The Creation Of Multiple Homozygous Mutants Of KCHs And Sutdy On KCHs Biochemical Function In Arabidopsis Thaliana

In a series of cell activity of animals and fungi,myosin and dynein can move along microtubule using the chemical energy from ATP hydrolysis.No dyneins have been found in plants,but terrestrial plants have a kinesin-14 family that could directionally move along microtubules as a form of dimers.The family has a specifical subfamily named KCHs(Kinesin Calponin Homology Domain),which contain a motor domain,two coiled –coil domain and one CH domain.According to present study,KCH1 is a linker between microfilament and microtubules,and has important biological function in rice roots and cotton fiber.Although seven KCHs have been identified in Arabidopsis,the knowledge of biological and biochemical functions of KCHs are very limited.Previously our laboratory found that KCHs proteins have serious functional redundancy in Arabidopsis.To explore the reason of functional redundancy,many multiple homozygous mutants of KCHs are needed.Here foure multiple homozygous mutants(MHMs)of KCHs were ontained by genetic hibridization or CRISPR/Cas9 gene editing techniques.Inaddtion,the biochemical functions of CH domain structure of KCHs was studied using high and low speed coprecipitation approach.The main research results are as follows:1.One seven-valent KCH MHM,KCH1/2/3/4/5/6/7,and two hexavalent KCH MHM,KCH1/2/3/5/6/7 and KCH1/2/3/4/6/7,have been created by artificial hybridization and PCR identification.In the KCH MHMs,KCH1,KCH2,KCH7,KCH4 and KCH6 appeared the best inhibition level,the expresions of which were almost completely inhibited,and both KCH3 and KCH5 showed a better inhibition level,the expreesion level of which were only 40% of WT.2.Using KCH1/2/3/6/7 MHM as a background material,a hexavalent KCH MHM,KCH1/2/3/4/6/7,was created by deleting base C of the 406 th position of KCH6 DNA using CRISPR/Cas9 technology.3.For studying the biochemical function of CH domain of KCHs,10 truncated KCH proteins,including KCH1(0-178),KCH2(0-139),KCH3(0-188),KCH4(0-162),KCH6(0-139),KCH7(0-185),KCH1(0-466)and KCH7(0-466)were prepared by expression vector construction,induction of KCH expressiion in E.coli as well as extraction and purification of KCH.4.High and low speed coprecipitation analysis showed that protein KCH1(0-466),KCH4(0-162),KCH6(0-466)could bind to F–actin and made F–actin form bunch.But the bundling function of KCH4(0-162)was the most strongest,and the bundling function of KCH1(0-466)and KCH6(0-466)are closed,and were weaker than that of KCH4(0-162).5.LSCM observation also confirmed that KCH1(0-466),KCH4(0-162)and KCH6(0-466)also could make F-actin form visible bundle-like structres.In summary,this study successfully created four KCHs multiple homozygous mutants,which provides reliable experimental materials for the genetics,cell biological and molecular biologic al research of KCHs.In addition this study firstly confirms that the proteins of KCHs family of Arabidopsis can bind with microfilament in vitro.