| Soybean(Glycine max)is an important grain and oil forage crop in the world.However,in recent years,China’s soybean imports have soared year by year,and the domestic soybean production safety has become seriously urgent.Intercropping is an effective way to increase the output of soybeans and other crops,and it is extremely important for solving the food crisis.However,in this system,soybean is subjected to the the canopy shade of high crops,which seriously affects the soybean yield under intercropping.The response of plants to shade stress is a very complicated process.Many detailed studies have been conducted on the shading regulation mechanism of the model plant Arabidopsis thaliana.Many Arabidopsis proteins have been identified to participate in the regulation of shade avoidance through expression levels and phosphorylation levels.The molecular mechanism of soybean crops in the shade avoidance is still unclear.Therefore,in order to study the early response of soybean seedlings at the protein molecular level under shade stress,the differentially expressed proteins and differentially phosphorylated proteins in response to shade stress were identified.In this study,two different soybean genotypes,Nan 032-4 and Nandou 12,were used as the test materials of relatively sensitive genotypes by variety screening with phenotypic analysis.Through proteomics and phosphoproteomics,proteins that respond to shading stress were selected.Combined with physiological experiments and q RT-PCR analysis,the effects of shading stress on sugar metabolism in soybean seedlings were further analyzed.The main results are as follows:1.In terms of phenotype,in order to screen shade-sensitive soybean varieties,we analyzed the phenotypes of two soybean varieties(Nan 032-4 and Nandou12)under shade stress.Compared with that in the white light,after the shading treatment,the seedlings of both varieties showed obvious shade avoidance,including increased plant height,petiole elongation,and internode length elongation.Nan 032-4 shows more severe shade response phenotypes and is a relatively ideal material for studying soybean shade avoidance.2.In order to screen soybean proteins involved in shade avoidance,we used proteomics and phosphoproteomics to screen the differential proteins.Tweenty-nine up-regulated and412 down-regulated proteins were obtained from proteomics,which were mainly located in chloroplasts and cytoplasm.Seventy-three up-regulated phosphorylated proteins and 155down-regulated phosphorylated proteins,which were mainly located in the nucleus,cytoplasm and chloroplast.Serine residues are its main phosphorylation site.These two proteomics results show that,after shading treatment of soybean,both up-regulated protein and up-regulated phosphorylated protein are involved in sugar metabolism-related pathways.3.To further study the effect of shade stress on sugar metabolism in soybean seedlings.We tested various sugar contents of soybean seedlings through physiological experiments.The results showed that after shading treatment,the various sugar contents(sucrose,reducing sugar,soluble total sugar,starch)of the two soybean varieties(Nan 032-4 and Nandou 12)showed a decreasing trend.On the other hand,q RT-PCR analysis showed that after shading treatment,the expression of carbohydrate metabolism-related genes was increasing.The results showed that after the shading stress,soybean seedlings increased the sugar metabolism by increasing the expression of genes related to sugar metabolism,leading to a decrease in the sugar content under the shade.4.For differentially phosphorylated proteins from the phosphorylation proteomics,we further screened 43 candidate proteins and amplified their genes from Nan032-4.All genes were constructed into an overexpression vector and transformed into Arabidopsis.Their over-expressing Arabidopsis plants were screened by herbicides,which provided potential research materials for further research on soybean shading response.In view of the practical problem facing agricultural production,this study screened proteins that responded to shade stress,and studied the response of soybean seedlings to shade.This provides a reference basis for related research of crop close planting or intercropping. |
