和面过程蛋白质行为对小麦品种面团流变学特性的影响
发布时间：2018-08-19 18:20
【摘要】：加工品质改良是小麦育种的主要任务之一。了解面团形成过程不同蛋白质的动态行为与面团流变学特性的关系是培育适合不同食品加工需要小麦品种的前提。利用和面过程面团中各类蛋白提取特性的差异,本研究综合利用SE-HPLC、2-DGE和质谱技术构建构建了一套用于面团蛋白行为跟踪检测的方法体系。随后,利用创建的方法对面团流变学特性存在差异的两组小麦品种的Mixolab面团样品进行了分析。第一对品种为Westonia和Wyalkatchem,后者具有更好的面条加工品质;第二对为直链淀粉含量存在差异的一对近等基因系Ventura-26(直链淀粉含量26%)和Ventura-19(直链淀粉含量19%)。两组品种和面过程面团蛋白行为的比较分析,为揭示蛋白行为与面团流变学特性的关系提供了重要信息,主要结果如下:1.构建了和面过程追踪检测面团蛋白质行为的方法体系首先,利用SE-HPLC对面团中各蛋白组分进行定量分析,明确其在加工过程中溶解性的动态变化。然后,通过2-DGE分析进一步获得不同品种特定阶段或同一品种不同阶段的差异蛋白点。最后,对得到的差异蛋白点进行质谱鉴定。在对面团蛋白质组的2-DGE分析中,非面筋蛋白用0.5M NaCl(pH 7.0)提取。面筋蛋白分别用两种裂解强度的提取液分离,0.3%SDS和15 mM DTT(SD)构成的提取液用于分离与面团基质结合较弱的蛋白;8 M尿素、4%CHAPS和60mM DTT(UCD)组成的提取液用于完全裂解后面筋蛋白的提取。面团蛋白的提取步骤为:先利用0.5 M NaCl分离面团中的清蛋白/球蛋白,再用SD将离心后剩余的残渣重悬,提取部分变性和还原后溶出的面筋蛋白;UCD提取的面团蛋白作为对照。2.C3/32 min(80℃)为面团流变学特性变化的关键时间点,一些大分子蛋白在该时间点参与了蛋白大聚体的形成对两组品种和面过程蛋白行为的分析均表明,y型HMW-GSs、大分子球蛋白、γ-醇溶蛋白、β-淀粉酶、丝氨酸蛋白酶抑制剂和一些大分子的代谢相关蛋白在C3/32 min(80℃)之后溶解性显著下降,表明其与面团基质的互作显著增强,开始参与面团内蛋白大聚体的形成。3.C4/38 min(85℃)处的面团强度差异与面团基质中蛋白的聚合/解聚密切相关在面条加工品质差异品种Wstonia与Wyalkatchem之间,C3/32 min(80℃)之前,品种Wstonia面团蛋白的溶解性高于Wyalkatchem,但之后正好相反;在淀粉糊化阶段,Westonia面团中的γ-醇溶蛋白、ALP-b、球蛋白-1、过氧化物酶和类烯醇酶在32min从面团基质中解聚后又在38 min重聚,导致其面团粘度在小幅下降后又重新增强;而在wyalkatchem面团中,球蛋白-3、球蛋白-1、醇溶蛋白、lmw-gss、alps和过氧物酶等蛋白在32min和38min持续溶出,导致其面团粘度在38min时大幅下降,表明c4/38min(85℃)处的面团强度差异与面团基质中一些蛋白的聚合/解聚密切相关。在品种westonia和wyalkatchem中,共鉴定出了33种与面团流变学特性差异相关的蛋白,编码基因主要位于1d、3a、4a、4b、4d、6a、6b、7a和7b染色体。4.面团形成过程蛋白与面团基质的互作特性受淀粉组成的影响在不同直链淀粉含量近等基因系ventura-26与ventura-19之间,淀粉糊化前后,ventura-26与ventura-19面团内的蛋白行为表现出显著差异。26min(56℃)后,逆境胁迫和代谢相关蛋白更趋向于与ventura-26的面团基质互作;醇溶蛋白、alp-b、lmw-gss和部分球蛋白与ventura-26的面团基质在c3/32min(80℃)的互作较强,然而,在c4/38min(85℃)和43min(80℃)时,这些蛋白与ventura-19面团基质的互作较强,表明面团形成过程蛋白的行为受淀粉组成影响。在整个和面过程中,鉴定出了46种与淀粉互作的蛋白,编码基因分别位于多条染色体,以第1、第4和第6同源群居多。5.与面团流变学特性相关蛋白按其在淀粉糊化阶段的行为模式可分为三类两对小麦品种和面过程共鉴定出92种差异蛋白,以贮藏蛋白(49.7%)、抗逆相关蛋白(24.4%)和碳水化合物代谢相关蛋白(12.1%)为主。其中,48种蛋白主要出现在淀粉糊化前,对面团流变学特性的影响作用有限;44种出现在淀粉糊化阶段。在淀粉糊化阶段的面团基质中,参与聚合/解聚和与淀粉互作的44种关键蛋白,按其行为模式可分为3类:18种蛋白兼具聚合/解聚和与淀粉的互作特性,主要为球蛋白、醇溶蛋白、alp-b和一些酶类,这些蛋白在淀粉糊化前与面筋蛋白结合形成相对稳定的聚合体;在淀粉糊化阶段,其中部分蛋白从聚合体解聚,参与与淀粉的互作,降低面团强度;hmw-gss、lmw-gss和少量α/β-醇溶蛋白等9种蛋白在淀粉糊化前参与面筋蛋白大聚体形成;淀粉糊化阶段,其中部分蛋白变性,与其他蛋白重新聚合成分子量更大但强度较低的大聚体,对维持面团强度的稳定有一定作用,一般不与糊化淀粉互作;17种代谢和抗逆相关的小分子蛋白在淀粉糊化前游离于面团基质内,淀粉糊化阶段与糊化淀粉高度亲和,通过参加或介导蛋白-淀粉互作影响面团流变学特性。6.小麦面团流变学特性相关蛋白基因的时空表达特性预测大部分面团流变学特性相关蛋白基因只在小麦籽粒中特异性表达,而且在籽粒中的表达时间和部位有所差别。根据与面团流变学特性相关的球蛋白、醇溶蛋白、alps和一些抗逆相关蛋白基因在籽粒灌浆阶段的表达特征,有望通过选育灌浆效率不同的品种、调节灌浆期氮肥和硫肥的使用量和比例、调整出粉率等途径有目的地改良小麦加工品质。本研究构建了用于面团蛋白质行为跟踪检测的方法体系;基于构建方法体系,从具有不同加工品质的两组小麦品种中,分别鉴定出了参与聚合/解聚和与淀粉互作的多种蛋白;通过对两组小麦和面各阶段蛋白行为数据的整合分析,提出了特定蛋白在整个和面过程的动态行为模式,借助小麦转录组共享数据库预测了与面团流变学特性相关的关键蛋白基因的表达特性。上述研究结果,为进一步分析小麦面团形成过程蛋白的动态行为提供了有效的方法,更新了人们对小麦加工品质相关蛋白的认识,为进一步开展品质分子生物学研究奠定了基础,也为优质专用小麦品种的育种、栽培和加工提供了理论依据。
[Abstract]:Improvement of processing quality is one of the main tasks in wheat breeding. Understanding the relationship between the dynamic behavior of different proteins during dough formation and dough rheological properties is the premise of breeding wheat varieties suitable for different food processing needs. Then, two groups of Mixolab dough samples with different dough rheological properties were analyzed by using the established method. The first pair of wheat varieties were Westonia and Wyalkatchem, which had better noodle processing quality. A pair of near-isogenic lines Ventura-26 (amylose 26%) and Ventura-19 (amylose 19%) with different amylose content were studied. The comparative analysis of protein behavior between the two groups of varieties and dough processing provided important information for revealing the relationship between protein behavior and dough rheological properties. The main results were as follows: 1. First, the protein components in dough were