Functional Study Of CpCK2 Kinase In Arabidopsis Chloroplast Localization

Plastid Transcription Kinase was present in chloroplast PEP complex,and it shared highly similarity with the catalytic subunit of casein kinase II(formerly known as CK2),and it was named cpCK2(Chloroplast-targeted CK2)or CK?4.The Arabidopsis genome has the other three genes(CKA1,CKA2 and CKA3)encoding ?subunits,besides the gene coding for the cpCK2,and these subunits have been shown to be localized in the cytoplasm,nucleus and also in chloroplasts.These CK2 have kinase activities.Nevertheless,the biological roles of these subunits have not been fully characterized yet.In this dissertation,we analyzed the mutants of cpck2 in the Arabidopsis,and investigate the role of cpck2 in plant development.In this work,we obtained the T-DNA insertion line of cpck2,CS355016.The T-DNA was inserted on the sixth nucleotide of the start code.RT-PCR analysed the transcripts of the CKA4(AT2G23070)in the homozygous mutant was barely detected,and the transcript amount of its proximity gene CKA3(AT2G23080)was decreased compared with that of the wild type.QRT-PCR results also confirmed these results.These results indicated that the T-DNA insertion of the CS355016 mutant has not only affected the expression of the CKA4 gene,but also influenced the expression of its neighbor gene CKA3.Hence,we named this mutant as cka3cka4-1.The cka3cka4-1 mutant displayed late flowering under long-day condition.RT-PCR analysed that the transcripts of the FLC gene were increased in the mutant,compared with those in wild type,and the expression levels of two floral pathway integrators genes,CONSTANT(CO)and FLOWERING LOCUS T(FT)were reduced.Genetic analysis showed that the fragment containing both CKA3 and CKA4 genomic sequence could complementary the phenotype,while either the genomics sequence of CKA3 or the genomics sequence of CKA4 could complementary the mutant phenotype.A construct of cpCK2:MYC or cpCK2:FLAG tag was produced ant introduced into cka3cka4-1mutant,and genetics analysis demonstrated that both cpCK2:MYC and cpCK2:FLAG could complementary cka3cka4-1 mutant,respectively.Here,we obtained single T-DNA insertion mutants for the three ? subunit genes ck?1,ck?2 and ck?3.The CKA1,CKA2 and CKA3 mutant displayed normal flowering phenotype under long-day condition,compared with wild type.Double mutation analysis showed that the cka1cka2,cka2cka3 and cka1cka3 mutant did not display delayed flowering underlong-day condition.Cellular localization analysis showed that cpCK2:EGFP localized in chloroplast as previous reports.Western blot experiments showed that cpCK2 protein is localized in the stroma and associated with thylakoid membrane.Sequence analysis showed that cpCK2 has high sequence similarity and conserved NLS sequence with the three other members.It seemed that EGFP:cp CK2 is localized in nuclear.Genetics analysis demonstrated that MYC:cpCK2 could complement ?4 triple mutants.Taken together,our results showed that both cpCK2 and CKA3 are functionaly reduntant in regulating the normal flowering of plants.