Isolation Of Soybean[Glycine Max(L.)Merr.] Gm2-MMP Promoter And Screening Of Gm2-MMP Interaction Proteins

Soybean[Glycine max(L.)Merr,]is the momentous food crop,it is the main source of the vegetable protein all over the world.In the South China,high temperature and humidity(HTH)stress is one of the major adverse factors which affect spring soybean seed physiological maturity(R6 and R7)in the field,pre-harvest seed deterioration often occur during this time,it easily leads seed vigor lower.Matrix metalloproteinases(MMPs)are a family of zinc-and calcium-dependent proteases belonging to the metzincin clan of metalloen-dopeptidases,EC subclass 3.4.24,MA(M)clan.They are widely distributed in animals and plants,and they can also degrade all the components of the extracellular matrix(ECM).Plant MMPs may be involved in many aspects,such as development regulation,immune responses and abiotic stress responses.Based on our previous proteomics study,we found that after HTH treatment,MMPs protein was up-regulated in Xiangdou No.3(pre-harvest seed deterioration resistant germplasm).In order to understand MMP functions in soybean growth and development and stress resistance.In this study,Gm2-MMP gene was selected as the research object,two soybean cultivars,Xiangdou No.3(pre-harvest seed deterioration-resistant germplasm)and Ningzhen No.1(sensitive)were used as experimental materials.The follow studies were carried out:(1)Isolation of Gm2-MMP promoter,and analyze the cis-acting elements within the Gm2-MMP promoter;(2)Identification of Gm2-MMP interactive proteins using yeast two-hybrid system;(3)Bioinformatics analysis and subcellular localization of Gm-PM31;(4)Tissue-specific expression analysis and expression characteristics analysis under high temperature and high humidity stress of Gm-PM31.The main results are as following:1.The promoter(1596 bp)sequence of Gm2-MMP was islolated and which contained numerous specific binding sites of stress-related transcriptional factors.In particular,it contains the elements of heat shock response(CCAATBOX1),dehydration and etiolation induced(ACGTATERD1),response drought,light regulated,and etc.The results of 5’-terminal Gm2-MMP promoter deletions suggested that the fragment from-359 bp to-223 bp is essential for the promoter activity in response to HTH stress.2.Using Gm2-MMP as bait,Gm2-MMP interacting proteins were screened by yeast two-hybrid(Y2H)from Glycine max(L.)Merr.seeds cDNA library.Thirty-nine proteins have been screened initial:globulin,oleosin,seed maturation protein,albumin,trypsin-inhibitor,sucrose-binding protein,aquaporin,glycine-rich protein,transport protein,transcription factor of MYB,60S ribosomal protein,calcium-binding protein,dehydrin protein,Ca2+ combined with EF hand protein and unknown function protein.3.Bioinformatics analysis showed that Gm-PM31 has the typical ACD-ScHsp26-like and IbpA domains,and Leguminosae had a closest relationship with Gm-PM31.Transient expression of the Gm-PM31-GFP protein in tobacco leaf cells showed that Gm-PM31 protein are localized in Golgi apparatus.The interaction between Gm2-MMP and Gm-PM31 protein in vivo was confirmed by yeast two hybrid technique and bimolecular fluorescence complementation assay(BiFC).4.The results of qRT-PCR showed that the levels expression of Gm-PM31 gene in mature seeds of cv.Xiangdou No.3 and cv.Ningzhen No.1 were the highest,while in other tissues,the levels were very low or almost undetectable.The expression of Gm-PM31 increased first and then decreased during the development of soybean seeds,the expression was the highest at 45 DAF(days after flowering).Compared to the corresponding control,Gm-PM31 was found to be markedly down-regulated in cv.Ningzhen No.1 at 96 h,and up-regulated at 168 h during HTH stress.Compared to the corresponding control,Gm-PM31 markedly up-regulated in cv.Xiangdou No.3 at 96 h and 168 h during HTH stress.