Isolation And Characterization Of A LMW-GS Gene From Taenitherum Nevski.

Low molecular weight glutenin subunits(LMW-GS) are the main storage proteins in wheat and wild relatives species.Although the LMW glutenins account for approximately 60%of the total glutenin components,they are important determinants of wheat grain processing quality because their composition affects the elasticity of wheat dough.Up to now,more and more LMW-GS genes were isolated and characterized from hexaploid wheat(Triticum aestivum L.).However,few homologous genes were obtained from its relative species,which limited our understanding of the relationships among them.Therefore,it is necessary to isolate LMW-GS homologous genes from wheat wild relative species.Taeniatherum Nevski.(2n=2x=14,TaTa) is a diploid annual genus that is far away from wheat and other wheat wild relative species in taxonomic position.In this genus,the separated gene pool was formed because their arid surroundings and absolute self-pollination character. Under this condition,there is little chance to make them exchange of LMW-GS genes between Taeniatherum and wheat as well as wheat wild relative species in its naturally condition.Therefore,Taeniatherum is a good plant material for studying the relationships among the LMW-GS genes derived from different Triticeae species.For better understanding and utilization of the LMW-GS genes from Taeniatherum,a gene sequence corresponding to LMW glutenin in this genus was isolated and characterized.The main results were as follows:(1) Identification and PCR amplification of LMW-GS gene from TaeniatherumBy using a pair of specific oligonucleotide primers for PCR amplification of Taenitherum accession PI220590 genomic DNA,a target DNA fragment,with nearly the same size of Triticeae LMW-GS genes and about 1000bp in size,was obtained.The DNA fragment was recovered from the agrose gel.The purified DNA fragment was further subjected ligate to the pMD18-T vector.The positive clones were selected and sequenced.A novel LMW-GS gene sequence was obtained and deposited in GeneBank database under accession number EU350087.Sequence alignment showed that this sequence contained only one complete ORFs and was a typical Triticeae LMW-GS homologous gene.The above result indicated that the LMW-GS gene was successfully isolated from Taenitherum. (2) Sequence alignments of LMW-GS genes from Taenitherum,wheat and wheat wild relativesThe LMW-GS gene sequence obtained from Taenitherum accession PI220590 was 1035bp in nucleotide size,and the deduced protein sequence contained 345 amino acid residues. Based on its deduced N-terminal amino acid sequence,it was a typical M-type LMW-GS gene.After alignment the gene sequence under the GenBank database using the signal peptide, N-terminal and C-terminal sequence,respectively,it was indicated that the gene sequence type obtained from Taenitherum was also existed in wheat and other wheat wild relatives.In the NCBI database,the gene nucleotide sequence of DQ822593 and its corresponding protein sequence ABI21861,showed the highest identities with EU350087 by 66%in amino acid sequence.The alignment between the two genes showed that their ORFs were nearly the same size but the gene sequence involved a large number of amino acid residues replacement, insertion and deletion.The result present here would give a reference to isolate LMW-GS homologous genes from Taenitherum as well as other wheat wild relatives.