The Research On Functional Of Actin Crosslinking Protein V-CROLINs In Arabidopsis Thaliana

The microfilament cytoskeleton is widely and conserved three-dimensional network structures in eukaryotes,it plays an important role in many cell physiological activities,such as maintaining cell morphology,cytokinesis,cell movement,and cell polarity establishment.Actin-binding proteins ensure the normal progression of these physiological activities by maintain orderly dynamics of the microfilaments cytoskeleton.In our team,a new type of plant-specific actin-binding protein family,CROLINs,was found in Arabidopsis thaliana,research on V-CROLINs will expand the people's understanding for plant-specific actin binding proteins.There are six members in this family,and it has been demonstrated that AtCROLIN1 is an actin bundling/cross-linking protein specifically expressed in pollen,which may be involved in pollen germination and polar growth of pollen tubes by regulating microfilament structures.Bioinformatics predicted that AtCROLIN2 is also specifically express in pollen,while the other four members are express in vegetative organs and named V-CROLINs,whose protein function and physiological function are unknown.This article focuses on V-CROLINs and the main results are as follows:1.The tissue expression pattern of V-CROLINs was obtained.The results of GUS staining showed that both CROLIN4 and CROLIN6 are express during the entire growth and development of Arabidopsis thaliana,and CROLIN4 is the major gene.CROLIN3 began to express in the cotyledons of 4 days old seedlings and CROLIN5 is express in the roots of 8 days old seedlings,and neither of them expressed in flower and pollen.2.T-DNA insertion mutants and V-CROLINs-RNAi lines of V-CROLINs were obtained.RTPCR demonstrated that crolin3 and crolin5 are completely inhibited,but crolin4 and crolin6 are partially inhibited;V-CROLINs-RNAi lines were screened out the RNAi-18,which has relatively high degree of down-regulation of four genes,and partially down-regulated RNAi-16 and RNAi-11 were also screened as follow-up experimental materials.3.The homozygous mutant of crolin3crolin5 was obtained and treated with UV-B radiation,the wild type recovered better than crolin3crolin5 after UV-B radiation.It was demonstrated that CROLIN3 and CROLIN5 are involved in Arabidopsis response to UV-B radiation stress.4.RNAi-18,RNAi-16,RNAi-11 have higher tolerant than the wild type under salt stress and osmotic stress;Meanwhile,RNAi-18 showed insensitivity to ABA-induced stomatal closure;RNAi-18,RNAi-16,RNAi-11 also have higher water loss rates than the wild type in dehydration experiments.It is speculated that V-CROLINs are involved in Arabidopsis response to salt stress,osmotic stress and drought stress.5.We obtained the homozygous mutants of crolin4,crolin6,crolin4/6,crolin3/4/5,crolin3/5/6,crolin3/4/5/6 by CRISPR technology.6.The crolin3/4/5/6 show longer root than the wild type,suggesting that V-CROLINs participate in Arabidopsis root development.7.The two sets of prokaryotic expression vectors pET30a-V-CROLINs and pGEX-4T1-VCROLINs were respectively transferred into BL21 for protein induction,it was found that pET30aCROLIN3,pET30a-CROLIN5 and pGEX-4T1-CROLIN5 induced the correct protein bands.In summary,the tissue expression pattern of Arabidopsis V-CROLINs was obtained in this study,and some necessary mutant materials were obtained,which laid the foundation for better research V-CROLINs;At the same time,it was initially demonstrated that V-CROLINs participate in the physiological processes of Arabidopsis root growth,UV-B stress,salt stress,osmotic stress,and drought stress.