小麦及其近缘种α-醇溶蛋白的鉴定与编码基因的分子克隆
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摘要
醇溶蛋白作为小麦胚乳中重要的贮藏蛋白，其组成和含量对小麦面粉的烘烤品质具有重要影响。普通小麦(Triticum aestivum L．，2n=6x=42，AABBDD)中的优质品种是研究小麦醇溶蛋白与优良品质关系的重要材料。粗山羊草(Aegilops tauschii，2n=2x=14，DD)是小麦D基因组的供体，具有丰富的贮藏蛋白等位变异，是小麦品质改良的重要基因资源。本研究以普通小麦优质品种和粗山羊草为材料，利用反相高效液相色谱(RP-HPLC)和基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)对α-醇溶蛋白进行分离和鉴定，并利用PCR方法对其编码基因进行分子克隆，进而分析这些α-醇溶蛋白基因的分子结构特点及醇溶蛋白基因家族的同源性，同时对部分α-醇溶蛋白编码基因进行原核表达研究。主要研究结果如下：
1．粗山羊草和普通小麦α-醇溶蛋白的分离与鉴定
利用RP-HPLC对粗山羊草T15、T43、T26和普通小麦优质品种中优9507、藁城8901的α-醇溶蛋白组分进行初步的分离鉴定。通过MALDI-TOF-MS对鉴定结果进行进一步的验证，并获得各组分的精确分子量。结果发现，在粗山羊草中α-醇溶蛋白组分较少，T15中约为4个、T43中约为3个、T26中约为6个；而普通小麦中α-醇溶蛋白组分较为复杂，而且其分子量很接近，中优9507和藁城8901中α-醇溶蛋白组分都在20个左右，表明六倍体普通小麦α-醇溶蛋白的多态性较高。
2．α-醇溶蛋白编码基因的分子克隆与序列比较分析
利用一对特异的α-醇溶蛋白基因引物，以粗山羊草T15、T43、T26的基因组DNA和普通小麦中优9507、藁城8901的cDNA为模板分别进行AS-PCR扩增，均获得了约850bp左右的单一条带，分别回收并克隆到pGEM-T Easy载体上，DNA测序后得到19个α-醇溶蛋白基因，其中T15和T43各1个、T26 2个、中优9507 8个、藁城8901 7个。这些基因命名后已登陆GenBank，编号为EF561270-EF561288。
基因序列分析表明，所得序列均为以ATG起始密码子开始、以TGA密码子结束的完整的α-醇溶蛋白编码基因，推导的氨基酸序列具有典型的α-醇溶蛋白的序列结构特征，由20个氨基酸的信号肽、N端重复区、多聚谷氨酰胺Ⅰ区、特征区Ⅰ、多聚谷氨酰胺Ⅱ区和特征区Ⅱ六部分组成，且部分基因推导出的分子量与质谱精确测定的分子量很接近，我们可以初步认为这些基因是质谱鉴定的蛋白的编码基因。氨基酸序列比对分析显示：这些基因与已知α-醇溶蛋白基因有很高的一致性，但也具有独有的特征。在Gli-At3和Gli-Z7的特征区Ⅱ前端发现了一个额外的半胱氨酸。半胱氨酸的增加有助于二硫键的形成，从而对面团特性产生影响。根据推导氨基酸序列所具有的四种T细胞抗原表位、多聚谷氨酰胺重复区的平均长度及同源性分析，我们对普通小麦中得到的基因进行了初步的染色体定位，将Gli-G2和Gli-Z3定位于6A染色体上；Gli-G1、Gli-G6、Gli-Z2、Gli-Z4和Gli-Z8定位于6B染色体上；Gli-G3、Gli-G4、Gli-G5、Gli-G7、Gli-Z1、Gli-Z5、Gli-Z7定位于6D染色体上。
3．克隆基因的原核表达
设计不扩增信号肽的表达引物，分别扩增T15、T43、T26、中优9507和藁城8901中的一个基因，扩增片断克隆到表达载体pGEX-4T-2上，转化到E．coli表达菌株BL-21(DE3)plysS。IPTG诱导后，收集菌液提取蛋白，进行SDS-PAGE分析，结果表明以上5个α-醇溶蛋白编码基因均在E．coli中获得了高效表达，表明我们克隆的这些基因在功能启动子的作用下具有表达活性。
本研究克隆的α-醇溶蛋白新基因以及在E．coli中的成功表达，为进一步研究它们的结构与功能奠定了基础，并有可能成为小麦品质改良的候选基因资源。
The gliadins are the major components of wheat storage proteins. It is well known that the composition and content of the gliadins play important roles in determining bread-making quality. Previous investigations showed that the related species of hexaploid wheat, such as Aegilops tauschii (DD, 2n=2x=14) possessed extensive storage protein variations, which could provide potential elite gene resources for wheat quality improvement. In this study, some specific α-gliadins from Aegilops tauschii and Triticum aestivum L. were detected by reversed-phase high performance liquid chromatographic (RP-HPLC) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The specific PCR primers were used to amplify, clone and sequence their encoding genes, and homologous analysis of storage protein gene family was carried out. Some of these α-gliadin genes cloned were further characterized by expressing in Escherichia coli. The main results were as the followings:
