Gene Cloning And Functional Studies Of Microtubule-binding Protein ZmKTN1 And Its Interaction Protein ZmSAV4 In Zea Mays

Microtubule(MT),one of the main components of cytoskeleton,is involved in the regulation of the synthesis and deposition of cellulose in plant cell walls and plays an important role in plant morphogenesis.Microtubule-severing enzyme Katanin is an important regulator of microtubule depolymerization and reunion,including a catalytic subunit P60 with ATPase activity and a regulatory subunit P80.Microtubule-binding protein KTN1 encodes the P60 subunit,which plays an important role in cellular activities including cell division,signaling and migration by regulating the dynamic changes of the microtubule.Currently,few proteins have been reported to interact with KTN1 directly,while SAV4 were found that can interact with KTN1 directly in Arabidopsis in our previous studies.In this study,maize homologous proteins KTN1 and SAV4 were cloned and functionally studied and the following experimental results were obtained:(1)Cloning and functional study of ZmSAV4 a in maizeFirst,by constructing the phylogenetic tree and comparing the homologous sequences of KTN1 in Arabidopsis and Zea mays,we found that there was a high homology between;Second,SAV4 was mainly expressed in newborn organs and elongated tissues;Third,the ZmSAV4 a overexpression in SAV4 mutant transgenic line was obtained by constructing a binary fusion expression vector.After obtaining the transgenic plant,the phenotype of SAV4 mutant was found to be reverted to the wild type;Finally,it was found that the localization of ZmSAV4 a was consistent with that of SAV4 in Arabidopsis.These results indicated that the function of SAV4 was conserved in Arabidopsis and Zea mays.(2)Cloning and functional study of ZmKTN1 a in maizeFirst,we found that KTN1 shares high homology in Arabidopsis and Zea mays by phylogenetic analysis and protein homology comparison;Second,GUS staining results showed that KTN1 was specifically expressed in roots,stems and leaves of plants;Third,the binary fusion expression vector of ZmKTN1 a was constructed to obtain the overexpressed transgenic line of ZmKTN1 a.Phenotype analysis showed that the ZmKTN1 a overexpression transgenic line presented the same phenotype as the KTN1 mutant and the overexpressed plant of KTN1;Finally,we found that ZmKTN1 a was located in the microtubule.These results suggest that KTN1 is functionally conserved in Arabidopsis and Zea mays.(3)The interaction study between ZmKTN1 a and ZmSAV4 a in maizeThe interaction between ZmKTN1 a and ZmSAV4 a was studied by Bi FC(bimolecular fluorescence complementation),which showed that there was a physical interaction between them.These results further confirmed that the functions of KTN1 and SAV4 were very conservative in Arabidopsis and Zea mays.