Trafficking and deposition of prolamins in wheat

The processing properties of wheat flour are largely determined by the structures and interactions of the grain storage protein, the so called “gluten proteins”. The synthesis, folding and deposition of the gluten proteins take place within the endomembrane system of the plant cell. Several studies have suggested that two trafficking pathways occur in wheat, with the proteins either being transported via the Golgi apparatus into the vacuole or assembling within the ER to form protein bodies that are subsequently internalized into vacuoles by a process analogous to autophagy. The precise routes of trafficking and deposition of individual gluten proteins remain unclear and so are the molecular and cellular mechanisms regulating their sorting in the two pathways.     

蛋白质相互作用技术在热带植物病毒研究中的应用

蛋白质-蛋白质相互作用(Protein-protein interaction)承载着生物体生命活动的各个过程,研究热带植物病毒中的蛋白质-蛋白质相互作用,不仅可以从分子水平揭示病毒蛋白质的功能,还可以揭示病毒在寄主细胞中的侵染机理,从而制定相关策略预防病毒的突然爆发或进行疾病治疗。而蛋白质相互作用研究方法已有50多年的历史,如早期的化学交联法;并且该方法的技术在不断地完善和更新,产生了一序列新的技术,包括酵母双杂交、GST pull-down,免疫共沉淀和串联亲和纯化等多种技术。由于本实验室在热带植物病毒研究中涉及了多种蛋白质相互作用技术,为此,本文总结热带植物病毒研究中常用的蛋白质相互作用技术。     

植物抗病基因工程育种策略及其在大豆上的应用

概述了植物抗真菌病害、细菌病害和病毒病害基因工程育种的主要策略,重点介绍了大豆转基因抗病育种的主要成果.植物抗病害转基因育种策略主要包括基于寄主-病原菌相互识别和信号传导体系的基因工程和基于抗菌蛋白的基因工程;植物抗病毒病害基因工程育种策略还包括病毒外壳蛋白基因介导的抗性、复制酶介导的抗性、运动蛋白介导的抗性、卫星RNA介导的抗性、RNA介导的抗性等.     

PRSV运动相关病毒蛋白的Sos恢复系统诱饵载体构建及自激活效应检测

为研究与番木瓜环斑病毒运动相关的病毒蛋白(CP、HC-Pro、CI)互作的寄主因子,构建以Sos恢复系统为原理的胞质酵母双杂交系统的3个病毒蛋白诱饵载体,并检测其自激活作用来验证是否适用于后续的文库筛选.通过RT-PCR从番木瓜环斑病毒海南分离物中扩增出这3个病毒蛋白的片段,按正确方向将目标片段分别插入诱饵载体pSos中,并将重组质粒导入酵母温度敏感酵母菌株cdc25H,检测其表达产物对酵母Sos恢复系统Ras信号通路的激活作用而产生的细胞温敏特性影响.结果表明,这3个含番木瓜环斑病毒运动相关的病毒蛋白的诱饵载体构建正确,对酵母菌株cdc25H的Sos恢复系统无激活作用,激活试验为阴性.     

Disease and pest resistance in grains of sorghum and millets

In this review available information on the mechanisms of resistance to insect pests and fungal pathogens in sorghum and millets is discussed. The primary source of resistance lies in the chemical and physical make up of the grain. Phenolic compounds such as ferulic acid and tannins present in some sorghums are potent inhibitors of pests and pathogens. Grain hardness is a major deterrent to infection and infestation in low tannin grains. The prolamins, the grain storage proteins of sorghum, are organized into protein bodies and provide a physical and a nutritional barrier since they are resistant to digestion by insect and fungal proteases. A plethora of proteins that belong to the ‘pathogenesis related protein’ group are distributed in various parts of the grain. Some of them are located in protein bodies. Notwithstanding, sorghum is still susceptible to insect pests and fungal pathogens. An understanding of the natural mechanisms of resistance in the grain is paramount for the development of durable resistance against pests and pathogens. The pyramiding of resistance genes and the development of transgenic lines based on this understanding are two sources of hope for the future protection of sorghum and millets.     

Lipids in bread making: Sources, interactions, and impact on bread quality

Lipids exhibit important functional properties in bread making, although they are present in lower levels than starch or protein. They originate from flour, in which they are endogenously present, or from added shortening and/or surfactants. This review discusses lipid sources and their interactions during the entire process of bread making from dough mixing to fermentation, proofing, baking and the stored product. The focus is on lipid interactions with starch and gluten proteins, their role in gas cell stabilisation and their impact on bread loaf volume, crumb structure and crumb firming. Widely accepted views on lipid functionality, although often opposing, are presented and critically discussed.     

