Characterization And Separation Of The Cysteine Proteinases Involved In Senescence Of Soybean Nodules

Soybeans are the most important legumes and one of the most important food crops.Soybeans can form a mutually beneficial symbiotic relationship with nitrogen-fixing bacteria.Utilizing the nodule nitrogen fixation characteristics of soybeans can not only increase the soybean yield,but also reduce the use of chemical fertilizers,which is conducive to the sustainable development of agriculture.The premature aging of root nodules caused by environmental stress during soybean planting will affect the yield.How to delay root nodule aging is an important issue for scientific research and production.At present,there are more and more researches on cysteine protease and root nodule senescence.The involvement of cysteine proteases in nodule development and aging has also been verified.In recent years,18 cysteine protease genes were found to be expressed in soybean nodule maturation and senescence by RNAseq technology,but it is not yet clear which of them are related to nodule senescence.Based on this background and premise,this study focused on the cysteine proteases specifically expressed in soybean nodules and explored the part of them involved in the senescence of soybean nodules,aiming to provide scientific basis and material for delaying the senescence of soybean root nodules and increasing nitrogen fixation efficiency.The main findings are as follows:1.Editing the soybean nodulation factor receptor gene by CRISPR-Cas9 system,so that soybean transgenic roots cannot form nodules,improve the soybean hair root transformation system,also successfully knocked out the gene using the CRISPRCas9 system in the soybean hair root transformation system.2.The cysteine protease genes specifically expressed in soybean nodules were designated Gm NCP1~18,respectively.They were divided into three groups by homology analysis,and then the CRISPR-Cas9 system vector was constructed to knock out three groups of genes,named CRISPR A,CRISPR B,and CRISPR C,respectively.After hair root transformation,the observation of nodule phenotype and determination of root nodule nitrogenase activity showed that CRISPR B could delay root nodule senescence.3.Dividing the genes involved in CRISPR B into three parts,we also constructed a CRISPR-Cas9 system vector knock-out three-part gene.The hairy root transformation phenotype indicates that knocking out the Gm NCP3 gene can delay nodules senescence.4.The genes of Gm NCP3,6,10,11 and 12 were expressed in Medicago truncatula.The results of hair root transformation showed that only the expression of Gm NCP3 and Gm NCP11 could cause premature aging of nodules.5.The yeast two-hybrid system was used to investigate the interactions between all proteins expressed by 18 cysteine protease genes and seven cystains(named Gm Cys1~7)that are specifically expressed in soybean nodules.It was shown that only two proteins expressed by the Gm NCP16(Gm NCP16.1 and Gm NCP16.2)could be verified to interact with Gm Cys1 and Gm Cys2 proteins in yeast,respectively.