Isolation And Expression Analysis Of GmSbh1 That Involved In Seed Deteriration

Soybean(Glycine max L.)is not only one of the world’s five top crops,but also the world’s most important oil crops and high-protein grain-forage crop,which plays an important role in planting structure adjustment in China.Spring soybean in southern China is one of the main producing areas,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,and pre-harvest seed deterioration has become the obstacle of soybean industry development in this area.Therefore,the mechanism research of pre-harvest seed deterioration is the prerequisite and foundation to carry to resistant breeding and to promote market competitiveness of soybean.Homeobox genes are playing an important role in regulating plant growth and development and responding to adversity stress or hormone induction.GmSbh1(L13663)was the first homeobox gene that cloned from soybean,GmSBH1 protein might involve in pre-harvest seed deterioration by proteomics research in our previous studies.In this study,two soybean cultivars,Xiangdou No.3(pre-harvest seed deterioration-resistant)and Ningzhen No.1(sensitive)were used as experimental materials.The follow studies were carried out.First,the identification of resistance to pre-harvest seed deterioration and the physiological mechanism study on pre-harvest seed deterioration in spring soybean.Second,the isolation and bioinformatics analysis of GmSbhl and it’s promoter.Third,the differential expression analysis of GmSbhl gene amnong differernt cultivars,tissues and organs,development and germination stage,leaf aging process and respond to HTH stress and hormone induction.Fourth,subcellular localization and transcriptional activation assy of GmSBHl protein.Fifth,the functional identification of GmSbhl gene involve in pre-harvest seed deterioration.Main results of this study are as follows.The aim of this study is to establish the basis to illustrate the mechanism of GmSbhl,which involve in pre-harvest seed deterioration,and the breedling theoretical basis of pre-harvest seed deterioration-resistant spring soybean cultivar.Effects of HTH stress(40℃/30℃,RH 100%/70%,10 h/14 h(day/night))on plant growth,seed vigor indexes,main nutritional components and coat anatomical structure of both the cultivars at physiological maturity stage(R7)were investigated.Growth mass of pod and stem leaf in both the cultivars were no significant difference when the stress time under 24 h,however,the growth of pod and stem leaf in cv.Ningzhen No.1 were restrained more serious than cv.Xiangdou No.3 when the stress time exceed 48h.HTH stress was found to cause the decrease of seed viability,germination energy,and germination percentage as well as the reduction of dehydrogenase and acid phosphatase activities,but increase seed cell membrane permeability as well as H+,soluble sugar and leucine levels in seed soaking liquid in the two cultivars.Moreover,the stress led to irregular change of seed oil and protein contents and alteration of anatomical structure of episperm and hilum in the two cultivars.Short term stress(less than 5 h)had no significant impact on seed vigor,but long term one(more than 48 h)caused rapid decrease of the indexes related to seed vigor.After stress,soybean cultivar Xiangdou No.3 was characteristics of compact seed coat and inact hilum and showed less decrease of seed germination energy,seed germination percentage and related enzyme activity and less increase of extravasation content in seed soaking liquid than soybean cultivar Ningzhen No.1.All these results may be partially the reasons why soybean cultivar Xiangdou No.3 is more resistant to pre-harvest seed deterioration than soybean cultivar Ningzhen No.1.The best condition to identify the pre-harvest seed deterioration resistance of spring soybean is 40℃/30℃,RH 100%/70%,10 h/14 h(day/night),and stress for 48 h.The sequences of cDNA,DNA and promoter of GmSbhl gene were isolated respectively,and the bioinformatics of those sequences were analyzed.The results showed that the intact ORF of GmSbh1 is 1137 bp(156～1293 bp)and it encoded a 42.37kDa protein with 379 amino acids.The GmSBH1 protein contains the KNOX1、KNOX2、ELK and Homeodomain(HD).Its DNA sequence has three introns.There were 18 homologous sequences with more than 80%identity.The promoter(2043bp)sequence of GmSbhl was isolated and which contains many cis acting elements such as 34 YACT,14 AAAQ 11 ARRland 9 AGAAA,the transcriptional start