Low-Molecular-Weight Glutenin Subunits (LMW-GS) Of Triticum Aestivum Cloning And Sequence Analysis

The quality of wheat is mainly affected by the storage proteins,glutenin and gliadin. At present,many studies focus on the high molecular weight glutenin(HMW-GS) of wheat,but low molecular weight gliadin and glutenin subunits(LMW-GS) are investigated less.This is mainly due to the variety of named systems of LMW-GS and the complexity of LMW-GS,which not only contains more subunits than HMW-GS,but also has closely linked subunits with gliadin genes.These factors largely obstructed the development of the study of LMW-GS.In this experiment,LMW-GS genes of wheat are cloned and analyzed to further enrich the LMW-GS gene family and form a strong foundation for the improvement of the quality of wheat.Four common wheat varieties with large quality discrepancies,R146,R97,MY11 and H-6 are selected.R146 and R97 are advanced strains cultivated by the State Key Laboratory of Breeding and Genetics.MY11 was widely promoted in Sichuan in 1980s. 12 pairs of specific primer(LMW-D1,LMW-A2,LMW-B3,LMW-B4,LMW-B5, LMW-B6,LMW-D7,LMW-D8,LMW-D9,LMW-10,LMW-B11,LMW-B12) are used for PCR amplification.LMW-D1,LMW-D7,LMW-D8 and LMW-D9 are located on chromosome D;LMW-B3,LMW-B4,LMW-B5,LMW-B6 and LMW-B12 are located on chromosome B;the location of LMW-10 cannot be determined until blasted with DNA sequences of LMW-GS in GenBank.DNA sequencing,sequence analysis,protein secondary structure prediction and phylogenetic analysis are also carried out in this experiment and the results are shown as follows:1.In 19 LMW-GS sequences obtained,there are 5 silencers,and the other 14 sequences have completed Reading Frames.Other than the LMW-GS sequences amplified with LMW-B4,the majority of the new cloned sequences of LMW-GS peptide have the complete structures:20 amino acids signal peptide(Sig),a 13 amino acids N-terminal conserved region of non-duplicate(Ⅰ),a high degree of central repeat region with 70～186 amino acids which are glutamine and proline-rich(Ⅱ),cysteine-rich region(Ⅲ), glutamine-rich region(Ⅳ) and C-end conserved region(Ⅴ).Except the primer,LMW-10, the other 11 pairs of primers produced highly consistent amplification sequences of LMW-GS among different species.Therefore,the LMW-GS sequences amplified by primers except LMW-10 are supposed to have nothing to do with the wheat qulity.The number of cysteines is also stable with the exceptions of LMW-D1 amplified products that are pseudogenes with more than 10 cysteins and LMW-B12 amplified products with 9 cysteines.All the other sequences have 8 cysteines and the locations of them are highly conserved.In the N-terminal conserved regionⅠ,theⅣDistrict,and the C-end conserved regionⅤ,there is one cysteine in each of these locations,and in the C-terminalⅢDistrict, there are five.The amount and the locations of cysteines will affect the formation of the inter-and-intra-molecular disulfide bond of LMW-GS,causing changes in the structure of proteins.Therefore,the LMW-B12 PCR products have significant protein conformation difference with the general LMW-GS with 8 cysteines.2.Compared with the published amino acid sequences of LMW-GS on NCBI,a new low molecular weight glutenin subunit R146-10 was found,and the similarity of the amino acid sequence was only 82%with other LMW-GS.Similar to the amplified products of primer LMW-10,R146-10 were compared with H-6-10,and MY11-10(R97-10 being a pseudogene was not involved in the analysis).InⅡDistrict,R146-10 lacks of two repeat units,but with an extra section of PPQQQQQQ.In regionⅣ,R146-10 has an additional CSFQQPQQQLGQQ.Judged by the conventional standards,the more duplicate units and Q quantity the better the subunit quality is.However,the opposite result is observed with MY11 and R146.Therefore,the quality of wheat must not be determined by the total number of duplicate units and Q quantity.It should also take into account the Q consecutive repeats,which implies that maybe not only DistrictⅡ,but also regionⅣ, could have significant impact to the wheat quality.The specific mechanism of the effect and actual impact however are pending further discovery.3.Made a protein structure prediction with SPIPRED secondary structure prediction method for the new gene R146-10,calculated the number ofα-helix,β-fold and analyzed the distribution and the content of them.In general,β-protein folding can increase the ability of anti-deformation for proteins.The main difference of the secondary structure between high-quality subunit and poor-quality subunit is the amount ofβ-fold.The subunit contains moreβ-fold manifests better quality.By comparing R146-10 with the high gluten quality AY263369,AY263369 has both largerβ-fold quantity and moreβ- fold content than R146-10,and R146-10 contains moreβ-fold than most other published LMW-GS sequences.Whereas the remarkable difference ofβ-fold quantity and content between R146-10 and AY263369,one probably should not rely solely on the basis of the number ofβ-fold to determine the merits of subunits;the distribution ofβ-fold should also be considered.In addition,the amount ofα-helix in AY263369 and R146-10 is also with great distinction,which may influence the quality of wheat as well.4.According to the mature LMW-GS proteins,or the first amino acid residue with the signal peptide removed,LMW-GS can be categorized into LMW-m,LMW-s and LMW-i, three types,which contain methionine,serine and isoleucine as the first amino acid.All sequences obtained in this experiment are LMW-m type.Based on the different locations of chromosomes LMW-GS can be divided into Glu-A3,Glu-B3,and Glu-D3.In this experiment,expect LMW-10,of which the chromosomal location of the amplified fragments cannot be determined prior to the experiment,the chromosomal locations of the rest amplified fragments are known,as stated before.The amplified products of the primer LMW-10,R146-10,H-6-10,MY11-10(R97-10 is a silent gene,no further analysis is performed) are compared with the sequences in GenBank,and are found most close to the Glu-D3 type of the LMW-GS sequence.Further phylogenetic analysis shows that LMW-10 amplified fragments and the Glu-D3-type LMW-GS genes tend to form one group.Therefore,preliminary conclusion is drawn that the specific amplification of the primer,LMW-10,is located in the Glu-D3 LMW-GS genes.5.After phylogenetic analysis,LMW-GS sequences on the same chromosome possess high consistency and have relatively close genetic relationship,namely,Glu-B3,Glu-D3 group into their own categories.Few Glu-B3 LMW-GS genes are found to assemble with Glu-D3,and individual Glu-D3 LMW-GS genes are seen to group with Glu-B3.The observation explains the LMW-GS genes on both B and D chromosomes have common original ancestor gene locus,but are distributed to different chromosomes in the process of time,and become relatively independent.Also,the silence LMW-GS genes have become significantly mutated from the current expression of LMW-GS.According to the cluster analysis map,LMW-GS genes differentiate in a relatively late stage,and in general,the LMW-GS gene sequences on different chromosomes can still maintain relatively high consistency.