| Matrix metalloproteinases(MMPs)are a family of zinc-and calcium-dependent endopeptidases.MMPs play an important role in plant growth,development and response to biotic and abiotic stress.Soybean[Glycine max(L.)Merr.]is the largest oilseed crop in the world,and plays an important role in planting structure adjustment in China.Spring soybean seeds often meet the hot and rainy summer during physiological maturation,seed deterioration occurred in the field before harvest and seriously restricted the yield and quality of spring soybean in South China.Gml-MMP protein accumulated in abundance under high temperature and humidity(HTH)stress(40 ?/100%RH),indicating it might involve in pre-harvest seed deterioration resistance by our previous proteomics study.In order to further study the function of soybean MMP.s in response to HTH stress.In this study,the Gm1-MMP and Gm2-MMP 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.The follow studies were carried out:(1)Isolation,bioinfonrmatics analysis and subcellular localization of Gml-MMP and Gm2-MMP,(2)Expression analysis of Gm1-MMP and Gm2-MMP gene among differernt tissues,seed development stage and respond to HTH stress(40 ?/100%RH),(3)Identification of Gm1-MMP interactive proteins using yeast two-hybrid system,(4)Isolation,bioinformatics analysis,validation and analysis of interaction between Gm2-MMP and GmMT-? and expression analysis of GmMT-?.(6)The functional identification of Gm1-MMP and Gm2-MMP genes involved in plant growth and development and HTH stress.The main results are as following:1.The cDNA sequence of Gm1-MMP gene contained a 915 bp open reading frame(ORF),it dose not carry any intron.Bioinformatics analysis showed that Gm1-MMP has the typical MMPs conserved domain:signal peptide,propeptide and catalytic domain.Interestingly,Gml-MMP had a lower phylogenetic relationship with MMPs in other species.The promoter(2116 bp)sequence of Gm1-MMP was islolated and which contained numerous specific binding sites of stress-related transcriptional factors.Two heat shock responsive elements,PRECONSCRHSP70A element and CCAAT box were also detected on the promoter,the transcriptional start site(TSS)was located upstream 78 bp of ORF.The results of 5'-terminal Gml-MMP promoters deletions suggested that the fragment from-399 to-299 is essential for the promoter activity in response to HTH stress.The cDNA sequence of Gm2-MMP gene contained a 1071 bp ORF,it also dose not carry any intron.Bioinformatics analysis showed that Gm2-MMP has the typical MMPs conserved domain,but also contained a transmembrane domain.Meanwhile,transient expression of the Gm1-MMP::GFP and Gm2-MMP::GFP protein in onion epidermal cells and tobacco leaf cells showed that Gml-MMP and Gm2-MMP protein are localized in cell membrane.2.qRT-PCR results showed that the levels expression of Gm1-MMP and Gm2-MMP gene in the different tissues of cv.Xiangdou No.3 and cv.Ningzhen No.1 were higher in mature leaves,old leaves and mature seeds,respectively.Gm1-MMP and Gm2-MMP expressed continuously from the early stage of seed development to the late stage of seed maturation,and reached higher levels in the middle and late stage of seed maturation in both the cultivars.Compared to the corresponding control,Gm1-MMP was found to be markedly up-regulated in the leaves of cv.Xiangdou No.3 at 48,96,and 168 h during HTH stress,while markedly down-regulated in the leaves of cv.Ningzhen No.1 at stress time points of 48,96,and 168 h.However,Gml-MMP in the leaves of cv.Ningzhen No.1 was found to be markedly up-regulated only at the stress time point of 12 h compared to the control.In the R7 seeds of cv.Xiangdou No.3,compared to the corresponding control,Gml-MMP was markedly up-regulated at 12,48,96,and 168 h during HTH stress,whereas its expression level in the seeds of cv.Ningzhen No.1 was found to be higher only at the stress time point of 12 h than under the control.Compared to the corresponding control,Gm1-MMP was found to be markedly up-regulated in the leaves of cv.Xiangdou No.3 at 24,48,96,and 168 h during HTH stress,while markedlyup-regulated in the leaves of cv.Ningzhen No.1 at 12 and 24 h during HTH stress.In the R7 seeds of cv.Xiangdou No.3,compared to the corresponding control,Gm2-MMP was markedly up-regulated at 96 and 168 h during HTH stress,whereas its expression in the seeds of cv.Ningzhen No.1 was found to be higher only at the stress time point of 168 h than under the control.3.Using Gm1-MMP as bait,its interactive proteins were screened by yeast two-hybrid(Y2H)from Glycine max(L.)Merr.seed cDNA library.Finally,14 proteins were creened by synthetic dropout nutrient medium and X-?-Gal staining.The 14 proteins could be divided into 7 groups according to NCBI and UniProt database.They are seed maturation proteins,seed storage proteins,metal ion binding proteins,GTP activating proteins,ubiquitin pathway protein,protease inhibitors and unknown function proteins.4.The full ORF sequence of GmMT-? is 255 bp,encoding 85 amino acids.The bioinformatics analysis showed that the conserved domain of GmMT-? contains 3 Cys rich regions,the GmMT-? protein belongs to the Type 4 MTs.In addition,phylogenetic tree analysis indicated that GmMT-? is clustered into the same clade with AhMT4 and AtMT4A.The interaction between Gm1-MMP and GmMT-? protein in vivo was confirmed by yeast two hybrid technique and bimolecular fluorescence complementation assay(BiFC).The propeptide region of Gml-MMP could bind to GmMT-? protein,When GmMT-? protein contains at least one Cys enrichment region,it could be combined with Gm1-MMP protein.The results of qRT-PCR showed that the expression levels of GmMT-? gene in 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 both the cultivars were increased firstly and then decreased in seed development,and the maximum at 45 days after flowering seed.In the R7 seeds of cv.Xiangdou No.3,compared to the corresponding control,GmMT-? was markedly up-regulated at 48,96,and 168 h during HTH stress,whereas its expression in the seeds of cv.Ningzhen No.1 was found to be higher only at the stress time point of 12 h than under the control.6.These results of heterologous expression indicated that over-expression of Gm1-MMP and Gm2-MMP could lead to the leaves of Arahidopsis grow stronger,with the cauline leaves changed morphologically,even some of which looked like rosette leaves.The paraffin section indicated a series of abnormal phenomena in transgenic plant leaves,such as sponge cells become larger,palisade cells arrange loosely,and node cells were distorted.In addition,the over-expression of Gm1-MMP and Gm2-MMP resulted in the decrease of stomatal length,stomatal density and stomatal opening.The damage degree of 4 weeks of Gm1-MMP and Gm1-MMP transgenic plants were lower than WT with HTH stress treatment,and still retained a certain stomatal opening and complete chloroplast structure.In addition,seed germination rates and vigor of Gm1-MMP and Gm2-MMP transgenic plants were significantly higher than that of WT with HTH stress treatment.We also found that the expression level of hydrogen peroxide(H2O2)and ROS in Gm1-MMP transgenic plants seeds were much lower than that of WT.All the results indicated that Gml-MMP and Gm2-MMP were involved in the development of leaves and seeds,and enhances tolerance to HTH stress.It will help us to understand the functions of MMPs of soybean in plant growth and development,and in response to high temperature and humidity stress. |
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