Preliminary Study On The Function Of Soybean GmCLV1 And GmCLV3 Genes In Root Nodule Development

As we all know,soybean is the main oil crop,and legumes provide nutrients to the ground through the symbiotic nitrogen fixation of root nodules.Nitrogen,an indispensable inorganic nutrient in plants,is one of components of essential amino acids,nucleic acids,chlorophylls,coenzymes and many plant secondary products.The nitrogen fixation of legumes is the main source of nitrogen in the natural and agricultural ecosystems.It has been reported that CLAVATA1（CLV1）and CLAVATA3（CLV3）genes in Arabidopsis thaliana play important roles in the shoot and root meristems,while their homologous genes MTSUNN and MTCLE12 in alfalfa promote downstream gene expression when induced by rhizobium and participate in the automatic regulation mechanism of nodulation,indicating MTSUNN and MTCLE12 inhibit nodule formation.Therefore,we predicted that Gm CLV1 and Gm CLV3,homologs of CLV1 and CLV3 in soybean,might play an important role in the nodulation.It is well known that soybeans are a major food crop.This study try to analyze the relationship between both Gm CLV1 and Gm CLV3 genes and nodulation and to pave a way to study further the function of these genes in nodulation.Our results are showed as follows:（1）Through bioinformatics analysis,Gm CLV1-2 and Gm CLV3-1 genes were identified as homologous genes of At CLV1 and At CLV3 in Arabidopsis thaliana,respectively.CLV1-2 proteins have two transmembrane domains and repeat motifs rich in leucine,and are composed of 38.74%αhelices and 44.34%random coilings.The stable spatial conformation of 11.93%elongates linkage and 4.99%β-angle.The CLV1-2 protein is a hydrophilic protein.CLV3-1 has a transmembrane domain,a stable spatial conformation consisting of 27.62%α-helix,58.1%random coil,12.38%elongation,and 1.90%β-angle.The CLV3-1 protein is also a hydrophilic protein.（2）The promoter sequence of Gm CLV1-2 and Gm CLV3-1 was amplified from the genome of soybean cultivar Tianlong 1,and the promoter sequence of approximate 3000 bps upstream of the transcription starting site of Gm CLV1-2 and Gm CLV3-1 was linked to the reporter gene GUS to obtain the expression vectors Gm CLV1-2_pro:GUS and Gm CLV3-1_pro:GUS.The gene was expressed in vascular tissues,epidermis,lateral root primordia and nodules of soybean.（3）CRISPR/Cas9 gene editing technology was used to construct the knockout vectors of Gm CLV1-2 and Gm CLV3-1 genes,and overexpression vectors of Gm CLV1-2 and Gm CLV3-1 genes were also be constructed.All vector were introduced into soybean hairy roots,respectively.The positive roots were screened and used to identify the gene edited hairy roots after inoculation of rhizobia.Finally,we found that there was only one target site being edited for Gm CLV1-2 with deletion of 4 bp or 5 bp,respectively,while both target sites of Gm CLV3-1 worked well and led to a 301 bp deletion.The phenotypes of the hairy root plants harbouring Gm CLV1-2 and Gm CLV3-1mutants or overexpressing Gm CLV1-2 or Gm CLV3-1 genes were analyzed,respectively.The mutation of Gm CLV1-2 and Gm CLV3-1 genes in hairy roots increases the number of nodules and promotes leaf development.By contrast,overexpressing plants reduce the number of nodules and inhibit the leaf development.Our results suggest that both genes negatively regulate nodule development and provide useful clues for the further study on the mechanism of nodule development in soybean and a new method for potentially improving biological nitrogen fixation in agricultural production.