1. Separation and identification of α-gliadins in Aegilops tauschii and common wheat
The components of α-gliadins in Aegilops tauschii accessions T15, T26, T43 and in common wheat cultivars Zhongyou 9507, Gaocheng 8901 were detected by RP-HPLC. Further characterization and the accurate molecular weights of these gliadins were determined by MALDI-TOF-MS. It was found that the components of α-gliadins in common wheat cultivars were much more complex than in Aegilops tauschii. There were about four α-gliadin fractions in T15, three in T43 and six in T26. But Zhongyou 9507 as well as Gaocheng 8901 contained about 20 different α-gliadins, suggesting that hexaploid wheat had higher polymorphism in gliadin compositions.
2. Cloning, characterization and homologous analysis of α-gliadin genes
Genomic DNA of Aegilops tauschii accessions T15, T43 and T26, and cDNA of common wheat cultivars Zhongyou 9507 and Gaocheng 8901 were used as templates, and a pair of AS-PCR primers for α-gliadin genes was used to amplify the coding regions of α-gliadin genes. Single strongly amplified band with about 850bp from all accessions were obtained, and then the amplified products were ligated into a pGEM-T Easy vector (Promega) and sequenced by primer walking. 19 complete code nucleotide sequences were obtained, including one in T15, one in T43, two in T26, eight in Zhongyou 9507, and seven in Gaocheng 8901. They were all named and deposited in GenBank with the accession number from EF561270 to EF561288.
All of these sequences contained no introns and ends at a stop codon TGA. The deduced amino acid sequences had typical characters of α-gliadin, including six domains in structure: signal of 20 amino acid residues, N-terminal repetitive domain, polyglutamine domain I, unique domain I, polyglutamine domain II and unique domain II. Furthermore, some of the deduced molecular weights of mature proteins were similar to the accurate molecular weights determined by MALDI-TOF-MS, suggesting that these α-gliadin commonents identified were well correspond to their coding genes cloned. Amino acid sequence analysis showed that they demonstrated a high similarity with other α-gliadin genes previously cloned, but the unique features were present. Particularly, Gli-At3 and Gli-Z7 contained an extra cysteine residue in domain II, which might affect the pattern of disulfide bond formation, and therefore both genes might be important in determining bread-making quality. We also analyzed the cloned genes distribution on the chromosomes of hexaploid bread wheat, Gli-G2 and GH-Z3 assigned to chromosome 6A; Gli-G1,Gli-G6,Gli-Z2,GH-Z4 and GH-Z8 assigned to chromosome 6B; Gli-G3, Gli-G4, Gli-G5, Gli-G7, Gli-Z1, Gli-Z5 and GH-Z7 assigned to chromosome 6D.
