Cloning And Characterization Of OAS-TL-Encoding Genes Involved In Soybean Sulfur-containing Amino Acid Biosynthesis Pathway

Soybean(Glycine max(L.) Merrill) is one of the most important plant protein sources for human diets and livestock feedings.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,but the precondition is to understand the molecular mechanism of sulfur-containing amino acid biosynthesis. Therefore,in this study we focus on cloning and characterization of the genes encoding O-acetylserine(thiol)lyase(OAS-TL) involved in sulfur-containing amino acid biosynthesis pathway which catalyzed the critical step reaction of cysteine synthesis.Firstly,comparisons about the molecular mechanism of cysteine synthesis accomplished by OAS-TL were made between wild soybean(Glycine soja Sieb.et Zucc.) and cultivated soybean[Glycine max(L.) Merrill].The results of conservation and specificity were obtained.The cDNA sequences of the reported soybean OAS-TL gene were obtained from the developing seeds of two wild soybean(ZYD4174 and ZYD3564) and two cultivated soybean(NN99-10 and 963069) materials.Sequence analysis showed that the gene from different templates was conserved on both the nucleotide and amino acid level.The genomic DNA sequence was also amplified from wild soybean ZYD4174 and it had 10 exons and 9 introns.Sulfur-containing amino acid content showed no significant difference between wild and cultivated soybean.In spite of the conservation described above,total activity in the corresponding seeds in four materials was obviously higher in wild soybean.RT-PCR analysis showed that in different organs of wild soybean ZYD4174 the gene transcripts were found lower in the roots and seeds than other tissues,while total enzyme activity was determined higher especially in the seeds.In contrast,in cultivated soybean NN99-10,OAS-TL was highly expressed in stems,leaves and seeds,while the enzyme activity in corresponding organs showed that higher level occurred in roots and seeds.In order to test the function of the OAS-TL gene,full-length cDNA from wild soybean ZYD4174 and cultivated soybean NN99-10 and three part-deletion fragments from the wild soybean template were expressed and identified in bacterial systems.The results showed that only the expression product of full-length cDNA from ZYD4174 could present the recombinant protein activity. Secondly,by combination of bioinformatic tools,RT-PCR and 3’ RACE-PCR reactions, six OAS-TL full-length cDNA sequences were obtained from cv.NN99-10.To confirm the identities of the isolated cDNA sequences,BLASTn and BLASTp subroutines were run against the non-redundant database in GenBank.On the amino acid level GmOAS-TL1 shared 89%identity with that of Citrullus vulgaris OAS-TL,and GmOAS-TL2 shared 89% identity with the reported soybean OAS-TL.GmOAS-TL3 shared 84%sequence identity with Malus x domestica CAS,and GmOAS-TL4 shared 86%identity with that of the putative chloroplast CSase from Nicotiana tabacum.GmOAS-TL6 and GmOAS-TL7 both presented an identity of 73%with that of Arabidopsis CSase.Phylogenetic analysis indicated that GmOAS-TL3 was classed into the CAS-like proteins and GmOAS-TL4 obviously showed more similarity to the platidic isoform of OAS-TL proteins,while the other four obviously showed higher similarity with those cytosolic isoforms from other plants.In order to identify and compare the genomic patterns of soybean OAS-TL genes, genomic DNA sequences of GmOAS-TL2,GmOAS-TL3 and GmOAS-TL4 were obtained.In the coding regions,both GmOAS-TL3 and GmOAS-TL4 had 10 exons and 9 introns with highly conserved exon sizes and strict intron locations.For GmOAS-TL2 it almost showed similar genomic structure pattern with the other two except that it had an extra short exon with the length 6 bp.All introns uniformly contained the canonical GT-AG binucleotide splice site junctions except the two flanked by the second exon of GmOAS-TL2.Southern hybridization showed that GmOAS-TL3 had 1-2 copies with small numbers of homologous genes in the genome and GmOAS-TL4 existed as one copy and had one highly homologous gene in the soybean genome.Thirdly,bacterial expression and RT-PCR analysis were perfomed on the soybean OAS-TL genes.In vitro expression results of 6 OAS-TL genes showed that the purified recombinant protein activity assessments were in agreement with the results of functional complementation in NK3 cys~- E.coli auxotroph.All OAS-TL cDNA expression products could present the activity except GmOAS-TL2 and GmOAS-TL7.RT-PCR comparison analysis was made on 7 OAS-TL genes(the reported one was named GmOAS-TL) in different organs of wild soybean ZYD4174 and cultivated soybean NN99-10.Both in wild and cultivated soybean organs,GmOAS-TL4 and GmOAS-TL7 had the similar constitutive expression patterns and both had a higher level in the leaves of cultivated soybean.In wild soybean organs GmOAS-TL,GmOAS-TL1 and GmOAS-TL3 were all nearly no detectable in seeds,while in cultivated soybean organs all the three were not expressed in the pod walls but with a relatively higher level in seeds.Expression of GmOAS-TL2 presented an obvious level in flowers of wild soybean and seeds of cultivated soybean.GmOAS-TL6 just had a higher expression level in the leaves of cultivated soybean.In four whorls of flower organs of cultivated soybean,GmOAS-TL2 was detected highly in the fourth whorl carpels,while GmOAS-TL6 was just detected in the second whorl petals.Expression distinctness of all these OAS-TL genes may indicate that each member had its distinct role involved in cysteine synthesis in different organs or under various environmental factors.During the seed filling process in the developing seeds of different developmental stages,almost all OAS-TL gene transcripts except GmOAS-TL3 showed the similar trend from the gradual increase at the early and medium stages to the later decrease and this presented the uniform with total enzymatic activity in the corresponding seed samples to some extent indicating that the OAS-TL gene members might play cooperative roles.The unexpected result that total cysteine content had a significantly negative correlation with the total OAS-TL enzyme activity throughout the developmental stages probably implied that cysteine synthesis in the developing seeds integratively should have a feedback inhibition to OAS-TL by its inherent regulation or the equilibrium mechanism controlled by certain regulation factors.Finally,the constructs of GmOAS-TL4 vectors were transformed into onion epidermal cells,which revealed that the subcelluar localization of GmOAS-TL4 was in the plastids. The pBI121-aP-GFP::OAS4F1 vector containing GmOAS-TL4 under the soybean 7S globulin a subunit gene promoter was constructed since a subunit gene promoter had the specificity of promoting gene’s expression at late stages of seed development.This may lay a foundation for improvement of soybean seed sulfur-containing amino acid content.