quantitatively analyzed by SE-HPLC to determine the dynamic changes of solubility in the process of dough processing. Then, 2-DGE analysis was used to obtain the different protein spots of different varieties at specific stages or different stages of the same variety. In 2-DGE analysis of dough proteome, non-gluten proteins were extracted with 0.5M NaCl (pH 7.0). Gluten proteins were separated by two kinds of lysis strength extracts, and the extracts of 0.3% SDS and 15 mM DTT (SD) were used to separate weak binding proteins with dough matrix; 8 M urea, 4% CHAPS and 60 mM DTT were used to separate proteins. The extract of T (UCD) was used to extract gluten protein after complete cleavage. The steps of extracting dough protein were as follows: separating albumin/globulin from dough by 0.5 M NaCl, then suspending the residual residue after centrifugation with SD, extracting partial denatured and reduced gluten protein; and using UCD as control.2.C3/32 min (8 min). Some macromolecular proteins were involved in the formation of protein macromers at this time point. The analysis of protein behavior of two groups of varieties and flour processing showed that Y-type HMW-GSs, macromolecular globulin, gamma-gliadin, beta-amylase, serine protease inhibitor and some macromolecular metabolic phases were involved. The solubility of Guan protein decreased significantly after C3/32 min (80 C), indicating that the interaction between Guan protein and dough matrix was significantly enhanced and began to participate in the formation of protein macromers in dough. 3. The difference of dough strength at C4/38 min (85 C) was closely related to the polymerization/depolymerization of proteins in dough matrix. Meanwhile, the solubility of protein in Wstonia dough was higher than that in Wyalkatchem before C3/32 min (80 ~C), but the latter was just the opposite; during starch gelatinization, gamma-gliadin, ALP-b, globulin-1, peroxidase and enolase in Westonia dough were depolymerized from the dough matrix at 32 min and then reunited at 38 min, resulting in a slight viscosity of the dough. In Wyalkatchem dough, the proteins of globulin-3, globulin-1, gliadin, lmw-gss, Alps and peroxidase were continuously dissolved in 32 min and 38 min, resulting in a significant decrease in dough viscosity at 38 min, indicating that the difference in dough strength at C 4/38 min (85 C) was closely related to the polymerization/depolymerization of some proteins in the dough matrix. 33 proteins related to the difference of dough rheological properties were identified in the cultivars westonia and wyalkatchem. The coding genes were mainly located on chromosomes 1d, 3a, 4a, 4b, 4d, 6a, 6b, 7a and 7b. 4. The interaction between dough forming proteins and dough matrix was affected by starch composition in the isogenic lines ventura-26 and ventura-26 with different amylose content. The protein behavior of ventura-26 and ventura-19 dough was significantly different before and after starch gelatinization. After 26 minutes (56), stress and metabolic related proteins tended to interact with ventura-26 dough matrix; gliadin, alp-b, LMW-GS and some globulins interacted with ventura-26 dough matrix at C 3/32 minutes (80). However, these proteins interacted strongly with ventura-19 dough matrix at C 4/38 min (85 C) and 43 min (80 C), suggesting that the behavior of the proteins during dough formation was affected by starch composition. According to the behavior pattern of dough rheological properties related proteins in starch gelatinization stage, 92 different proteins were identified in three types of wheat varieties and flour processes, including storage proteins (49.7%), stress resistance related proteins (24.4%) and carbohydrate metabolism related proteins (12.1%). Before starch gelatinization, the effect on dough rheological properties was limited; 44 kinds of proteins appeared in starch gelatinization stage. In starch gelatinization stage, 44 kinds of key proteins involved in polymerization/depolymerization and starch interaction could be divided into three types according to their behavior patterns: 18 kinds of proteins had both polymerization/depolymerization and starch interaction characteristics, mainly globular eggs. Albumin, gliadin, alp-b, and some enzymes, these proteins bind to gluten proteins before starch gelatinization to form relatively stable polymers; during starch gelatinization, some proteins are depolymerized from the polymers and participate in the interaction with starch to reduce dough strength; nine proteins, such as hmw-gss, lmw-gss and a small amount of alpha/beta-gliadin, are used as precursors to starch gelatinization. In starch gelatinization stage, some of the proteins denatured and some of the other proteins were reunited with larger molecular weight but lower strength polymers, which played a certain role in maintaining the stability of dough strength, and generally did not interact with gelatinized starch; 17 kinds of small molecular proteins related to metabolism and stress resistance were free before starch gelatinization. In the dough matrix, the starch gelatinization stage is highly compatible with the gelatinized starch, and affects dough rheological properties by participating in or mediating protein-starch interaction. 6. Temporal and spatial expression characteristics of wheat dough rheological properties-related protein genes predict that most of the dough rheological properties-related protein genes are specifically expressed only in wheat grains, and in seeds. According to the expression characteristics of globulin, gliadin, Alps and some stress-resistance-related protein genes related to dough rheological properties, it is hopeful to adjust the amount and proportion of nitrogen and sulfur fertilizers used in grain filling stage and flour yield by breeding varieties with different grain filling efficiency. In this study, a method system was constructed to detect the protein behavior of dough. Based on the method system, the proteins involved in polymerization/depolymerization and starch interaction were identified from two groups of wheat varieties with different processing qualities. The integrated analysis of protein behavior data provides a dynamic behavior model of specific proteins in the whole and dough process, and predicts the expression characteristics of key protein genes related to dough rheological properties with the help of wheat transcriptome shared database. The effective methods have renewed people's understanding of wheat processing quality-related proteins, laid a foundation for further research on quality molecular biology, and provided a theoretical basis for breeding, cultivation and processing of high-quality wheat varieties.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS213.2
，
本文编号：2192448