Minor components and wheat quality: Perspectives on climate changes

Minor compounds of cereal grains such as lipids and cell wall polysaccharides play an important role in their milling properties, their transformation into baked cereal products and their nutritional properties. These effects are mainly due to their interactive properties, such as their ability to absorb large amounts of water for polysaccharides or to interact with starch polymers and proteins for lipids. Environmental stress induced by climate change and regulations for environmentally friendly agriculture can interfere with the biosynthesis of these minor grain compounds as well as the main compounds starch and protein. Indeed, the metabolic networks of all components of the endosperm of grains are closely associated. Therefore, in the context of climate change and agricultural sustainability, lipids and cell wall polysaccharides may be affected. Contrasting with a plethora of studies reporting the impact of environment and fertilizer on cereal proteins, there is a real lack of information on this particular topic for these minor compounds. However, our knowledge of the physico-chemical properties and biosynthesis of these minor compounds allows us to assess the likely strong impact of environmental and agronomic constraints on the relationships between cell wall polysaccharides, lipids and cereal quality.     

Microbial Diversity and Phage–Host Interactions in the Georgian Coastal Area of the Black Sea Revealed by Whole Genome Metagenomic Sequencing

Viruses have the greatest abundance and highest genetic diversity in marine ecosystems. The interactions between viruses and their hosts is one of the hot spots of marine ecology. Besides their important role in various ecosystems, viruses, especially bacteriophages and their gene pool, are of enormous interest for the development of new gene products with high innovation value. Various studies have been conducted in diverse ecosystems to understand microbial diversity and phage–host interactions; however, the Black Sea, especially the Eastern coastal area, remains among the least studied ecosystems in this regard. This study was aimed at to fill this gap by analyzing microbial diversity and bacteriophage–host interactions in the waters of Eastern Black Sea using a metagenomic approach. To this end, prokaryotic and viral metagenomic DNA from two sampling sites, Poti and Gonio, were sequenced on the Illumina Miseq platform and taxonomic and functional profiles of the metagenomes were obtained using various bioinformatics tools. Our metagenomics analyses allowed us to identify the microbial communities, with Proteobacteria, Cyanobacteria, Actinibacteria, and Firmicutes found to be the most dominant bacterial phyla and Synechococcus and Candidatus Pelagibacter phages found to be the most dominant viral groups in the Black Sea. As minor groups, putative phages specific to human pathogens were identified in the metagenomes. We also characterized interactions between the phages and prokaryotic communities by determining clustered regularly interspaced short palindromic repeats (CRISPR), prophage-like sequences, and integrase/excisionase sequences in the metagenomes, along with identification of putative horizontally transferred genes in the viral contigs. In addition, in the viral contig sequences related to peptidoglycan lytic activity were identified as well. This is the first study on phage and prokaryote diversity and their interactions in the Eastern coastal area of the Black Sea using a metagenomic approach.     

Seaweed Protein Hydrolysates and Bioactive Peptides: Extraction,Purification, and Applications

Seaweeds are industrially exploited for obtaining pigments, polysaccharides, or phenolic compounds with application in diverse fields. Nevertheless, their rich composition in fiber, minerals, and proteins, has pointed them as a useful source of these components. Seaweed proteins are nutritionally valuable and include several specific enzymes, glycoproteins, cell wall-attached proteins, phycobiliproteins, lectins, or peptides. Extraction of seaweed proteins requires the application of disruptive methods due to the heterogeneous cell wall composition of each macroalgae group. Hence, non-protein molecules like phenolics or polysaccharides may also be co-extracted, affecting the extraction yield. Therefore, depending on the macroalgae and target protein characteristics, the sample pretreatment, extraction and purification techniques must be carefully chosen. Traditional methods like solid–liquid or enzyme-assisted extraction (SLE or EAE) have proven successful. However, alternative techniques as ultrasound- or microwave-assisted extraction (UAE or MAE) can be more efficient. To obtain protein hydrolysates, these proteins are subjected to hydrolyzation reactions, whether with proteases or physical or chemical treatments that disrupt the proteins native folding. These hydrolysates and derived peptides are accounted for bioactive properties, like antioxidant, anti-inflammatory, antimicrobial, or antihypertensive activities, which can be applied to different sectors. In this work, current methods and challenges for protein extraction and purification from seaweeds are addressed, focusing on their potential industrial applications in the food, cosmetic, and pharmaceutical industries.     

Seed proteins ofPisum mutants and recombinants

The seed protein production of more than a hundredPisum mutants and recombinants was determined. There is a negative correlation between seed production and protein content of the seed meal in the material tested. Geneipc of thePisum genome increases the protein content of the seed flour by about 20%, but the mutant carrying this gene and the recombinants available so far produce only low amounts of protein, due to their poor seed production. The biochemical methods used allow the detection of the genetic diversity as expressed in altered gene products in closely related genotypes. Many mutants and recombinants, derived from the same mother variety, are available in our collection showing not only quantitative and qualitative differences in their seed proteins, but also other characters of agronomic interest such as higher yield or adaptation to distinct climatic conditions. They represent a suitable material for analyzing the regulatory mechanisms controlling the incorporation of proteins into seeds.     

