和面過程蛋白質(zhì)行為對小麥品種面團(tuán)流變學(xué)特性的影響
發(fā)布時間：2018-08-19 18:20
【摘要】：加工品質(zhì)改良是小麥育種的主要任務(wù)之一。了解面團(tuán)形成過程不同蛋白質(zhì)的動態(tài)行為與面團(tuán)流變學(xué)特性的關(guān)系是培育適合不同食品加工需要小麥品種的前提。利用和面過程面團(tuán)中各類蛋白提取特性的差異,本研究綜合利用SE-HPLC、2-DGE和質(zhì)譜技術(shù)構(gòu)建構(gòu)建了一套用于面團(tuán)蛋白行為跟蹤檢測的方法體系。隨后,利用創(chuàng)建的方法對面團(tuán)流變學(xué)特性存在差異的兩組小麥品種的Mixolab面團(tuán)樣品進(jìn)行了分析。第一對品種為Westonia和Wyalkatchem,后者具有更好的面條加工品質(zhì);第二對為直鏈淀粉含量存在差異的一對近等基因系Ventura-26(直鏈淀粉含量26%)和Ventura-19(直鏈淀粉含量19%)。兩組品種和面過程面團(tuán)蛋白行為的比較分析,為揭示蛋白行為與面團(tuán)流變學(xué)特性的關(guān)系提供了重要信息,主要結(jié)果如下:1.構(gòu)建了和面過程追蹤檢測面團(tuán)蛋白質(zhì)行為的方法體系首先,利用SE-HPLC對面團(tuán)中各蛋白組分進(jìn)行定量分析,明確其在加工過程中溶解性的動態(tài)變化。然后,通過2-DGE分析進(jìn)一步獲得不同品種特定階段或同一品種不同階段的差異蛋白點。最后,對得到的差異蛋白點進(jìn)行質(zhì)譜鑒定。在對面團(tuán)蛋白質(zhì)組的2-DGE分析中,非面筋蛋白用0.5M NaCl(pH 7.0)提取。面筋蛋白分別用兩種裂解強度的提取液分離,0.3%SDS和15 mM DTT(SD)構(gòu)成的提取液用于分離與面團(tuán)基質(zhì)結(jié)合較弱的蛋白;8 M尿素、4%CHAPS和60mM DTT(UCD)組成的提取液用于完全裂解后面筋蛋白的提取。面團(tuán)蛋白的提取步驟為:先利用0.5 M NaCl分離面團(tuán)中的清蛋白/球蛋白,再用SD將離心后剩余的殘渣重懸,提取部分變性和還原后溶出的面筋蛋白;UCD提取的面團(tuán)蛋白作為對照。2.C3/32 min(80℃)為面團(tuán)流變學(xué)特性變化的關(guān)鍵時間點,一些大分子蛋白在該時間點參與了蛋白大聚體的形成對兩組品種和面過程蛋白行為的分析均表明,y型HMW-GSs、大分子球蛋白、γ-醇溶蛋白、β-淀粉酶、絲氨酸蛋白酶抑制劑和一些大分子的代謝相關(guān)蛋白在C3/32 min(80℃)之后溶解性顯著下降,表明其與面團(tuán)基質(zhì)的互作顯著增強,開始參與面團(tuán)內(nèi)蛋白大聚體的形成。3.C4/38 min(85℃)處的面團(tuán)強度差異與面團(tuán)基質(zhì)中蛋白的聚合/解聚密切相關(guān)在面條加工品質(zhì)差異品種Wstonia與Wyalkatchem之間,C3/32 min(80℃)之前,品種Wstonia面團(tuán)蛋白的溶解性高于Wyalkatchem,但之后正好相反;在淀粉糊化階段,Westonia面團(tuán)中的γ-醇溶蛋白、ALP-b、球蛋白-1、過氧化物酶和類烯醇酶在32min從面團(tuán)基質(zhì)中解聚后又在38 min重聚,導(dǎo)致其面團(tuán)粘度在小幅下降后又重新增強;而在wyalkatchem面團(tuán)中,球蛋白-3、球蛋白-1、醇溶蛋白、lmw-gss、alps和過氧物酶等蛋白在32min和38min持續(xù)溶出,導(dǎo)致其面團(tuán)粘度在38min時大幅下降,表明c4/38min(85℃)處的面團(tuán)強度差異與面團(tuán)基質(zhì)中一些蛋白的聚合/解聚密切相關(guān)。在品種westonia和wyalkatchem中,共鑒定出了33種與面團(tuán)流變學(xué)特性差異相關(guān)的蛋白,編碼基因主要位于1d、3a、4a、4b、4d、6a、6b、7a和7b染色體。4.面團(tuán)形成過程蛋白與面團(tuán)基質(zhì)的互作特性受淀粉組成的影響在不同直鏈淀粉含量近等基因系ventura-26與ventura-19之間,淀粉糊化前后,ventura-26與ventura-19面團(tuán)內(nèi)的蛋白行為表現(xiàn)出顯著差異。