation sites. Through phylogenetic analysis, same as AtCDPK(Arabidopsis thaliana) gene family,AvCDPKs were divided into four subfamilies. The genes in the same subfamily had similar gene struc- ture and motifs. In addition, we identified 15 conserved motifs, of which motif10 was distributed in sub- families III and IV, but not in II and some members of subfamilies I. Motif15 exists in subfamilies Iand III, while subfamilies II and IV are absent. Motif14 only exists in subfamily II. AvCDPK genes have obvious tissue-specific expression. For example, AvCDPK11 was highly expressed in leaves, butAvCDPK29, 41 and 63 were low expressed; AvCDPK43 was highly expressed in petioles, but low ex- pressed in sepals and flowers; AvCDPK36 was highly expressed in pedicels, and AvCDPK36, 38, 43 and53 were low expressed in petals. These results suggest that different AvCDPK gene may play different roles in growth and development of Actinidia valvata genotype KR5. The expression levels of AvCD- PK6, 11, 28, 44, 45, 49 and 61 were up-regulated under salt stress, and the expression levels of AvCD- PK36, 41, 44, 45, 46, 47, 48, 49 and 50 were up-regulated in waterlogging test. The expression levels ofAvCDPK44, 45 and 49 were up-regulated in both salt stress and waterlogging stress (all up-regulated more than 2 folds). The expression levels of AvCDPK2, 14, 21, 31 and 38 were down regulated by more than 10 folds under salt stress, and the expression levels of AvCDPK28, 30 and 31 were down-regulated by more than 5 folds under waterlogging stress. The expression levels of AvCDPK31 were significantly down-regulated in both stresses. These results indicated that the different AvCDPK genes played differ- ent roles in the process of salt stress and waterlogging stress in Actinidia valvata. In addition, there were different expression patterns of the same gene under two stresses. The expression of AvCDPK28 in- creased significantly under waterlogging stress, but decreased under salt stress, indicating that AvCD- PK28 may participate in different regulatory pathways under different stresses. 【 Conclusion】A total of 63 CDPK genes were identified in Actinidia valvata. Phylogenetic tree showed that these genes andAtCDPK genes were highly conserved in evolution. There were significant differences in the expression of AvCDPKs among different tissues. Three members were highly expressed, which were induced by salt and waterlogging stresses, indicating that these members may play an important role in the response to salt and waterlogging tolerance in Actinidia valvata.