Novel LMW-GS （low molecular weight glutenin subunit）,α- and γ-gliadin from Triticum macha accessions were characterized via genomic PCR, which can do favor to improve the wheat quality. The complete coding regions...Novel LMW-GS （low molecular weight glutenin subunit）,α- and γ-gliadin from Triticum macha accessions were characterized via genomic PCR, which can do favor to improve the wheat quality. The complete coding regions of two α-gliadin, two γ-gliadin and two LMW-GS gene sequences, which designed as Gli-Mal, Gli-Ma2, Gli-Mrl, Gli-Mr2, Glu-LM1 and Glu-LM2, encoded the mature proteins with 307, 241, 348, 302, 474 and 377 amino acid residues, respectively. Gli-Mal and Gli-Ma2 were recognized as pseudogenes due to the in-frame stop codons. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α- or γ-gliadin or LMW- m type proteins with the exception of Gli-Ma2. Phylogenetic analysis showed Gli-Mal was closely related to those from T. aestivum, whereas Gli-Ma2 seemed to be more homologous with the gene sequences from Dasypyrum breviaristatum. Gli-Mrl was closely related to those from T. turgidum ssp. dicoccoides, while Gli-Mr2 was the nearest to those from T. aestivum. Glu-LM1 was closely related to those from Aegilops tauschii, whereas GIu-LM2 seemed to be more homologous with those from T. durum.展开更多
Gliadins are the major components of storage proteins in wheat and play an important role in determining the extensibility properties of dough.In the present work,six novel full-length γ-gliadin genes were cloned fro...Gliadins are the major components of storage proteins in wheat and play an important role in determining the extensibility properties of dough.In the present work,six novel full-length γ-gliadin genes were cloned from the C genome of Aegilops markgrafii using a PCR-based strategy.Analysis of the deduced amino acid sequences showed that the cloned genes had primary structures that were similar,but not identical,to published γ-gliadins from other wheat-related species.The lengths of the open reading frames(ORFs)ranged from 909 to 963 bp,and the repetitive and glutamine-rich domains were mainly responsible for the size of the proteins.An extra cysteine residue was present in the repetitive domain of sequence JX566513.All amino acid sequences of γ-gliadin genes from Ae.markgrafii were searched for the five peptides identified as T cell stimulatory epitopes in celiac disease(CD)patients.Peptide Gliγ-3 was present in sequences JX566513 and JX566514.Peptide Gliγ-5 was present only in JX566513.The otherγ-gliadins contained no toxic epitopes.These results provide information to better understand the use of Ae.markgrafii in wheat breeding and the evolutionary relationship of theγ-gliadin genes in Ae.markgrafii and other Triticeae species.展开更多
