Isolation And Characterization Of Genes Encoding Key Enzymes In Soybean Sulfur-Containing Amino Acid Biosynthesis Pathway

Soybean (Glycine max (L.) Merr) is one of the most important plant protein sources for human diets and livestock feedings and contains essential amino acids for human dietary needs. The protein content of soybean exceeds that of the most commonly consumed food sources. Although soybean is an excellent source of protein, its nutritional quality is compromised by a low concentration of the sulfur amino acids methionine and cysteine. One of the potent ways to improve soybean sulfur-containing amino acid content by genetic engineering is to manipulate the genes encoding critical enzymes involved in sulfate assimilation pathway. Genes involved in sulfur-containing amino acid biosynthesis pathway have already been identified, whereas little is known about that of sulfur-containing amino acid biosynthesis pathway of soybean. In this study, we focus on the identification and characterization of the sulfur-containing amino acid biosynthesis pathway genes from soybean. The successful cloning of sulfur-containing amino acid biosynthesis pathway genes may provide valuable information for fully understanding sulfur-containing amino acid biosynthesis pathway and its molecular mechanisms; such information could also be of critical importance in genetic manuscription of sulfur-containing amino acid biosynthesis pathway. The results were listed as follows:Firstly, full-length cDNA GmCBL was cloned from the soybeans cultivation NN88-31, and sequence analysis, expression analysis and functional identification of GmCBL were carried out. Sequence analysis showed that, the amino acidssequence encoded by GmCBL has high homology with other plant species, C-terminal was very conservative, and GmCBL belonged to pyridoxal-5-phosphate-dependent enzymeγ-subfamily ofα-family, and contained the conserved N-terminal chloroplast transit peptide, pyridoxal-5’-phosphate binding site, substrate-cofactor binding pocket and homodimer interface, CGS_like specific hits, AAT_likesuper-families, Cys_Met_meta_PP multi-domains. searched soybean genome database by GmCBL as probe and found that GmCBL was located in Gm03, the structure of the gene in genome included 13 exons and 12 introns. Phylogenetic analysis indicated that GmCBL belonged to the dicotyledon plant cluster.QRT-PCR tissue expression analysis, GmCBL was detected highly in the pod shell of 15d and 45d, followed by 50d pod shell, and was very low in other pod shell. In the developing seeds of different developmental stages, GmCBL was detected the highest expression in 45d. In roots, stems, leaves, flowers, GmCBL was the highest expression in roots and flowers. GmCBL was induced by salt, insects, hurt and heavy metals cadmium stresses. Southern blot analysis indicated that a single copy of GmCBL gene existed in transgenic tobacco genome. Methionine contents of transgenic tobacco plants were significantly higher than that of wild-type plant. There was no difference in phenotype between transgenic plants and wild-type.Secondly, GmOASTL isolated from soybean was overexpressed in tobacco by two defferent promoter. The results show that, expression levels and OASTL activity of GmOASTL4 were different under two different promoters, under 35S promoter, expression levels and OASTL activity of GmOASTL4 were significantly higher than that under ap promoter. Southern Blot analysis revealed that GmOASTL4 is present in the tobacco genome with single-copy form. In seeds, the content of cysteine, methionine and total free amino acidap promoter>wild type>35S promoter in the transgenic tobacco seed.35S promoter of the transgenic tobacco plants were further analysised. The phenotype and endogenous protective enzyme analysis indicated that transgenic plants have resisitence to drought, salt, heavy metals (cadmium, copper) stresses. The cysteine content of the transgenic plants leaves was higher than wild-type plants.Upon heavy metals stress, the content of cysteine, methionine and total free amino acid of the transgenic plants leaves was significant higher than wild-type plants. With the exception of mannitol stress, methionine content of the transgenic plants was higher than that of wild-type plants, is much higher especially upon cadmium stress. QRT-PCR analysis revealed that the expression of transgenic plants under stress is lower than that of non-stress, but significantly higher than wild-type plants.Thirdly, we isolated and characterized MtCBL from medicago truncatula. MtCBL was 85% homology with GmCBL from soybean, belonged to pyridoxal-5’-phosphate-dependent enzymeγ-subfamily ofα-family, and contained the conserved N-terminal chloroplast transit peptide, had encoding proteins domain of GmCBL. MtCBL tertiary structure of proteins matched 82.32% with the Arabidopsis cystathionine-γ-lyase crystal structure, which had 10α-helix and 12β-pleated sheet composition. There were a number of MtCBL protein phosphorylation sites. Phyogenetic tree analysis showed that MtCBL had closest relationship with MsCBL, was divided in dicotyledon type. Tissue expression analysis showed that in young leaves and old leaves MtCBL significantly expressed and expressed no difference in the roots, stems, flowers. Expression was the highest leaves in the young leaves. Analysis QRT-PCR, MtCBL stablely expressed and have larger differences in different transgenic tobacco plants. There was no difference in Met content between transgenic plants and wild-type.