Study On The Function Of Soybean Cystatin Genes In The Symbiotic Nodulation Of Lotus Japonicas

Nodules are the key organs in legumes that convert nitrogen from the air into usable nitrogen for themselves.It is very important to improve soil fertility and crop yield and reduce the use of industrial nitrogen fertilizer by nitrogen fixation of root nodules.The formation of nitrogen fixation nodules in leguminous plants involves a series of reactions such as the early infection of rhizobia,the occurrence of nodule organs and the coordination of root development during nodule formation.However,the symbiotic nodule mechanism of legumes themselves is not fully understood.Cystatin(CYS)plays an important role in nodule and nodule development and senescence.In this study,we cloned soybean cysteine protease inhibitor genes GmCYS2 and GmCYS20.The biochemical function was revealed by the inhibition test of protease activity in vitro.Using Lotus japonicus as the carrier,and hairy root transformation technology mediated by Agrobacterium rhizogenes,the function of GmCYS2 and GmCYS20 genes in the symbiotic signal transduction process was confirmed by identifying the symbiotic nodulation phenotype of the complex plants of Lotus japonicus.The main research achievements during the postgraduate period are as follows:1.Two soybean CYS genes GmCYS2 and GmCYS20 were cloned.Amino acid sequence alignment and evolutionary tree analysis showed that GmCYS2 and Cajanus cajan CYS had the highest similarity.The similarity between soybean GmCYS20 protein and wild soybean(Glycine soja)cystatin protein was 99%.2.Expression and purification of GmCYS2 protein in vitro.Through the enzyme activity inhibition identification of recombinant protein GmCYS2,it was found that this protein had inhibitory effect on class L,class H and class B cathepsin,and the activity of class L and class B was higher than that of class H cathepsin.In addition,the inhibition activity of the enzyme activity of the protein inoculated for 30 days was significantly higher than that of the protein inoculated for 60 days.3.The plant expression vectors of GmCYS2 and GmCYS20 genes were constructed,and the overexpression complex plants of GmCYS2 and GmCYS20 genes were obtained by the method of hairy root transformation technology mediated by Agrobacterium rhizogenes.4.After 4 weeks of inoculation with rhizobia,the number of nodule in transgenic plants with GmCYS2 and GmCYS20 genes was significantly higher than that in empty vector control.5.The expression level of three nodule related genes(NIN,ENOD40-1 and ENOD40-2)in overexpressed plants was detected by real-time fluorescence quantitative PCR.The results showed that the transcription level of the three genes was significantly higher than that of control group.6.Further statistics on the infection phenotypes of rhizobia in the inoculated 10-d complex plants showed that the overexpression of GmCYS20 gene did not affect the infection process of rhizobia,but played a positive regulatory role in the initiation and development of root nodules.These results suggest that GmCYS2 protein has certain inhibition of enzyme activity and positively regulates the process of root nodules.GmCYS20 gene can regulate the process of symbiotic nodule formation by promoting the initiation and development of nodule.It provides important genetic information and theoretical basis for revealing the mechanism of symbiotic nodule formation in legumes.