26min(56℃)后,逆境脅迫和代謝相關(guān)蛋白更趨向于與ventura-26的面團(tuán)基質(zhì)互作;醇溶蛋白、alp-b、lmw-gss和部分球蛋白與ventura-26的面團(tuán)基質(zhì)在c3/32min(80℃)的互作較強,然而,在c4/38min(85℃)和43min(80℃)時,這些蛋白與ventura-19面團(tuán)基質(zhì)的互作較強,表明面團(tuán)形成過程蛋白的行為受淀粉組成影響。在整個和面過程中,鑒定出了46種與淀粉互作的蛋白,編碼基因分別位于多條染色體,以第1、第4和第6同源群居多。5.與面團(tuán)流變學(xué)特性相關(guān)蛋白按其在淀粉糊化階段的行為模式可分為三類兩對小麥品種和面過程共鑒定出92種差異蛋白,以貯藏蛋白(49.7%)、抗逆相關(guān)蛋白(24.4%)和碳水化合物代謝相關(guān)蛋白(12.1%)為主。其中,48種蛋白主要出現(xiàn)在淀粉糊化前,對面團(tuán)流變學(xué)特性的影響作用有限;44種出現(xiàn)在淀粉糊化階段。在淀粉糊化階段的面團(tuán)基質(zhì)中,參與聚合/解聚和與淀粉互作的44種關(guān)鍵蛋白,按其行為模式可分為3類:18種蛋白兼具聚合/解聚和與淀粉的互作特性,主要為球蛋白、醇溶蛋白、alp-b和一些酶類,這些蛋白在淀粉糊化前與面筋蛋白結(jié)合形成相對穩(wěn)定的聚合體;在淀粉糊化階段,其中部分蛋白從聚合體解聚,參與與淀粉的互作,降低面團(tuán)強度;hmw-gss、lmw-gss和少量α/β-醇溶蛋白等9種蛋白在淀粉糊化前參與面筋蛋白大聚體形成;淀粉糊化階段,其中部分蛋白變性,與其他蛋白重新聚合成分子量更大但強度較低的大聚體,對維持面團(tuán)強度的穩(wěn)定有一定作用,一般不與糊化淀粉互作;17種代謝和抗逆相關(guān)的小分子蛋白在淀粉糊化前游離于面團(tuán)基質(zhì)內(nèi),淀粉糊化階段與糊化淀粉高度親和,通過參加或介導(dǎo)蛋白-淀粉互作影響面團(tuán)流變學(xué)特性。6.小麥面團(tuán)流變學(xué)特性相關(guān)蛋白基因的時空表達(dá)特性預(yù)測大部分面團(tuán)流變學(xué)特性相關(guān)蛋白基因只在小麥籽粒中特異性表達(dá),而且在籽粒中的表達(dá)時間和部位有所差別。根據(jù)與面團(tuán)流變學(xué)特性相關(guān)的球蛋白、醇溶蛋白、alps和一些抗逆相關(guān)蛋白基因在籽粒灌漿階段的表達(dá)特征,有望通過選育灌漿效率不同的品種、調(diào)節(jié)灌漿期氮肥和硫肥的使用量和比例、調(diào)整出粉率等途徑有目的地改良小麥加工品質(zhì)。本研究構(gòu)建了用于面團(tuán)蛋白質(zhì)行為跟蹤檢測的方法體系;基于構(gòu)建方法體系,從具有不同加工品質(zhì)的兩組小麥品種中,分別鑒定出了參與聚合/解聚和與淀粉互作的多種蛋白;通過對兩組小麥和面各階段蛋白行為數(shù)據(jù)的整合分析,提出了特定蛋白在整個和面過程的動態(tài)行為模式,借助小麥轉(zhuǎn)錄組共享數(shù)據(jù)庫預(yù)測了與面團(tuán)流變學(xué)特性相關(guān)的關(guān)鍵蛋白基因的表達(dá)特性。上述研究結(jié)果,為進(jìn)一步分析小麥面團(tuán)形成過程蛋白的動態(tài)行為提供了有效的方法,更新了人們對小麥加工品質(zhì)相關(guān)蛋白的認(rèn)識,為進(jìn)一步開展品質(zhì)分子生物學(xué)研究奠定了基礎(chǔ),也為優(yōu)質(zhì)專用小麥品種的育種、栽培和加工提供了理論依據(jù)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TS213.2
，
本文編號：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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TS213.2
，
本文編號：2192448
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