Functional Study Of Arabidopsis Thaliana DUF761 Family

The cytoskeleton of plants are consists of microtubules and microfilaments,which perform a large number of functions essential for cell division,maintaining cytoplasmic integrity,intracellular trafficking,and cell morphology.The dynamic transition between monomeric actin(GF)and filamentous actin(AFs)is the basis of its function and is finely regulated by a series of actin binding proteins(ABPs).Recently,a new type of microfilament binding protein GhCFEl,which in cotton contains two conserved unknown functional domains(DUFs),DUF761(293-330 residues)and DUF4408(42-75 residues).As a new family of actin binding proteins,CFEs are widespread in other plants and their physiological and biochemical functions in Arabidopsis thaliana is remain unknown.In this paper,Arabidopsis thaliana as a research material,through bioinformatics,molecular biology and biochemistry and other methods,the CFEs protein family(in this paper in Arabidopsis thaliana named DUF761 family)to conduct research,and obtained the following results:1)It was found that the DUF761 protein family may originate from gymnosperms and is widely present in vascular plants.According to the sequence analysis of the DUF761 family homologues from 61 species of algae to angiosperms,DUF761 homologues only exist in gymnosperms and angiosperms,and there are homologues in the analyzed vascular plants exist.2)The organization of the DUF761 family of proteins was obtained.Through RT-qPCRand GUS chemical staining analysis,it was found that DUF761-7 was removed and Arabidopsis DUF761 family members were all expressed in Arabidopsis thaliana tissues.It was speculated that the family was widely involved in the growth and development of Arabidopsis thaliana.3)Identification of a loss-of-function mutant of the DUF761 family gene.Through the genotypic analysis and RT-qPCR analysis of the T-DNA insertion mutants,it was confirmed that the T-DNA insertion mutants 766 and 337 were the genes DUF761-1,458 and the genes DUF761-5,006 and 583 were the gene DUF761-7 loss-of-function mutants.And further edited mutants of other DUF761 family genes were obtained by CRISPR-Cas9 gene editing technology.4)The subcellular localization and expression vectors of DUF761 family proteins and prokaryotic expression vectors were constructed.The in vitro prokaryotic expression induction and purification conditions of this family protein were explored,which laid the foundation for further elucidating the characteristics of the family proteins.