Functional Study And Generation Of Polyclonal Antibody Of GmMS1 In Soybean

The utilization of heterosis is one of most effective ways to improve crop yield.To apply heterosis in soybean,it is urgent to establish a stable male sterility system.Identifying stable and reliable genic male sterile genes,studying the molecular mechanism of causing male sterility,and taking advantage of the male sterile genes to improve the male sterile lines have great value in agricultural productivity.The ms1(male sterile 1)mutant is a nuclear male sterile line caused by a spontaneous mutation in soybean.The vegetative growth of ms1 mutant showed no obvious phenotype,but it only produces empty pods and is an ideal male sterile mutant.Our laboratory recently cloned the coding gene of the soybean ms1 locus,named GmMS1,which encodes a kinesin protein.We provided preliminary evidence in cell biology that gmms1 may affect fertility by regulating the depolymerization of phragmoplast.To further study the molecular and biochemical function of gmms1,the following experiments was carried out in this study:1.To explore the molecular mechanism of GmMS1 in regulating male fertility,we produced the motor domain(MD)and His-tag fusion proteins of GmMS1 and its mutant urms1 with single amino acid replacement(GmMS1A263L)in motor domain,named GmMS1MD-His and urms1MD-His,respectively.In vitro microtubule binding experiment revealed that the binding ability of urms1 to microtubules was significantly reduced,indicating that the arginine at position 263 of GmMS1 protein is the key amino acid involved in microtubule binding,Therefore,the male sterile phenotype of urms1 was assumed to weaken the microtubule binding ability.2.To reveal the function of GmMS1 in regulating plant male fertility at protein level,the polyclonal antibody immunized in rabbit with the antigen of GmMS1(425-495aa)was generated.The western blot showed that the Anti-GmMS1 polyclonal antibody could recognize the endogenous GmMS1 protein in soybean.3.To compare whether GmMS1 and its Arabidopsis homologous gene AtNACK2(NPK1activating kinesin)have functional differentiation during evolution,the AtNACK2 motor domain loss of function mutant was generated using CRISPR/cas9 technology.The fertility results showed that the loss of function led to semi-sterile phenotype.Therefore,we first revealed that GmMS1 and AtNACK2 have gene functional differentiation.4.A total of 157 kinesin genes were identified in soybean.The functions of soybean kinesins were predicted according to the gene expression pattern from public database,and we speculated that the genes may be related to soybean male fertility.This study further analyzed the molecular and biochemical functions of GmMS1,and generated the anti-GmMS1 polyclonal antibody,providing the rational and material support to reveal the mechanism of soybean male sterility and accelerating the application of soybean heterosis.