The Research On Molecular Mechanism Of Calcium-dependent Protein Kinase CDPK31 Regulates Arsenite Influx Through Interacting With Arsenic Transporter AtNIP1;1 In Arabidopsis Thaliana

The contamination of arsenic is widespread in the global,becauseof humans' industrial activities,such as direct discharge the industrial sewage,the offshore and soil had accumulated arsenic and other heavy metals is also very serious.Arsenic pollution of the offshore and soil had seriously threatened the agricultural production,ecological environment and animal and humans' life and health.One of the major ways of arsenic enrichment to the human body is through the food chain.Therefore,to clarify the molecular mechanism of plant on the absorption,transport and metabolism regulation pathways of arsenic is of great significance to control the arsenic to influx to the food chain.Previous studies had confirmed that NIP1;1 is one of the family members of AQP and is permeable to As(?).In Arabidopsis thaliana,there are 38 genes encoding AQP.So far,the regulation mechanism of arsenic channel activity had not been reported in detail.In this study,we found that the regulation of the activity of NIP1;1 was closely related to the arsenic tolerance in plants.By yeast two hybrid technology and bimolecular fluorescence complementation technology,we conformed that there was interaction between the CDPK31 and NIP1;1 in the cytoplasm membrane.The mutant cpk31 and nip1;1 both had arsenic tolerance phenotype,both shoot and root of the mutant cpk31 and nip1;1 accumulated less arsenic than the wild type.In addition,we found tissue expression pattern of CDPK31was consistent with NIP1;1,especially in root vascular.During arsenic stress environment the expression of CDPK31 and NIP1;1 both decreased,therefore,CDPK31 and NIP1;1 were likely to be in the same metabolic pathways.Under As(?)stress conditions,likely could reduce arsenic absorption and root-to-shoot translocation by reducing the expression ofCDPK31 to reduce the activation of NIP1;1 mediated by CDPK31.In this study,we used the mutant nip1;1 and cpk31 as experimental meterial,In this dissertation work,we studied the mechanism of how did CDPK31 regulate the activity of As(?)transporter AtNIP1;1.The main results were summarized as follow:1.The yeast two hybrid screening libraries experiments showed the interaction between NIP1;1 and CDPK31,bimolecular fluorescence complementation technology combined with subcellular localization technology further proved that the interaction of NIP1;1 and CDPK31 happened in plasma membrane.2.Phenotypic analysis experiment showed cpk31 and nip1;1 both had arsenic resistant phenotype,the double mutantcpk31 nip1;1 had more strong tolerance to arsenic than nip1;1.It implied that CDPK31 may regulate the activity of NIP1;1.Detection arsenic by ICP-OES after treating Col-0,nip1;1 and cpk31with 10 ?MAs(?)for different time,cpk31 and nip1;1 both accumulated less arsenic than Col-0.The same phenotype and arsenic accumulation pattern showed CDPK31 and NIP1;1 were likely in the same metabolic pathways.3.GUS staining analysis of tissue expression pattern shows:CDPK31 has a similar expression pattern with NIP1;1,and negatively responses to As(?)stress,both in the roots of vascular CDPK31 expression in leaves are mainly concentrated onmesophyll cells and stomata,CDPK31 decreased expression to reduce arsenic absorption and transportation,CDPK31 expression in flower and siliques might affect the As(?)accumulation in the seeds and the long distance transport of As(?).4.Real-time quantitative PCR analysis,the expression of NIP1;1 and CDPK31 in root reduced after arsenite treatment,but the expression of NIP1;1 increased in leaf.NIP1;1 and CDPK31 mainly expressed in the roots in three-week-old seedling,which meaned that Arabidopsis thaliana not only through reducing the expression ofNIP1;1 to resist arsenic stress,but also lowering the expression of CDPK31 to reduce the activation of NIP1;1,Arabidopsis thalinana might prefer reducing the expression of CDPK31 to against arsenic stress.5.Subcellular localization technology analysis of the NIP1;1 expression changes of nip1;1-mCherry seedling,which NIP1;1 was combined with RFP and the expression of NIP1;1 could be observed by RFP in laser confocal microscope,We found that NIP1;1 expressed mainly in roots under normal conditions.However,the expression pattern changed when we treated nip1;1-mCherry seedling with As(?)and/or Ca2+,its expression mainly in cytoplasm without As(?),the expression location transferred to the vacuole membrane and the plasma membrane when we treated nip1;1-mCherry seedling with As(?)and Ca2+ together,and the expression increased.This meant that calcium might enhance the influx ofAs(?)to vacuole.In summary,according to the results of real-time quantitative PCR and the arsenic accumulation pattern ofmutants,we concluded that the physiological function of CDPK31 was enhancing As(?)influx through interacting with arsenic transporter AtNIP1;1 in Arabidopsis thaliana.The expression of NIP1;1 waspositively regulated by CDPK31 by cutting the expression ofCDPK31 or by reducing the activity of CDPK31to enhance theAs(?)tolerance of Arabidopsis thaliana.