Research About Drought Tolerance Mechanism Of Soybean Phospholipase C GmPLC10

As sessile organism on land,plants growth and development will inevitably be affected by the surrounding environment.Put it another way,the growth and development of plants will be affected by abiotic stress,and then the seed setting rate of plants will be influenced.If crops suffer from abiotic stress,it will lead to a large reduction in crop yield.Consequently,it is a new trend of crop cultivation to breed new crop lines that can resist abiotic stress and improve crop yield.Soybean is one of the three major crops in China.Due to the large volume of demand,half of the aggregate soybean on the market depends on imports.With the exacerbation of the environment,the trend of land desertification is becoming more and more serious,and there is less and less high-quality land for soybean growth.Hence,it is necessary to cultivate new drought resistant soybean lines.In recent years,it has been proved that many genes are involved in the response of soybean to drought stress.Despite there are also testimony that PLC is involved in the response of soybean to drought stress,there are relatively few studies.Through the study on the function of GmPLC10,it was proved that GmPLC10 could improve the tolerance of soybean to drought stress.The specific research contents are as follows:1.By constructing the recombinantexpression vector p CAMBIA3301-GmPLC10,the soybean plants overexpressing GmPLC10 were obtained by agrobacterium mediated genetic transformation of soybean cotyledon node and soybean hairy root transformation.The drought stress phenotype was observed.It was preliminarily proved that GmPLC10 can improve the drought tolerance of soybean.2.Under drought stress,the physiological and biochemical indexes of over expressing GmPLC10 soybean plants were detected.Physiological and biochemical indexes include fresh weight,dry weight,water content,malondialdehyde（MDA）content,hydrogen peroxide（H₂O₂）content,proline（Pro）content,soluble sugar content,soluble total protein content,catalase（CAT）activity,peroxidase（POD）activity and superoxide dismutase（SOD）activity.Through the detection of physiological and biochemical indexes and DAB and NBT staining of soybean leaves,it is further proved that GmPLC10 can improve the drought tolerance of soybean.3.The enzyme activity of GmPLC10 was determined by ELISA.It is proved that GmPLC10 has PI-PLC activity and can hydrolyze PIP₂.And further detect the contents of DAG,IP₃ and PA of GmPLC10 hydrolysates under drought stress.It shows that GmPLC10 can regulate the response of soybean to drought stress.4.GmPLC10 was heterologously expressed by INVSc1 yeast cells.The transgenic yeast grew well under mannitol simulated osmotic stress,and then the drought tolerance of GmPLC10 was determined.5.Under drought stress,the K⁺content and stomatal closure of soybean leaves overexpressing GmPLC10 were detected,and using q PCR detection and lipid-protein coprecipitation,it was proved that the product PA of GmPLC10 could bind to Gm AKT2 and regulate stomatal movement,Then,gmplc10 and gmakt2 soybean plants were obtained by VIGS method.By observing the drought phenotype,it was finally proved that GmPLC10 regulates stomatal movement through the combination of PA and Gm AKT2,so as to improve the drought tolerance of plants.6.Yeast two hybrid technique（Y2H）,bimolecular fluorescence complementary experiment（Bi FC）and in vitro protein Pull-down experiment were used to prove the direct combination of GmPLC10 and Gm IPK2.Soybean plants overexpressing Gm IPK2 were obtained through agrobacterium mediated soybean hairy roots transformation system,and their drought phenotypes were observed to prove that GmPLC10 combined with Gm IPK2 to improve soybean drought tolerance.