γ-Gliadins are an important component of wheat seed storage proteins. Four novel γ-gliadin genes （Gli-ngl to Gli-ng4） were cloned from wheat （Triticum aestivum） and Aegilops species. The novel γ-gliadins were m...γ-Gliadins are an important component of wheat seed storage proteins. Four novel γ-gliadin genes （Gli-ngl to Gli-ng4） were cloned from wheat （Triticum aestivum） and Aegilops species. The novel γ-gliadins were much smaller in molecular size when compared to the typical γ-gliadins, which was caused by deletion of the non-repetitive domain, glutamine-rich region, 3＂ part of the repetitive domain, and 5＇ part of the C-terminal, possibly due to illegitimate recombination between the repetitive domain and the C-terminal. As a result, Gli-ngl and Gli-ng4 only contained two and three cysteine residues, respectively. Gli-ngl, as the representative of novel γ-gliadin genes, has been sub-cloned into an Escherichia coli expression system. SDS- PAGE indicated that the both cysteine residues of Gli-ngl could participate in the formation of intermolecular disulphide bonds in vitro. Successful cloning of Gli-ngl from seed cDNA of T. aestivum cv. Chinese Spring suggested that these novel γ-gliadin genes were normally transcribed during the development of seeds. Phylogenic analysis indicated that the four novel γ-gliadin genes had a closer relationship with those from the B （S） genome of wheat.展开更多
为进一步研究γ-醇溶蛋白1基因调控小麦面粉品质的分子机制,以郑麦158(低蛋白质含量、高面团强度)为研究材料,克隆小麦γ-醇溶蛋白1基因,构建诱饵载体pGBKT7-γ-醇溶蛋白1,利用酵母双杂交技术从郑麦158的cDNA文库中筛选γ-醇溶蛋白1的...为进一步研究γ-醇溶蛋白1基因调控小麦面粉品质的分子机制,以郑麦158(低蛋白质含量、高面团强度)为研究材料,克隆小麦γ-醇溶蛋白1基因,构建诱饵载体pGBKT7-γ-醇溶蛋白1,利用酵母双杂交技术从郑麦158的cDNA文库中筛选γ-醇溶蛋白1的候选互作蛋白,并通过回转验证试验进一步验证其互作关系。结果表明,成功克隆了γ-醇溶蛋白1基因,其cDNA全长为1 020 bp。利用酵母双杂交技术共筛选到117个蓝色单克隆菌落,经菌液PCR检测、测序以及NCBI的BLAST比对分析,共获得10个与γ-醇溶蛋白1互作的候选蛋白,它们分别是富含半胱氨酸和跨膜结构域蛋白1(XM_044546254.1)、α-淀粉酶抑制剂(XM_044509330.1)、特异性内切-1,3:1,4-β-D-葡聚糖酶2(XM_044550285.1、XM_044594213.1)、蔗糖果糖基转移酶(XM_044554537.1)、支链淀粉酶1(XM_044577151.1)和半胱氨酸蛋白酶B-like(XM_044577994.1),还有与植物生殖生长尤其是果实或者籽粒生长有关的细胞数目调控因子CNR8-like(XM_044483337.1、XM_044491902.1)、CNR2-like(XM_044468782.1)以及参与蛋白质修饰的泛素结构域蛋白DSK2b-like(Dominant suppressor of KAR 2b like,XM_044546840.1)和CIP73(CCa MK-interacting protein of 73 ku,XM_044554473.1)。候选互作蛋白的回转验证试验结果表明,富含半胱氨酸和跨膜结构域蛋白1、α-淀粉酶抑制剂、特异性内切-1,3:1,4-β-D-葡聚糖酶2和半胱氨酸蛋白酶B-like等4个蛋白质可能与γ-醇溶蛋白1存在互作关系。推测γ-醇溶蛋白1可能与这些蛋白质相互作用参与小麦种子中贮藏蛋白(如半胱氨酸)和淀粉的合成与降解等过程。展开更多
基金supported by the National High Technology Research and Development Program of China(863 Program, 2006AA10Z179, 2006AAl0Z1F8)the Key Technologies R&D Program (2006BAD01A02,2006 BAD13B02)
文摘Novel LMW-GS （low molecular weight glutenin subunit）,α- and γ-gliadin from Triticum macha accessions were characterized via genomic PCR, which can do favor to improve the wheat quality. The complete coding regions of two α-gliadin, two γ-gliadin and two LMW-GS gene sequences, which designed as Gli-Mal, Gli-Ma2, Gli-Mrl, Gli-Mr2, Glu-LM1 and Glu-LM2, encoded the mature proteins with 307, 241, 348, 302, 474 and 377 amino acid residues, respectively. Gli-Mal and Gli-Ma2 were recognized as pseudogenes due to the in-frame stop codons. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α- or γ-gliadin or LMW- m type proteins with the exception of Gli-Ma2. Phylogenetic analysis showed Gli-Mal was closely related to those from T. aestivum, whereas Gli-Ma2 seemed to be more homologous with the gene sequences from Dasypyrum breviaristatum. Gli-Mrl was closely related to those from T. turgidum ssp. dicoccoides, while Gli-Mr2 was the nearest to those from T. aestivum. Glu-LM1 was closely related to those from Aegilops tauschii, whereas GIu-LM2 seemed to be more homologous with those from T. durum.
