Functional Analysis Of Soybean GmLEA,GmFAD2-1B And GmPM27 Genes In Seed Vigor Formation Under High Temperature And High Humidity

Soybean[Glycine max(L.)Merr.]is one of the most important high-protein grain-forage crops in the world.Spring soybean in southern China is one of the main producing areas.However,physiological maturation period of soybean seed is the time of high temperature and humidity(HTH)in southern China,pre-harvest seed deterioration often occur in this time.The seed vigor,yield and quality will decrease after pre-harvest seed deterioration.Late embiyogenesis abundant protein(LEA)exists widely in seeds of higher plants.It has functions of binding metal ions,scavenging reactive oxygen radicals,protecting enzyme activity and enhancing plant's ability of dehydration.Delta 12 fatty acid desaturases(FAD2)is distributed in several species of Cruciferae.In many higher plants,It has been proved that FAD2 can adapt to temperature stress through regulating the synthesis of unsaturated fatty acids.Seed maturity protein(PM27)is a kind of protein related to seed maturity,and it is one of the potential target genes of miR164c related to seed vigor.In previous studies,we found that GmCDPKSK5 was related to the formation of seed vigor of spring soybean under high temperature and high humidity stress.A yeast two hybrid library of spring soybean seed membrane protein was constructed under high temperature and high humidity stress,and GmCDPKSK5 was used as bait to screen proteins interacting on cell membrane,the protein GmLEA,GmFAD2-1B and GmPM27 interacted with GmCDPKSK5 were preliminarily determined.In this study,the following studies were carried out:(1)Verification of the interaction between three proteins(GmLEA,GmFAD2-1B and GmPM27)and GmCDPKSK5,respectively;(2)The isolation of GmLEA,GmFAD2-1B and GmPM27 gene and the subcellular localization of they encoded protein;(3)The expression pattern analysis of three genes of GmLEA,GmFAD2-1B and GmPM27 in the seed of two soybean cultivars Xiangdou No.3(pre-harvest seed deterioration-resistant)and Ningzhen No.1(sensitive);(4)The effects of overexpression of GmLEA and GmFAD2-1B on seed vigor in Arabidopsis thaliana.These results will helpful to understand the functions of GmLEA,GmFAD2-1B and GmPM27 involved in the formation of soybean seed vigor and response to abiotic stress.These results of the study are as follows:1.The results of yeast two-hybrid rotation verification showed that:GmCDPKSK5 has specific interactions with GmLEA,GmFAD2-1B and GmPM27 in yeast,respectively.In addition,bimolecular fluorescence complementation(BiFC)experiment showed that the three proteins(GmLEA,GmFAD2-1B and GmPM27)could interacted with GmCDPKSK5 on cell membrane of tobacco leaf cells.2.The cDNA sequence of GmLEA gene contains a 1377 bp open reading frame(ORF),and the DNA sequence is 1879 bp,which consists of two exons and one intron.The subcellular localization result showed that the encoded protein was located on the cell membrane;GmFAD2-1B gene contains a long 1164 bp ORF,and the DNA sequence was 1164 bp,which had no introns.The subcellular localization result showed the encoded protein was located on the cell membrane;GmPM27 gene contains a long 1020 bp ORF,and the DNA sequence was 1135 bp,which consists of two exons and an intron,The subcellular localization result showed the encoded protein was located on the cell membrane and nucleus.3.Real-time quantitative PCR analysis showed that GmLEA gene was mainly expressed in the mature seeds of the cv.Xiangdou No.3 and cv.Ningzhen No.1.The expression level of GmLEA was increased first and then decreased during the development of cv.Xiangdou No.3 seeds.During the process of seed development of cv.Ningzhen No.1,the level of GmLEA expression was on the rise and reached the maximum at 60 days after flowering;After high temperature and high humidity(HTH)stress,the expression of GmLEA was decreased at 96 h in the cv.Xiangdou No.3,and the other time points were increased;in cv.Ningzhen No.1,the expression was decreased at 24 h.GmFAD2-1B was mainly expressed in mature seeds of both of the cvs.During the seed development,expression of GmFAD2-1B was increased first and then decreased,and reached the maximum at the 20 day after flowering;After HTH stress,compared with the control,the expression of GmFAD2-1B was up-regulated at all time points in the cv.xiangdou No.3.However,in cv.Ningzhen No.1,GMFAD2-1B was only up-regulated at 24 h and 168 h,and was down-regulated at 48 h and 96 h.GmPM27 was also mainly expressed in mature seeds.During the process of seed development,the expression of GmPM27 was increased first and then decreased,and reached the maximum at 50 days after flowering.After HTH stress,compared with the control,in cv.Xiangdou No.3,GmPM27 was up-regulated at 24 h,96h,and 168 h.However,in cv.Ningzhen No.1,GmPM27 was up-regulated at all the time points,but the expression of GmPM27 in the seeds of cv.Xiangdou No.3 was much higher than that in cv.Ningzhen No 1.The experimental results showed that these three genes were differentially expressed in soybean seeds before and after high-temperature and high-humidity stress,presumably participating in the formation of seed vigor under high temperature and high humidity stress.4.Compared with the control,after HTH stress,the germination rate,germination potential and germination index of WT,pBI121-GUS transgenic lines,GmLEA transgenic lines and GmFAD2-1B transgenic lines were decreased in Arabidopsis thaliana,However,the germination rate,germination potential and germination index of GmLEA transgenic lines and GmFAD2-1B transgenic lines were better than those of WT and pBI121-GUS transgenic lines.In addition,the average germination days of GmLEA transgenic lines and GmFAD2-1B transgenic lines were lower than those of WT and pBI121-GUS transgenic lines;Moreover,it was shown that the seed vigor of GmLEA transgenic lines and GmFAD2-1B transgenic lines were higher than that of WT and pBI121-GUS transgenic line.It is suggested that over expression of GmLEA and GmFAD2-1B could enhance the vigor of Arabidopsis thaliana seed.