3. Expression of cloned genes in E. coli
A pair of AS-PCR primers was designed without signal to amplify some of genes cloned. The amplified products were ligated into expression vector pGEX-4T-2 and then transformed into E. coli strain BL-21 (DE3) plysS. After induced by isopropyl-β-D-thiogalactoside (IPTG), cells were harvested, and then the expressed proteins were extracted and identified by SDS-PAGE. All 5 α-gliadin genes were expressed successfully in E. coli. This indicated that these genes can express under the control of the functional promoter.
1．粗山羊草和普通小麦α-醇溶蛋白的分离与鉴定
利用RP-HPLC对粗山羊草T15、T43、T26和普通小麦优质品种中优9507、藁城8901的α-醇溶蛋白组分进行初步的分离鉴定。通过MALDI-TOF-MS对鉴定结果进行进一步的验证，并获得各组分的精确分子量。结果发现，在粗山羊草中α-醇溶蛋白组分较少，T15中约为4个、T43中约为3个、T26中约为6个；而普通小麦中α-醇溶蛋白组分较为复杂，而且其分子量很接近，中优9507和藁城8901中α-醇溶蛋白组分都在20个左右，表明六倍体普通小麦α-醇溶蛋白的多态性较高。
2．α-醇溶蛋白编码基因的分子克隆与序列比较分析
利用一对特异的α-醇溶蛋白基因引物，以粗山羊草T15、T43、T26的基因组DNA和普通小麦中优9507、藁城8901的cDNA为模板分别进行AS-PCR扩增，均获得了约850bp左右的单一条带，分别回收并克隆到pGEM-T Easy载体上，DNA测序后得到19个α-醇溶蛋白基因，其中T15和T43各1个、T26 2个、中优9507 8个、藁城8901 7个。这些基因命名后已登陆GenBank，编号为EF561270-EF561288。
基因序列分析表明，所得序列均为以ATG起始密码子开始、以TGA密码子结束的完整的α-醇溶蛋白编码基因，推导的氨基酸序列具有典型的α-醇溶蛋白的序列结构特征，由20个氨基酸的信号肽、N端重复区、多聚谷氨酰胺Ⅰ区、特征区Ⅰ、多聚谷氨酰胺Ⅱ区和特征区Ⅱ六部分组成，且部分基因推导出的分子量与质谱精确测定的分子量很接近，我们可以初步认为这些基因是质谱鉴定的蛋白的编码基因。氨基酸序列比对分析显示：这些基因与已知α-醇溶蛋白基因有很高的一致性，但也具有独有的特征。在Gli-At3和Gli-Z7的特征区Ⅱ前端发现了一个额外的半胱氨酸。半胱氨酸的增加有助于二硫键的形成，从而对面团特性产生影响。根据推导氨基酸序列所具有的四种T细胞抗原表位、多聚谷氨酰胺重复区的平均长度及同源性分析，我们对普通小麦中得到的基因进行了初步的染色体定位，将Gli-G2和Gli-Z3定位于6A染色体上；Gli-G1、Gli-G6、Gli-Z2、Gli-Z4和Gli-Z8定位于6B染色体上；Gli-G3、Gli-G4、Gli-G5、Gli-G7、Gli-Z1、Gli-Z5、Gli-Z7定位于6D染色体上。
3．克隆基因的原核表达
设计不扩增信号肽的表达引物，分别扩增T15、T43、T26、中优9507和藁城8901中的一个基因，扩增片断克隆到表达载体pGEX-4T-2上，转化到E．coli表达菌株BL-21(DE3)plysS。IPTG诱导后，收集菌液提取蛋白，进行SDS-PAGE分析，结果表明以上5个α-醇溶蛋白编码基因均在E．coli中获得了高效表达，表明我们克隆的这些基因在功能启动子的作用下具有表达活性。
本研究克隆的α-醇溶蛋白新基因以及在E．coli中的成功表达，为进一步研究它们的结构与功能奠定了基础，并有可能成为小麦品质改良的候选基因资源。
The gliadins are the major components of wheat storage proteins. It is well known that the composition and content of the gliadins play important roles in determining bread-making quality. Previous investigations showed that the related species of hexaploid wheat, such as Aegilops tauschii (DD, 2n=2x=14) possessed extensive storage protein variations, which could provide potential elite gene resources for wheat quality improvement. In this study, some specific α-gliadins from Aegilops tauschii and Triticum aestivum L. were detected by reversed-phase high performance liquid chromatographic (RP-HPLC) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The specific PCR primers were used to amplify, clone and sequence their encoding genes, and homologous analysis of storage protein gene family was carried out. Some of these α-gliadin genes cloned were further characterized by expressing in Escherichia coli. The main results were as the followings:
1. Separation and identification of α-gliadins in Aegilops tauschii and common wheat
