Expression Analysis Of Cysteine Protease During Nodule Senescence In Soybean

The nitrogen fixation of root nodules is of great significance to the growth of leguminous plants.The premature aging of root nodules will negatively affect plant growth.Therefore,we need to understand more about the development and senescence of nodules.In the process of senescence of legume nodules,a series of proteolytic enzymes including cysteine proteases are activated in plants.Plant cysteine proteases are mainly classified into papain-like cysteine proteinases(C1)and legumeins(C13 the legumains).Studies have shown that cysteine proteases are involved in root senescence.In Astragalus sinicus,silencing the cysteine protease gene As NODF32 by RNA interference also delays the senescence of nodules.A large number of nodule-specific genes were found in soybean root nodules by EST database and c DNA microarray techniques.The expression level of cysteine protease AN03008D06 was significantly increased in nodule senescence.Studies have shown that transcription of eighteen cysteine proteases in the development of soybean root nodules have changed through analysis of transcriptome data.In this study,in order to screen out proteases that play a role in the process of senescence of soybean nodules,promoter expression analysis,Real-time PCR analysis and fluorescence assay were used to investigate the role of some cysteine proteases in soybean nodule development and senescence.The main findings of the study are as follows:1.Eighteen cysteine protease promoter sequences were amplified and ligated to the vector with the reporter gene GUS.Agrobacterium-mediated hair root transformation was used to transfer the recombinant plasmid into soybean and the expression of the promoter was detected by observing the expression of GUS.At present,transgenic plants transformed with GmNCP2 pro and GmNCP17 pro have been successfully obtained.The results showed that GmNCP2 was expressed in root nodules at 4 weeks,8 weeks and 12 weeks of inoculation.From 4 weeks to 8 weeks,the expression of GmNCP2 in root nodules increased,and the expression level in root nodules decreased from 8 weeks to 12 weeks.The results showed that the expression of GmNCP2 was decreased in the later period of root nodule and may not be involved in the senescence of root nodules;GmNCP17 was also expressed in root nodules at 4weeks,8 weeks and 12 weeks of inoculation.However,unlike GmNCP2,the expression of GmNCP17 in root nodules continued to increase from 4 weeks to 12 weeks,indicating that the expression of GmNCP17 was increased in the late root nodule and may be related to the aging of root nodules.2.The expression of these eighteen cysteine protease genes in root nodules was analyzed by Real-time PCR.The results showed that the expression of GmNCP1,GmNCP2,GmNCP4,GmNCP8,GmNCP9,GmNCP11,GmNCP12,GmNCP13,GmNCP14,GmNCP15,GmNCP16 and GmNCP18 increased in root nodules from 4weeks to 8 weeks,and the expression decreased in root nodules from 8 weeks to 12weeks;The expression of GmNCP3,GmNCP5,GmNCP6,GmNCP7,GmNCP10,GmNCP17 in root nodules increased continuously from 4 weeks to 12 weeks;The Real-time PCR results of GmNCP2 and GmNCP17 are consistent with the promoter results.In addition,compared with other studies of cysteine proteases,GmNCP3 showed the largest increase in the expression of aged nodules,suggesting that GmNCP3 is most likely associated with root nodule senescence;3.In order to detect the cysteine protease activity of GmNCP3 and GmNCP17,GmNCP3-A protein and GmNCP17-A protein with GmNCP3 and GmNCP17 corresponding inhibitory domains removed were expressed in prokaryotic cells.The protease activity was measured by fluorescence.The results showed that: GmNCP3-A and GmNCP17-A can decompose the substrate;when the cystatin E-64 was added,the protease activity of GmNCP3-A and GmNCP17-A was inhibited.GmNCP3 and GmNCP17 were shown to have cysteine protease activity.