Analysis Of DcaCIPKs Family And Molecular Mechanism Of DcaCIPK3 In Response To Drought Stress In Dendrobium Catenatum

How to sense and response environmental stress is a basic biological function of plants.CIPK（Calcineurin B-like-interacting protein kinase）plays an important role in signal transduction under drought stress.Dendrobium catenatum is an epiphytic plant which sticks to tree bark and naked rocks in the wild.To adapt to these harsh habitats,D.catenatum has envolved succulent storage-organs,such as pseudobulbs,thick leaves,and crassulacean acid metabolism（CAM）pathway.The CIPK family had been analyzed in many C3 and C4 plants,but comprehensive information about CAM plants is still limited.Studying a highly drought-resistant plant that was shaped by natural selection is the most direct and effective way to understand drought resistance mechanisms during drought stress procedure.This study for the first time analyzed CIPK gene family in CAM plant.We identified CIPK family members and analyzed gene structures and phylogenetic evolution.Dca CIPKs’expression patterns and alternative splicing was analyzed based on RNA-seq data produced in different tissues and in response to both drought and cold stresses.Furthermore,we identified a protein Dca ABR1which can interact with Dca CIPK3 and negatively regulates in ABA signaling transduction under drought treatment.The results are as follows:1.I identified 24 Dca CIPK family members in D.catenatum based on the Hidden Markov Model of NAF and Pkinase domain.The results indicated that this gene family which both belong to monocot and dicot plant can be divided into intron-rich and intron-poor types.It is presumed that plants drought resistance was enhanced by abandoning inrons.2.The RNA-seq data（different tissues,cold treatment,and drought treatment）and q PCR analysis suggested that the At CIPK14’s homologous gene Dca CIPK14 was abundantly expressed in leaves and pseudobulbs,which are rich in polysaccharides.Dca CIPK14 can also be induced by cold and drought stress.It presumed that Dca CIPK14 may act in connecting and regulating stress induction and energy metabolism.Dca CIPK3,12 and 16 can be significantly induced by drought treatment both at day and night,which suggested that they may play important roles in drought resistance.3.The alternative splicing analysis indicated that Dca CIPKs may be influenced by the circadian clock.The q PCR results suggested that Dca CIPK1,7 and 15 significantly influenced by day and night rhythm.Dca CIPK1 significantly elevated during the dark phase,while Dca CIPK7 and 15 were active in the day time.The overall abundance of Dca CIPK3 transcripts was not significantly influenced by the circadian clock,but the expression levels of main transcripts suggested that,under drought stress,the most highly expressed transcript variant of Dca CIPK3 tended to be transcript5 during the daytime and transcript2 at nighttime.The control treatment had a negative effect on the sum expression abundance of transcripts but did not change the routine preference of transcript5 during the day and transcript2 at night.The protein domain in main expressed transcripts analysis indicated that Dca CIPKs may use different domain combinations by alternative splicing to improve the functional diversity of gene to adapt the circadian influence and the CAM metabolism pathway under drought stress.4.I did in-depth research of Dca CIPK3 which was presumed to play important roles under drought treatment.I found a protein Dca ABR1 which can be interactive with Dca CIPK3in the nucleus and negatively regulated in ABA signaling transduction under drought stress.The Arabidopsis was weaker and advance to the reproductive growth by overexpression of Dca CIPK3.The downstream genes in ABA signaling pathway were inhibited by overexpression of Dca CIPK3.Dca CIPK3’s overexpression also disrupted the H₂O₂’s balance of generation and dissipation.These results indicated that the overexpression of Dca CIPK3 put the Arabidopsis in an abiotic stress-like state.The Arabidopsis prematurely step into the reproductive stage to reduce the stress damage and assure the success of the next generation.In conclusion,alternative splicing improves the gene functional diversity by recombinant exons.The results indicated that alternative splicing was influenced by circadian rhythm.Dca CIPKs take different splicing ways in different time to adapt CAM metabolize and drought stress.Positive and negative control in metabolize pathway is very important to maintain steady state.The disruption of balance would lead to disorder of the plant’s metabolism.The negative control of Dca CIPK3 competed with positive genes and formed an elastic gene network.This is the vital mechanism of maintaining a steady state.The overexpression of Dca CIPK3 in Arabidopsis leads to an up-regulation of the interactive protein ABR1.Cascade amplification inhibited the downstream genes.The generation and elimination of H2O2 system were destroyed,which resulted in Arabidopsis shortened the time of life cycle to ensure the reproduction.