[Abstract]:Improvement of processing quality is one of the main tasks in wheat breeding. Understanding the relationship between the dynamic behavior of different proteins during dough formation and dough rheological properties is the premise of breeding wheat varieties suitable for different food processing needs. Then, two groups of Mixolab dough samples with different dough rheological properties were analyzed by using the established method. The first pair of wheat varieties were Westonia and Wyalkatchem, which had better noodle processing quality. A pair of near-isogenic lines Ventura-26 (amylose 26%) and Ventura-19 (amylose 19%) with different amylose content were studied. The comparative analysis of protein behavior between the two groups of varieties and dough processing provided important information for revealing the relationship between protein behavior and dough rheological properties. The main results were as follows: 1. First, the protein components in dough were quantitatively analyzed by SE-HPLC to determine the dynamic changes of solubility in the process of dough processing. Then, 2-DGE analysis was used to obtain the different protein spots of different varieties at specific stages or different stages of the same variety. In 2-DGE analysis of dough proteome, non-gluten proteins were extracted with 0.5M NaCl (pH 7.0). Gluten proteins were separated by two kinds of lysis strength extracts, and the extracts of 0.3% SDS and 15 mM DTT (SD) were used to separate weak binding proteins with dough matrix; 8 M urea, 4% CHAPS and 60 mM DTT were used to separate proteins. The extract of T (UCD) was used to extract gluten protein after complete cleavage. The steps of extracting dough protein were as follows: separating albumin/globulin from dough by 0.5 M NaCl, then suspending the residual residue after centrifugation with SD, extracting partial denatured and reduced gluten protein; and using UCD as control.2.C3/32 min (8 min). Some macromolecular proteins were involved in the formation of protein macromers at this time point. The analysis of protein behavior of two groups of varieties and flour processing showed that Y-type HMW-GSs, macromolecular globulin, gamma-gliadin, beta-amylase, serine protease inhibitor and some macromolecular metabolic phases were involved. The solubility of Guan protein decreased significantly after C3/32 min (80 C), indicating that the interaction between Guan protein and dough matrix was significantly enhanced and began to participate in the formation of protein macromers in dough. 3. The difference of dough strength at C4/38 min (85 C) was closely related to the polymerization/depolymerization of proteins in dough matrix. Meanwhile, the solubility of protein in Wstonia dough was higher than that in Wyalkatchem before C3/32 min (80 ~C), but the latter was just the opposite; during starch gelatinization, gamma-gliadin, ALP-b, globulin-1, peroxidase and enolase in Westonia dough were depolymerized from the dough matrix at 32 min and then reunited at 38 min, resulting in a slight viscosity of the dough. In Wyalkatchem dough, the proteins of globulin-3, globulin-1, gliadin, lmw-gss, Alps and peroxidase were continuously dissolved in 32 min and 38 min, resulting in a significant decrease in dough viscosity at 38 min, indicating that the difference in dough strength at C 4/38 min (85 C) was closely related to the polymerization/depolymerization of some proteins in the dough matrix. 33 proteins related to the difference of dough rheological properties were identified in the cultivars westonia and wyalkatchem. The coding genes were mainly located on chromosomes 1d, 3a, 4a, 4b, 4d, 6a, 6b, 7a and 7b. 4. The interaction between dough forming proteins and dough matrix was affected by starch composition in the isogenic lines ventura-26 and ventura-26 with different amylose content. The protein behavior of ventura-26 and ventura-19 dough was significantly different before and after starch gelatinization. After 26 minutes (56), stress and metabolic related proteins tended to interact with ventura-26 dough matrix; gliadin, alp-b, LMW-GS and some globulins interacted with ventura-26 dough matrix at C 3/32 minutes (80). However, these proteins interacted strongly with ventura-19 dough matrix at C 4/38 min (85 C) and 43 min (80 C), suggesting that the behavior of the proteins during dough formation was affected by starch composition. According to the behavior pattern of dough rheological properties related proteins in starch gelatinization stage, 92 different proteins were identified in three types of wheat varieties and flour processes, including storage proteins (49.7%), stress resistance related proteins (24.4%) and carbohydrate metabolism related proteins (12.1%). Before starch gelatinization, the effect on dough rheological properties was limited; 44 kinds of proteins appeared in starch gelatinization stage. In starch gelatinization stage, 44 kinds of key proteins involved in polymerization/depolymerization and starch interaction could be divided into three types according to their behavior patterns: 18 kinds of proteins had both polymerization/depolymerization and starch interaction characteristics, mainly globular eggs. Albumin, gliadin, alp-b, and some enzymes, these proteins bind to gluten proteins before starch gelatinization to form relatively stable polymers; during starch gelatinization, some proteins are depolymerized from the polymers and participate in the interaction with starch to reduce dough strength; nine proteins, such as hmw-gss, lmw-gss and a small amount of alpha/beta-gliadin, are used as precursors to starch gelatinization. In starch gelatinization stage, some of the proteins denatured and some of the other proteins were reunited with larger molecular weight but lower strength polymers, which played a certain role in maintaining the stability of dough strength, and generally did not interact with gelatinized starch; 17 kinds of small molecular proteins related to metabolism and stress resistance were free before starch gelatinization. In the dough matrix, the starch gelatinization stage is highly compatible with the gelatinized starch, and affects dough rheological properties by participating in or mediating protein-starch interaction. 6. Temporal and spatial expression characteristics of wheat dough rheological properties-related protein genes predict that most of the dough rheological properties-related protein genes are specifically expressed only in wheat grains, and in seeds. According to the expression characteristics of globulin, gliadin, Alps and some stress-resistance-related protein genes related to dough rheological properties, it is hopeful to adjust the amount and proportion of nitrogen and sulfur fertilizers used in grain filling stage and flour yield by breeding varieties with different grain filling efficiency. In this study, a method system was constructed to detect the protein behavior of dough. Based on the method system, the proteins involved in polymerization/depolymerization and starch interaction were identified from two groups of wheat varieties with different processing qualities. The integrated analysis of protein behavior data provides a dynamic behavior model of specific proteins in the whole and dough process, and predicts the expression characteristics of key protein genes related to dough rheological properties with the help of wheat transcriptome shared database. The effective methods have renewed people's understanding of wheat processing quality-related proteins, laid a foundation for further research on quality molecular biology, and provided a theoretical basis for breeding, cultivation and processing of high-quality wheat varieties.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS213.2
，
本文编号：2192448
本文链接：https://www.wllwen.com/shoufeilunwen/gckjbs/2192448.html