基金financially supported by the National Natural Science Foundation of China (nos. 31520103911, 31071405, and 31471488)National Basic Research Program of China (no. 2014CB138100)+1 种基金National Key Research and Development Program of China (no. 2016YFD0100102)Genetically Modified Organism Breeding Major Projects (no. 2016ZX08002003)
文摘Gliadins are the major components of storage proteins in wheat and play an important role in determining the extensibility properties of dough.In the present work,six novel full-length γ-gliadin genes were cloned from the C genome of Aegilops markgrafii using a PCR-based strategy.Analysis of the deduced amino acid sequences showed that the cloned genes had primary structures that were similar,but not identical,to published γ-gliadins from other wheat-related species.The lengths of the open reading frames(ORFs)ranged from 909 to 963 bp,and the repetitive and glutamine-rich domains were mainly responsible for the size of the proteins.An extra cysteine residue was present in the repetitive domain of sequence JX566513.All amino acid sequences of γ-gliadin genes from Ae.markgrafii were searched for the five peptides identified as T cell stimulatory epitopes in celiac disease(CD)patients.Peptide Gliγ-3 was present in sequences JX566513 and JX566514.Peptide Gliγ-5 was present only in JX566513.The otherγ-gliadins contained no toxic epitopes.These results provide information to better understand the use of Ae.markgrafii in wheat breeding and the evolutionary relationship of theγ-gliadin genes in Ae.markgrafii and other Triticeae species.
基金finically supported by the National Natural Science Foundation of China (31230053)
文摘γ-Gliadins are an important component of wheat seed storage proteins. Four novel γ-gliadin genes （Gli-ngl to Gli-ng4） were cloned from wheat （Triticum aestivum） and Aegilops species. The novel γ-gliadins were much smaller in molecular size when compared to the typical γ-gliadins, which was caused by deletion of the non-repetitive domain, glutamine-rich region, 3＂ part of the repetitive domain, and 5＇ part of the C-terminal, possibly due to illegitimate recombination between the repetitive domain and the C-terminal. As a result, Gli-ngl and Gli-ng4 only contained two and three cysteine residues, respectively. Gli-ngl, as the representative of novel γ-gliadin genes, has been sub-cloned into an Escherichia coli expression system. SDS- PAGE indicated that the both cysteine residues of Gli-ngl could participate in the formation of intermolecular disulphide bonds in vitro. Successful cloning of Gli-ngl from seed cDNA of T. aestivum cv. Chinese Spring suggested that these novel γ-gliadin genes were normally transcribed during the development of seeds. Phylogenic analysis indicated that the four novel γ-gliadin genes had a closer relationship with those from the B （S） genome of wheat.
文摘为进一步研究γ-醇溶蛋白1基因调控小麦面粉品质的分子机制,以郑麦158(低蛋白质含量、高面团强度)为研究材料,克隆小麦γ-醇溶蛋白1基因,构建诱饵载体pGBKT7-γ-醇溶蛋白1,利用酵母双杂交技术从郑麦158的cDNA文库中筛选γ-醇溶蛋白1的候选互作蛋白,并通过回转验证试验进一步验证其互作关系。结果表明,成功克隆了γ-醇溶蛋白1基因,其cDNA全长为1 020 bp。利用酵母双杂交技术共筛选到117个蓝色单克隆菌落,经菌液PCR检测、测序以及NCBI的BLAST比对分析,共获得10个与γ-醇溶蛋白1互作的候选蛋白,它们分别是富含半胱氨酸和跨膜结构域蛋白1(XM_044546254.1)、α-淀粉酶抑制剂(XM_044509330.1)、特异性内切-1,3:1,4-β-D-葡聚糖酶2(XM_044550285.1、XM_044594213.1)、蔗糖果糖基转移酶(XM_044554537.1)、支链淀粉酶1(XM_044577151.1)和半胱氨酸蛋白酶B-like(XM_044577994.1),还有与植物生殖生长尤其是果实或者籽粒生长有关的细胞数目调控因子CNR8-like(XM_044483337.1、XM_044491902.1)、CNR2-like(XM_044468782.1)以及参与蛋白质修饰的泛素结构域蛋白DSK2b-like(Dominant suppressor of KAR 2b like,XM_044546840.1)和CIP73(CCa MK-interacting protein of 73 ku,XM_044554473.1)。候选互作蛋白的回转验证试验结果表明,富含半胱氨酸和跨膜结构域蛋白1、α-淀粉酶抑制剂、特异性内切-1,3:1,4-β-D-葡聚糖酶2和半胱氨酸蛋白酶B-like等4个蛋白质可能与γ-醇溶蛋白1存在互作关系。推测γ-醇溶蛋白1可能与这些蛋白质相互作用参与小麦种子中贮藏蛋白(如半胱氨酸)和淀粉的合成与降解等过程。
