| CDPK(Calcium-dependent protein kinase)protein kinases are widely found in a variety of plants and play a key role in plant signal transduction.34 CDPK family members and 8 CRK(CDPK-related kinase)family members have been identified in Arabidopsis.CDPKs play important roles in plant growth and development,secondary metabolism and stress response,but little is known about the biological functions played by CRKs in plants.In this paper,we found that CRK8,a member of the CRK family,is specifically highly expressed in the early stages of roots and seeds,and thus it is hypothesized that CRK8 may be involved in the growth and development of plant roots and seeds.The specific biological functions played by CRK8 are still unclear,and no studies on CRK8 in root and seed growth and development within plants have been reported so far.Therefore,the present study was conducted to explore the biological functions of CRK8 initially,and the following results were obtained.The sequence analysis of CRK8 showed that it contained 606 amino acid residues.Subcellular localization and transmembrane domain analysis revealed that CRK8 is present in the nucleus without transmembrane structural domains.Through the analysis of physical and chemical properties,it was found that CRK8 was a hydrophilic and unstable weak acidic protein.Advanced structural analysis showed that the protein contained the highest percentage of irregular coiled-ups,at 44.06%.Phylogenetic analysis revealed that CRK8 in Arabidopsis is highly homologous to CRK2 and is related to rice,tomato,melon,tribulus alfalfa and pepper.Expression profiling showed that CRK8 was expressed in most plant tissues,and was particularly highly expressed in roots and seeds.The reporter gene expression vector was successfully constructed,and the expression of crk8 in the whole root and some leaves was successfully located by Gus staining.Under the microscope,it can be clearly found that the expression of crk8 in the endothelial layer and middle column is higher than that in other parts.Stress analysis of reporter plants revealed no significant difference in CRK8 expression after 6h of 150 m M Na Cl stress treatment,while CRK8 expression was significantly down-regulated after 6h of 5μM ABA(abscisic acid),5μM GA(gibberellin)and 100μM Me JA(methyl jasmonate)stress treatments.The differences between the CRK8 and CRK2 mutants and the wild-type phenotypes were investigated separately and no significant differences were found between the three phenotypes.Statistical results showed that seed weight was significantly increased in the case of re-expression compared to the wild type,while seed weight was lower and significantly down-regulated in the case of gene silencing,indicating that the CRK8 gene may be involved in early seed development but not in the seed germination process.The phenotypes of the overexpression plants were not significantly different from those of the wild type;the phenotypes of the gene silenced plants were weaker than those of the wild type,and the differences were highly significant.Stress tolerance results showed that using GNS-treated wild-type seedlings and DMSO treatment as controls,the phenotypes revealed that root length and root crafters were severely affected in GNS-treated seedlings,suggesting that GNS may affect multiple protein activities in CRKs.Under 50 m M Na Cl treatment,OE plants showed more primary root length,lateral root number and seedling severity than the wild type,while gene silencing was not significantly different from the wild type,indicating that OE plants were more tolerant under 50 m M Na Cl treatment;Under 25 n M ABA treatment,there was no significant difference in the number of lateral roots among the three,and no significant difference in root length and fresh weight between wild-type and gene silenced plants,while their root length and fresh weight were significantly lower compared to the overexpression plants,thus indicating that the overexpression plants were more tolerant under 25 n M ABA treatment.The transcriptome sequencing analysis of wild-type,overexpression and gene silencing plants showed that there were significant differences between overexpression and gene silencing plants at the cell level through go function enrichment.It is speculated that crk8 gene may be involved in early cell division.The results of gene silencing and WT differential expression gene enrichment showed that there were significant differences in transcriptional regulatory activities,especially DNA transcriptional regulatory activities Therefore,it is speculated that the silencing of crk8 / crk2 may lead to the difference of DNA transcriptional activity and DNA synthesis between cell divisions.The enrichment results of KEGG metabolic pathway showed that there were significant differences in light energy synthesis between gene silenced plants and wild-type plants.Therefore,it is speculated that crk8 / 2 may be involved in the regulation of light energy synthesis;Overexpression plants and wild-type plants have the most significant differences in plant hormone signal transduction and metabolic pathways,with 45 genes enriched,indicating that their hormone receptors and components are different,resulting in two different characters between overexpression plants and wild-type plants.Therefore,it is speculated that this may be due to the differences in hormone levels between overexpression plants and wild-type plants.The results of KOG enrichment analysis showed that there were significant differences in signal transduction mechanism between gene silenced plants and wild-type plants.It is speculated that the silencing of crk8 and crk2 may lead to the weakening of the signal transduction pathway involved by crks.In addition,comparing gene silencing plants,overexpression plants and wild-type plants,it was found that there were significant differences in the synthesis,transportation and catabolism of secondary metabolites.Therefore,it is speculated that crk8 may also be involved in multiple secondary metabolism processes. |
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