ABSTRACT
Young leaves of Kabuli chickpea as well as soybean Xiangdou No.3, which are the current plants that studied in our laboratory were selected as materials. Effects on protoplasts yield and survival rate of different enzyme combination, concentration of D-Mannitol in enzyme combinations, pH of enzyme combinations and enzymolysis time are detected. The results showed that, the best condition for Xiangdou No.3 leaf protoplasts isolation is to rotate the cut materials for 6 hours in enyzme solution under temperature of 27 ℃ and rotate speed of 45 r/min for 6 h. Onozuka R-10 (0.5%), Hemicellulase (0.8%), Macerozyme R-10 (0.8%) in combination with Pectolyase Y-23 (0.4%) dissolving in CPW solution with MES (0.1%) and Mannitol (10%), pH 6.0 was found best for protoplasts isolation of Xiangdou No.3 leaves.The best condition for protoplasts isolation of Kabuli chickpea is to put the cut materials into enzymatic hydrolysate enzymolyse for 7 to 8 hours under temperature of 27 ℃ and rotate speed of 45 r/min on water bath shaker, the optimum combination of enzyme consists of Onozuka R-10 (0.5%), Hemicellulase (0.8%), Macerozyme R-10 (0.8%), MES (0.1%) and Mannitol (10%) dissolved in CPW solution with pH 4.8. The protoplasts prepared with the methods above are used in subcellular location and the effects show well.
ABSTRACT
High temperature and humidity stress during seed growth and development of spring soybean can result in seed deterioration in South China. We isolated two genes (GmSBP and GmSBPL) encoding putative SBP proteins from soybean (Glycine max (L.) Merr.) to study their biological functions and response to abiotic stress,. The two SBP proteins are hydrophilic and incomplete membrane ones. Real-time quantitative (RT-PCR) analysis reveals that the expression of the two genes in the developing seeds of the seed deterioration resistant cultivar Xiangdou No. 3 and sensitive cultivar Ningzhen No. 1 was significantly affected by high temperature and humidity treatment. Meanwhile, the levels of sucrose and soluble sugar in the developing seeds of both cultivars were also affected under high temperature and humidity stress. During seed growth and development, the expression of the two genes as well as the levels of sucrose and soluble sugar reached the highest at 30 days after flower. GmSBP2 and GmSBPL were found to be differentially expressed in different soybean tissues. Sub-cellular localization indicated that two genes were located in cytoplasm and cell membrane. Our results indicate that GmSBP2 and GmSBPL might be involved in the response to abiotic stress, which will enrich our understanding of pre-harvest seed deterioration and resistance in soybean from one side.