| Soybean (Glycine max (L.) Merr.) is one of the important oil crops and also strategic resources and occupies an important place in adjusting the structure of planting industry and development of nationality seed industry. 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 southern China. Plant transcription factors (for example,TALE superfamily members, KNOTTED-like homeobox (KNOX) and BEL1-like homeobox (BELL or BLH) ) play an important role in plant growth, development,and response to biotic and abiotic stress. Previous studies found that the KNOX family member GmSBH1 could involve in the regulation of plant seedlings morphogenesis, development and resistance to pre-harvest seed deterioration in our laboratory. In this study, seeds of pre-harvest seed deterioration-resistant cv. Xiangdou No. 3 treated with high temperature and humidity (HTH) stress were used as the materials to carry out the HTH yeast two-hybrid library construction. Meanwhile, 15 interacting proteins were identified to associate with pre-harvest seed deterioration based on GmSBH1 as bait protein. Further analysis showed that a BELL family transcription factors, GmBLH4 has an interactive relationship with GmSBH1 protein by using GST pull-down and bimolecular fluorescence complementation (BiFC)assay from in vitro and in vivo, respectively. The role and evolution characteristics of BELL family were preliminary elucidated during soybean seed developing process by phylogenetic analysis, protein structure and function analysis. Meanwhile, the potential role of GmBLH4 invovled in the plant morphogenesis and pre-harvest seed deterioration process were clarified using real-time fluorescence quantitative PCR(RT-qPCR), subcellular localization, promoter analysis and function verification of transgenic plants. There results will provide a theoretical basis for the regulation networks of TALE family involved in resistance to pre-harvest seed deterioration in spring soybean. The main results were as follows:1. Pre-harvest seed deterioration -resistant cv. Xiangdou No, 3 used as the yeast two-hybrid cDNA library construction material. Seeds in maturation stage (R7 stage)were treated with HTH stress (relative humidity: 95±5%,day /70±5% night,10h day/14h night), and harvested at 0, 6, 12, 24,48,64,96,168 h, respectively. Based on SMART technique and homologous recombination method, the spring soybean yeast two-hybrid cDNA library was constructed in yeast strains using both of Oligo (dT),8 primer and Random primer, respectively. In this study, the constructed cDNA library had better diversity and homogeneity, and its efficiency was greater than 1.0×106.These results showed that the constructed cDNA library was well-suited to large-scale screen protein-protein interactions for us.2. Pre-harvest seed deterioration-related transcription factor,GmSBH1 was isolated and identified, and used as bait protein for the constructed cDNA library. A total of 49 positive clones were screened by synthetic dropout nutrient medium and X-gal staining. The plasmid of 49 positive clones was extracted and transformed into E.coli for strains propagation,the sequencing was performed. Finally,15 proteins were obtained and proved to have relationship with GmSBH1 protein. The fifteen proteins could be divided into five groups according to NCBI and UniProt database.They were unknown function proteins,seed maturation proteins,seed storage proteins,development-associated proteins and protease inhibitors.3. The BEL 1-LIKE HOMEODOMAIN 4 (GmBLH4) associated with plant development was selected from 15 suspected interactions and used as the focus of this study. Phylogenetic analysis indicated that the soybean genome contained 34 BELL family genes, and GmBLH4 protein showed high homology relationship with the identified BLH4 (SAW2) in Arabidopsis. Expression profile assay showed that GmBELLs exhibited different expression patterns and strong tissues-specific expression in different tissues which were leaf, root, stem, flower, pod, nodules, root hair, seed and shoot apical meristem (SAM), respectively. According to CDS sequence of GmBLH4 in NCBI, its complete coding region was isolated from the soybean cDNA genome. Sequence analysis showed that the full length ORF of GmBLH4 was 2022bp,encoding 674 amino acids. Protein conserved domains analysis displayed that GmBLH4 contains three conserved domain structure, SKY domain, typical BELL domain and Homeodomain, respectively. SignalP website projections indicated that GmBLH4 has no signal peptide. The secondary structure predictions showed that a-spiral (38.58%), ?-corner (6.68%), extended strand(10.39%) and random coil (44.36%) were present in GmBLH4 protein sequences.Meanwhile, the cloned GmBLH4 protein fused to a transient expression vectors pA7-GFP for viewing its subcellular localization by tobacco epidermal cells and soybean protoplast transformation system, the results showed that GmBLH4 was located in the nucleus of two plant cells.4. The relationship between GmBLH4 and GmSBH1 protein in vitro or in vivo was identified by using the bimolecular fluorescence complementation (BiFC) and GST pull-down as well as Western blot assay. The results indicated that GmBLH4 could specifically bound to GmSBH1 protein in vitro and in vivo.5. The deletion fragments of two proteins were constructed to analysis the impact of conserved domains for GmBLH4 and GmSBHl interaction and further determine the binding sites by yeast two-hybrid system. The results indicated that GmSBH1 conserved domains, MEINOX (KNOX1 and KNOX2) and Homeodomain have a capability of binding GmBLH4 protein. However, the highest interacting activity was the presence of GmSBH1 complete coding region. Meanwhile, the SKY, BEL and Homeodomain were beneficial for GmBLH4 and GmSBH1 protein interaction. These results demonstrated that the conserved domains of TALE family are important for KNOX/BELL heterodimeric complexes formation.6. The expression level analysis of GmBLH4 in roots, stems, leaves, flowers,developing seeds and ripening seeds were completed in soybean using FQ-PCR assay and promoter expression characterization analysis. The results showed that GmBLH4 expression pattern was consistent between pre-harvest seed deterioration-sensitive soybean cv. Ningzhen No. 1 and -resistant cv. Xiangdou No. 3.The pattern showed the highest expression level in plant flower organs, followed by in roots, and little expression in seeds. Likewise, GUS gene staining results driven under by GmBLH4 gene upstream promoter revealed that GUS gene was highly expressed in floral organs, and roots of seedlings and apical meristem in Arabidopsis. However,the expression level was not detected in pods, mature leaves and seeds. The expression level of GmBLH4 showed significant up-regulation until 48h HTH stress treatment in developing seeds of soybean cv. Ningzhen No. 1, but it showed significant up-regulation until 168h treatment in -resistant cv. Xiangdou No. 3. These results indicated that GmBLH4 might play roles in maintaining the shoot apical meristem (SAM) and flower organs development, and also involve in pre-harvest seed deterioration process.7. These results of heterologous expression indicated that over-expression of GmBLH4 lead to curled leaf, shorter of siliques and reduced production in Arabidopsis. The paraffin sections results revealed that a series of abnormal phenomena in transgenic plant leaves, such as the cells arranged loosely, cells gap is large, shorter and thinner of palisade cells, irregular distribution of the sponge cells,smaller and more tightness adaxial and abaxial cell, especially the abaxial cells. Seed germination rates and vigor of GmBLH4 transgenic plants were significantly higher than that of wild-type and mutant blh4 plants with HTH stress treatment, and showed the similar to GmSBHl transgenic seeds. Therefore, we speculated that GmBLH4 might also involved in response to high temperature and humidity stress and the regulation of pre-harvest seed deterioration process. By the above experiments, we speculated that GmBLH4 could interact with GmSBHl and form a heterodimeric complex, and played a key role in plant development and involving in pre-harvest seed deterioration process. |
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