The components of α-gliadins in Aegilops tauschii accessions T15, T26, T43 and in common wheat cultivars Zhongyou 9507, Gaocheng 8901 were detected by RP-HPLC. Further characterization and the accurate molecular weights of these gliadins were determined by MALDI-TOF-MS. It was found that the components of α-gliadins in common wheat cultivars were much more complex than in Aegilops tauschii. There were about four α-gliadin fractions in T15, three in T43 and six in T26. But Zhongyou 9507 as well as Gaocheng 8901 contained about 20 different α-gliadins, suggesting that hexaploid wheat had higher polymorphism in gliadin compositions.
2. Cloning, characterization and homologous analysis of α-gliadin genes
Genomic DNA of Aegilops tauschii accessions T15, T43 and T26, and cDNA of common wheat cultivars Zhongyou 9507 and Gaocheng 8901 were used as templates, and a pair of AS-PCR primers for α-gliadin genes was used to amplify the coding regions of α-gliadin genes. Single strongly amplified band with about 850bp from all accessions were obtained, and then the amplified products were ligated into a pGEM-T Easy vector (Promega) and sequenced by primer walking. 19 complete code nucleotide sequences were obtained, including one in T15, one in T43, two in T26, eight in Zhongyou 9507, and seven in Gaocheng 8901. They were all named and deposited in GenBank with the accession number from EF561270 to EF561288.
All of these sequences contained no introns and ends at a stop codon TGA. The deduced amino acid sequences had typical characters of α-gliadin, including six domains in structure: signal of 20 amino acid residues, N-terminal repetitive domain, polyglutamine domain I, unique domain I, polyglutamine domain II and unique domain II. Furthermore, some of the deduced molecular weights of mature proteins were similar to the accurate molecular weights determined by MALDI-TOF-MS, suggesting that these α-gliadin commonents identified were well correspond to their coding genes cloned. Amino acid sequence analysis showed that they demonstrated a high similarity with other α-gliadin genes previously cloned, but the unique features were present. Particularly, Gli-At3 and Gli-Z7 contained an extra cysteine residue in domain II, which might affect the pattern of disulfide bond formation, and therefore both genes might be important in determining bread-making quality. We also analyzed the cloned genes distribution on the chromosomes of hexaploid bread wheat, Gli-G2 and GH-Z3 assigned to chromosome 6A; Gli-G1,Gli-G6,Gli-Z2,GH-Z4 and GH-Z8 assigned to chromosome 6B; Gli-G3, Gli-G4, Gli-G5, Gli-G7, Gli-Z1, Gli-Z5 and GH-Z7 assigned to chromosome 6D.
3. Expression of cloned genes in E. coli
A pair of AS-PCR primers was designed without signal to amplify some of genes cloned. The amplified products were ligated into expression vector pGEX-4T-2 and then transformed into E. coli strain BL-21 (DE3) plysS. After induced by isopropyl-β-D-thiogalactoside (IPTG), cells were harvested, and then the expressed proteins were extracted and identified by SDS-PAGE. All 5 α-gliadin genes were expressed successfully in E. coli. This indicated that these genes can express under the control of the functional promoter.
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