site(TSS)be located upstream 751bp of ROF,TATAbox at-27 bp,and CAATbox at-144 bp,respectively.GmSBH1 was a hydrophilic protein without signal peptide and contains 10%Ser,9.8%Asn and 8.4%Leu,the phosphorylation sites be located at Ser(13),Tys(2)and Thr(1),respectively.Secondary structure prediction results show that the GmSBHlprotein contains alpha-helix(39.31%),beta turn(5.28%),extended strand(8.44%)and random coil(46.97%),but without transmembrane helical structure.The probability of GmSH1 interact with RPL,BEL1,AT4G32980.1-P,BLH2 and BLH3 was higher,and coexpression with BEL1 in Arabibidopsis.Real-time quantitative PCR analysis showed that GmSbhl was expressed in root,stem,leaf,flower,green pod and mature seed of soybean,and especially high in flower in Ningzhen No.1,and in green pod in Xiangdou No.3.The expression in Glycine soja was higher than another cultivars,and we found that the expression of GmSbhl relevant to resistance,the expression of pre-harvest seed deterioration-resistant cultivar higher than sensitive cultivar.The expression of GmSbhl increased with the development and maturation of soybean seed,and the maximum at full ripe stage(R8).The expression increased with seed germination,there has an increasement when seed imbibition 24～48 h,and then decreased.Firstly,the expression of GmSbhl increased and then decreased at leaf aging process,and the maximum at function leaf stage.The expression increased with HTH stress time extended,especially when the stress time exceeded 48 h,the expression was up-regulated compared with control in Ningzhen No.1,but down-regulated in Xiangdou No.3 under THT stress at the physiological maturation stage.There was no significant expression difference with 100μM ABA,the leaf expression of GmSbhl in Ningzhen No.1 decreased significantly with 100μM MeJA,and increased in Xiangdou No.3,the expression of both the cultivars increase firstly and then decrease with 100μM GA3,and declined with 20μM IAA.To determine the subcellular localization of GmSBHl protein,we introduced GmSBH1-GFP,N-terminal(KNOX1 and KNOX2)and C-terminal(ELK and HD)constructs into soybean and Arabidopsis mesophyll protoplasts.The confocal laser scanning microscopy showed that the GmSBH1-GFP and C-terminal fusion protein exhibited clear signals in the nucleus in both the plant cell types,indicating that GmSBH1 was a nuclear protein with NLS in the C-terminal.To identify and characterize the transcriptional activation activity of GmSBH1,its full-length and different truncated ORF were cloned into the pGBKT7 plasmid,these constructed plasmids were then transformed into the yeast strain AH109,the transformed yeast cells were grown on the His-deficient medium to check the transcriptional activation activity according to LacZ staining and relative quantitative assay ofβ-galactosidase activity.The results indicated that GmSBH1 had the transcriptional activation activity with transcriptional activation sites located in the HD domain and N-terminal nonconservative region,and KNOX1 domain was found to play an obvious role in suppressing transcriptional activation activity of GmSBH1.To characterize the biological function of the GmSbh1 gene in plant growth and development,we transformed it into Arabidopsis driven by the 35S promoter and homozygous transgenic lines were used for further analysis.The morphology of transgenic lines was dramatically changed compared with the wild type,such as linear seedlings cotyledon,broaden petiole,wrinkled and clustered leaves and thick root hair,growth stage of transgenic line was extended,and with cluster inflorescence and buckle silique.The result of TTC testing showed that seed viability under HTH stress(40℃,RH 100%,8 h)of transgenic lines was higher than wild type,and MDA contents decreased significantly.We found that the green seedlings of transgenic lines were significantly lower than wild type plants after 10 days treatment with 0.5μM and 1μM ABA,and root growth of transgenic lines were inhibited with 30μM and 50μM ABA.The expression of AtRD29A,AtRD29B and AtABI3 that were typical marker genes induced by ABA significantly up-regulated with 100μM ABA treatment.These results may be indicated that transcription regulation of GmSBHl was induced by ABA signal.The expression of HTH responsive genes,AtHsfA2 and AtHsfB2A,significantly up-regulated compared with wild type under HTH stress.Overall result indicated that GmSbhl gene was likely to involve in pre-harvest seed deterioration.