GmHMADP And GmHP61 Involved In Seed Vigor Formation Of Soybean Under High Temperature And Humidity Stress And The Function Analysis Of GmHMADP Responsive To Copper And Cadmium Stress

Soybean(Glycine max[L.]Merrill)is one of the most important economic and food crop in the world,and it is also the strategic material of China.Abiotic stresses such as high temperature and high humidity(HTH),heavy metal stress are important factors that restrict the production of soybean and affect the development and quality of seed.Spring soybean in South China was usually in a high temperature and rainy season during the physiological maturity of seeds(R6-R7),and seed deterioration occurred in the field before harvest,reduced the vigor of seed and restricted the production of spring soybean severely.In addition,heavy metal pollution is one of environmental problems which cannot be ignored at present.It not only has a serious impact on the growth and development of crops,but also can endanger human health through the food chain.In previous studies,we found that GmSBH1 was involved in the response to high temperature and humidity stress and the formation of seed vigor.A yeast two hybrid library of spring soybean seed was constructed under high temperature and high humidity stress,and GmSBH1 was used as bait to screen proteins interacting on nucleus,the protein GmHMADP,and GmHP61 screened.In order to further study the function of soybean GmHMADP and GmHP61 in response to abiotic stress.In this study,the GmHMADP and GmHP61 genes were selected as the research object,two soybean cultivars,Xiangdou No.3(pre-harvest seed deterioration-resistant)and Ningzhen No.1(sensitive)were used as experimental materials.In this study,the following studies were carried out:(1)Isolation of GmHMADP and GmHP61 genes,and verification of the interaction of GmHMADP and GmHP61 with GmSBH1,respectively;(2)Expression analysis of GmHMADP and GmHP61 among differernt tissues,seed development stage of soybean and respond to HTH stress(40?/100%RH);(3)The functional identification of GmHMADP and GmHP61 genes involved in seed vigor formation under high temperature and high humidity stress in Arabidopsis;(4)Expression analysis of GmHMADP gene under heavy metal copper and cadmium stress,and functional identification of its role under copper and cadmium stress in Arabidopsis.The main findings are as following:1.The cDNA sequence of GmHMADP gene contained a 996 bp open reading frame(ORF),and the DNA sequence was 1814 bp,which consisted of four exons and three intron.By multiple alignment of the amino acid sequences of proteins with high homologous to GmHMADP,it was found that all of them contained the HMA domain and two metal ion binding site CXXC motifs.The cDNA sequence of GmHP61 gene contained a 468 bp open reading frame(ORF).Meanwhile,transient expression of the 35S::GmHMADP-GFP and 35S::GmHP61-GFP protein in tobacco mesophyll cells showed that GmHMADP and GmHP61 protein are localized in cell membrane and nucleus.The results of yeast two-hybrid and bimolecular fluorescence complementation assay(BiFC)experiments showed that GmSBH1 could interact with GmHMADP and GmHP61 in vivo,respectively.2.The results of qRT-PCR showed that the expression levels of GmHMADP gene were the highest in the mature and developing seeds of cv.Ningzhen No.1 and cv.Xiangdou No.3,respectively.During the process of seed development,the expression of GmHMADP gene increased first and then decreased,and reached the highest on the 40th day after flowering.Compared to the corresponding controls,the expression level of GmHMADP gene was significantly(p<0.01)increased in seeds of cv.Ningzhen No.l at the HTH stress time points 48,96,and 168h;the expression level of GmHMADP gene was significantly(p<0.01)increased in seeds of cv.Xiangdou No.3 at the HTH stress time points 24,48,96,and 168 h;In addition,GmHMADP was significantly(p<0.01)increased under Cu2+ and Cd2+treatment with different concentrations in seedling root of cv.Xiangdou No.3,while in that of cv.Ningzhen No.1,the expression level of GmHMADP gene was significantly(p<0.01)increased only under 50 ?mol·L-1 Cu2+and 200 ?mol·L-1 Cd2+ treatment.The expression levels of GmHP61 gene were the highest in the mature and developing seeds of cv.Ningzhen No.1 and cv.Xiangdou No.3,respectively.During the process of seed development,the expression level reached the highest on the 40th day after flowering in cv.Ningzhen No.1 and the highest on the 30th day after flowering in cv.Xiangdou No.3.Compared to the corresponding controls,the expression level of GmHP61 gene was significantly(p<0.01)increased in seeds of cv.Ningzhen No.1 at the HTH stress time points 48 and 168 h;the expression level of GmHP61 gene was significantly(p<0.01)increased in seeds of cv.Xiangdou No.3 at the HTH stress time points 24,96,and 168 h.The above results indicated that GmHMADP and GmHP61 genes responded to high temperature and high humidity at the transcriptional level,and GmHMADP gene could also respond to copper and cadmium stress at the transcriptional level.3.GmHMADP and GmHP61 overexpressed Arabidopsis strains were obtained by Agrobacterium inoculation,respectively.The seed germination rate,germination potential,germination index and vigor index of GmHMADP and GmHP61 transgenic plants were all better than that of WT under HTH stress.It is suggested that overexpression of GmHMADP and GmHP61 could enhance the seed vigor of Arabidopsis.In addition,the root length and dry weight of GmHMADP transgenic Arabidopsis were significantly(p<0.05)higher than those of the wild type under copper and cadmium stress.It will help us to understand the functions of GmHMADP and GmHP61 of soybean in plant growth and development,and in response to high temperature and humidity stress.