Functional Studay Of Soybean [Glycine Max(L.)Merr.] HMADP Genes Under Cu And Cd Stress

Soybean(Glycine max(L.)Merrill)is one of the most important economic crops and strategic material in China.However,in recent years,high temperature and high humidity(HTH)stress and heavy metal pollutionhave become increasingly serious,and it has become a key factor restrict in agricultural development.High temperature and high humidity stress is extremely easy to seriously affect the normal growth and development of plants and their metabolism,and is a key environmental factor limiting plant growth,distribution and productivity(Gulen H,2004;Ghouil H,2003;Chaitanya KV,2002).Heavy metals are difficultly biodegrad and easily accumulate in soil and water,which not only seriously affects crop growth and development,but also endangers human health through the food chain(Zhang et al.,2018).Heavy-metal-associated domain(HMAD)is a conserved domain containing approximately 30 amino acids and was found in some heavy metal ion transport and detoxification proteins.Heavy metal transporting ATPases(HMAs)contain Heavy-metal-associated domain(HMA),which can promote the trans-membrane movement of heavy metal ions to achieve detoxification(Arguello et al.,2007;Williams and Mills et al.,2005).At present.the research of HMAs is mainly concentrated in rice and arabidopsis,and less research is carried out in soybeans(Mikkelsen et al.,2012;Yeh et al.,2007).When the heavy metal content in plants is excessive,HMA promotes transmembrane movement by utilizing the energy released by ATP hydrolysis to achieve detoxification.1.In this paper,the high-vigor genetics germplasm Xiangdou No.3 was used as the material,and the Cu、Cd stress were carried out in the hydroponic period before the physiological maturity stage and the physiological maturity to the full maturity stage.Mainly carry out the following research:(1)effects and expression patterns of GmHMADP gene expression in soybean seedling stage and reproductive growth stage after Cu and Cd stress treatment;(2)measured the accumulation of Cu/Cd in different tissues of soybean after different concentrations of Cu/Cd stress;(3)determination of physiological indexes of GmHMADP overexpressing Arabidopsis lines after Cu、Cd stress;(4)determination of physiological indexes of GmHMADP overexpressing Arabidopsis lines after HTH stress.The aim is to discover the role of soybean GmHMADP gene in response to heavy metal Cu、Cd stress at the molecular level;exploring the application value of soybean in the utilization of Cu、Cd contaminated soil,to provide excellent candidate genes for plant resistance breeding,And provide theoretical basis and practical basis for cultivating new soybean resistant varieties,and make efforts for the safe production of soybeans under the conditions of heavy metal pollution in farmland in China.The main findings are as follows:After Cu、Cd stress in soybean seedling growth period,the expression of GmHMADP decreased first and then increased in roots,and the expression reached the maximum after 24h,significantly down-regulated at 48h of stress and significantly up-regulated at 168h;in stems,GmHMADP showed that almost no expression at 24h after stress treatment,but significantly up-regulated at 48h after stress treatment and significantly down-regulated at 96h;in leaves,Compared with the control,the expression of GmHMADP increased first,then decreased and then increased.GmHMADP was almost not expressed at 24h after stress treatment,significantly up-regulated at 48h,significantly down-regulated at 96 h,and significantly increased at 168h.2.qRT-PCR results after soybean Cu、Cd stress treatment in reproductive growth stage showed that:Compared to the control,the expression of GmHMADP in roots and leaves increased first and then decreased,and reached the maximum in R4 phase and decreased significantly in R5 phase.In the grain,the target gene was significantly up-regulated in R8 phase,and The higher the treatment concentration,the higher the amount of gene expression.3.Inductively coupled plasma mass spectrometry method showed that:after short-term Cu、Cd treatment,the accumulation of Cu、Cd in soybean seedling roots was significantly higher than that of stems and leaves.At the entire growth period of soybean plants,It is accumulate in soybean roots,stems,leaves,pods and grains,and the accumulation of Cd is higher than that of Cu.Cd accumulated the highest amount in the roots,followed by the grain;Cu accumulated the highest in the roots,followed by the leaves.4.Physiological indicators of GmHMADP overexpressing Arabidopsis lines Cu、Cd stress showed that:compare with the control,the activities of antioxidant enzymes(SOD,POD,CAT)were significantly increased(p<0.01)and the MDA content was significantly decreased in different transgenic Arabidopsis after Cu、Cd stress.These phenomena preliminarily indicate that the GmHMADP gene is resistant to Cu、Cd stress.5.Physiological indicators of GmHMADP overexpressing Arabidopsis lines HTH stress showed that:compare to the control,the activities of antioxidant enzymes(SOD,POD,CAT)were significantly increased(p<0.01)and the MDA content was significantly decreased in transgenic Arabidopsis lines after stress,and the H2O2 and ROS content is less than wild type Arabidopsis.These phenomena preliminarily indicate that the GmHMADP gene is resistant to HTH stress.