Cloning And Functional Analyses Of Gmsk1 And Gmbzip99 In Soybean

Soybean(Glycine Max L.)is an important food and economic crop with rich protein in seeds.It is not only major source of human protein and lipid,but also the important feed and industrial raw materials.In the process of soybean growth and development,many kinds of stress conditions,such as diseases,high temperature,low temperature,drought,waterlogging,salinization of soil,etc,seriously impact on soybean growth by reducing the yield and quality,causing huge losses to the soybean production.Genetic improvement and breeding of resistant soybean can improve soybean tolerantance to various stresses and is one of important ways for high yield and good quality of soybean.Under stress,on the one hand,related protein turnover is regulated and abnormal proteins are degraded by protein degradation system and so dynamic balance of life activities is kept.Ubiquitin-26S proteasome pathway is the most important and highly efficient proteinaceous selective degradation pathway in eukaryotic cell.On the other hand,plants regulate the expression of defense genes related to stress through regulation of protein,then adapt to the new environment.Transcriptional regulation is completed by transcription factors.Therefore,Learning more about the mechanism of ubiquitin-26s protein degradation pathway and regulation gene transcription,is of great significance to genetically improve tolerance to different kinds of stress in plants.SKP1(S-phase kinase-associated-protein 1)protein family generally exists in eukaryotes as a kind of small molecular weight proteins.SKP1,cullin1/Cdc53,Rbx1/Rocl/Hrtl and F-box protein form SCF complex which is involved in ubiquitin-mediated protein degradation processes.Based on soybean genome database,we obtained 15 soybean SKP1 genes,which are distributed on 11 chromosomes in soybean.They are divided into 2 subfamilies according to phylogenetic analysis.Expression patterns of SKP1 gene of soybean in different tissues such as roots,stems,leaves,flowers,pods and seeds are analyzed using gene chip data and real-time fluorescent quantitative PCR.The result indicated that most,if not all,of SKP1 gene in soybean are constitutively expressed.However,different soybean SKP1 gene has its unique expression pattern.We isolated a SKP1 homologous gene in soybean,named GmSK1(Glycine max SKP1-like1).The results showed that the full length of GmSK1 cDNA sequence is 1125bp.GmSK1 contains two exons and one intron,located on chromosome 11.DNA hybridization showed that GmSK1 has multiple copies in soybean genome.GmSK1 encodes a predicted protein of 155 amino acids,which has the typical characteristics of the SKP1 protein with structure characteristic of SKP1 protein,and highly homologous to multiple SKP1 protein in other plants.Phylogenetic analysis showed that GmSK1 belongs to the subfamily I.Subcellular localization revealed GmSK1 protein is localized in the cytoplasm and nucleus.Expression of GmSK1 is up-regulated after the induction of ABA,JA and SA,suggesting that GmSK1 is involved in plant hormone signal transduction.Expression of GmSK1 were induced by drought(20%PEG6000),low temperature(4?)and high salt(150 mM NaC1 solution),suggesting that GmSK1 is also involved in the drought,cold,salt stress reaction process.Heterologous overexpression of GmSK1 in tobacco,transgenic plants(T2 gernation)show short plant height,small leaf blade and slow growth compared to wild-type tobacco.Further,transgenic tobacco plants were treated with 8%PEG6000 and 150 mM NaCl,it was found that overexpressing of GmSK1 in transgenic tobacco can significantly enhance the tolerance to drought and high salt in tobacco plants.Basic leucine zipper(basic region/leucine zipper motif,bZIP)transcription factor is one of the largest families of transcription factors and is widely distributed in eukaryotes.We isolated a gene encoding bZIP transcription factor in soybean,GmbZIP99.The full-length cDNA sequence of this gene is 1767bp in length.GmbZIP99 gene encodes a predicted protein of 469 amino acids,with a typical bZIP domain and the D class transcription factor characteristic motifs.GmbZIP99 has high homology with bZIP gene in Arabidopsis,alfalfa,tobacco,rice,corn and other plants.Phylogenetic analysis indicated that GmbZIP99 belongs to the D class bZIP transcription factor.DNA hybridization showed that this gene is low-copy existing in soybean genome.The results of subcellular localization showed that the protein is localized to the nucleus.Yeast one-hybrid experiment showed that the protein has transcriptional activation activity.Real-time fluorescent quantitative analysis showed that GmbZIP99 can express in root,stem,leaf,flower and pod,but the expression level is varied in different tissues.For example,GmbZIP99 is highly expressed in flower but its expression level is very low in seed.Further experiments showed that expression of GmbZIP99 is up-regulated after the induction of ABA,JA and SA,suggesting that GmbZIP99 is involved in plant hormone signal transduction.Expression of GmbZIP99 is increased after treated with drought(20%PEG6000),low temperature(4?)and high salt(150 mM NaCl solution),indicated GmbZIP99 may be related to drought,salt and cold stress and can enhace the planttolerance to drought,salt and chilling of soybean.