Cloning And Drought Resistance Verification Of Soybean E3 Ligase GmPLR-2 Gene

Soybean is an important economic crop and food crop in China,and it will inevitably be affected by various stresses during its growth and development.In order to adapt to adversity stress,the gene expression pattern of plants will change,which will affect related metabolic pathways and produce corresponding physiological changes.Drought,saline-alkali and other abiotic stresses that are not conducive to plant growth are important reasons for its decline in yield and quality.Therefore,the resistance to abiotic stress is enhanced by digging soybean-related regulatory genes,identifying gene functions,and exploring regulatory mechanisms.It will promote soybean resistance breeding.Related studies have shown that E3 ligase plays an important role in improving plant drought resistance in rice,Arabidopsis,tobacco and other plants,but there are few reports on E3ligase-related genes and their drought resistance in soybean.In this study,the differentially expressed E3 ligase gene Gm PLR-2 was obtained by screening transcriptome sequencing.The soybean mutant M18 was used as the test material.The homologous PCR technology was used to clone the Gm PLR-2 gene and perform bioinformatics analysis.Real-time fluorescence quantification was used.PCR method was used to detect the change of transcription level of Gm PLR-2 gene under PEG simulated drought stress;the plant overexpression vector p CAMBIA3301-Gm PLR-2 and RNA interference expression vector p CAMBIA3301-Gm PLR-2-RNAi were constructed,and soybean Jinong 38 was transformed by Agrobacterium-mediated method,and breed in the greenhouse for additional generations.Finally,T₂transgenic plants were molecularly tested and identified for drought resistance.The results are as follows:1.The cloned Gm PLR-2 gene was obtained.The gene fragment was 324 bp in length and encoded 107 amino acids.The encoded protein has a typical RING-finger domain,which is located between amino acids 14 to 56 and is a RING-H2（C3H2C3）Type protein with 100%homology to the protein GLYMA＿17G213300.2.Gm PLR-2 gene is expressed in soybean roots,stems,and leaves,and the relative expression level in leaves is the highest,which is about 2.5 times that in roots;the relative expression level in stems is the second,about 1.7 times in the root,the expression level from high to low is:leaf>stem>root.Under simulated drought stress of PEG6000,the Gm PLR-2 gene showed a trend of rising first and then decreasing,reaching the highest value at 6 hours,and then decreasing.3.The results of double digestion showed that the plant overexpression vector p CAMBIA3301-Gm PLR-2 and the RNA interference expression vector p CAMBIA3301-Gm PLR-2-RNAi were successfully constructed and transformed into Jinong 38,and T₂generation transgenic plants were obtained by breeding in the greenhouse.4.After PCR detection,12 T₂overexpression positive plants were obtained,and 11 RNA interference-positive plants were obtained.Southern Blot analysis showed that the genes had been integrated into the genome of the recipient Jinong 38,and different hybridization bands appeared in different plants,indicating that the gene introduction integration sites were different.Quantitative fluorescent detection showed that the expression level of Gm PLR-2 gene in over-expressed plants was significantly higher than that in untransformed plants,which was about 2.37 times that of untransformed plants.The expression level in RNA interference-positive plants was significantly lower than that of untransformed plants,which was about 0.5 times of transformed plants.5.After seven days of drought stress,overexpression plants,RNA interference plants,and untransformed plants all experienced varying degrees of wilting.Overexpression plants were less wilted than untransformed plants,while RNA interference plants were more wilted than untransformed plants.The dry matter content in roots of over-expressed plants was significantly higher than untransformed plants,the dry matter content in roots of RNA interference plants was significantly lower than untransformed plants.The relative water content,POD,SOD,Pro content in over-expressed plants were higher than untransformed plants.The content of RNA interference plants is lower than that of untransformed plants.On the other hand,the relative conductivity and MDA content of overexpressed plants are lower than those of untransformed plants,while the content of RNA interference plants is higher than that of untransformed plants,and the differences are very significant,indicating that the expression of Gm PLR-2 gene is related to changes in soybean drought-resistance physiology and root morphology,which affects soybean drought resistance.