暹罗炭疽菌中脂滴包被蛋白Cap20与乙酸激酶CsAck的互作研究

暹罗炭疽菌（Colletotrichum siamense）是一种重要的病原真菌,是我国橡胶树炭疽病（Colletotrichum leaf disease, CLD）的田间优势病原种,也是热带、亚热带地区许多其他农林作物炭疽病的主要病原种。脂滴是细胞的中性脂,存在于绝大多数真核细胞中,是细胞内甘油三酯的主要贮存形式。脂滴在细胞内可以参与脂质代谢、膜转运、蛋白降解和信号传导等。包被蛋白（perilipin）是脂滴表面最丰富的脂质相关蛋白,主要存在于动植物体内,对细胞内脂滴代谢起着重要的调控作用。Cap20是暹罗炭疽菌中包被蛋白的同源蛋白,前期证实Cap20是一个致病相关蛋白,它参与了炭疽菌附着胞膨压形成、脂滴数量和致病性。了解其互作蛋白和互作的生物学意义可能为深入了解脂滴包被蛋白参与的致病分子机制具有重要意义。课题组前期通过酵母双杂交技术在暹罗炭疽菌cDNA文库中筛选到一个与Cap20互作的候选蛋白乙酸激酶,本研究克隆了该乙酸激酶的编码基因,并进行了蛋白结构和同源蛋白聚类分析。结果表明,乙酸激酶编码基因的DNA大小为1312 bp,包括一个内含子,cDNA为1248 bp,编码415个氨基酸,含有一个乙酸激酶结构域,命名为CsAck。然后构建含6×his标签的原核表达载体PET32a-CsAck,利用其和含GST标签的pGEX6p-1-Cap20（先前构建）进行蛋白表达和纯化,获得含有6×His标签的融合蛋白CsAck和含有GST标签的Cap20。通过Pull down验证了Cap20和CsAck蛋白之间的体外互作。最后,构建含有潮霉素转移酶基因HPH的表达载体PXY203-CsAck-S和含有氯嘧磺隆抗性基因ILV1的表达载体pCB1532-GFP-Cap20,将它们共同转化暹罗炭疽菌野生型菌株获得转化子。Co-IP（Co-immunoprecipitation）的结果证实Cap20和CsAck在暹罗炭疽菌中可以相互作用。该结果证实了致病性相关蛋白Cap20和乙酸激酶CsAck在体外和体内均可发生蛋白互作,可为进一步研究Cap20在暹罗炭疽菌中的致病功能和调控机制奠定基础。     

水稻瘤矮病病毒和矮缩病病毒的核苷酸结合蛋白和核酸结合蛋白及其结合能力分析

采用α-32P标记的三磷酸核苷酸与病毒粒子共浴的方法及Northwestern分子杂交方法,研究证明了RGDV结构蛋白中,P1蛋白可能是依赖于RNA的RNA聚合酶, P5蛋白可能具有RNA鸟苷酰转移酶功能,P6蛋白可能是核酸结合蛋白,是RGDV复制包装中的关键蛋白。与RGDV结构蛋白具有相同生物功能的RDV的核酸结合蛋白与核酸的结合能力最强,依赖于RNA的RNA聚合酶与核酸的结合能力次之,RNA鸟苷酰转移酶与核酸的结合能力最弱。结果暗示了可以根据与核酸的结合能力,辅助判断RDV或RGDV这3个结构蛋白的生物功能。     

The S-poor prolamins of wheat,barley and rye: Revisited

The last review of the S-poor prolamins was published in 1995. Since then there has been a considerable increase in out knowledge of this interesting and unique group of proteins. The advances in the understanding of genetics and polymorphisms of the proteins are discussed including the available gene sequences and their alignments and consensus sequences. This group of prolamins are implicated as major allergens in WDEIA and wheat allergy and as immunodominant proteins in coeliac disease. The epitopes and their distribution throughout the protein sequences are reviewed. Their structure and physical chemistry is discussed in relation to their functional properties.     

小麦贮藏蛋白分子结构及其研究技术进展

小麦贮藏蛋白不仅是人类植物蛋白的重要来源,也是面粉加工品质的决定因素.尽管人们对面团中二硫键的形成模式有了初步的认识,但仍无法完整、准确、系统地阐述面团的产生机制.为了给相关研究提供参考,笔者介绍了蛋白质分子结构研究的主要技术,如圆二色光谱技术、傅立叶-红外光谱技术、扫描隧道显微镜检测技术、原子力显微镜、核磁共振波谱技术和差示扫描量热技术的原理及其特点,综述了近年来这些技术应用于小麦贮藏蛋白分子结构研究方面的进展,并讨论了存在问题及其发展前景.     

Rheological and thermal properties of masa as related to changes in corn protein during nixtamalization

Traditional nixtamalization process produces a masa (dough) with appropriate cohesiveness and adhesiveness. Masa is considered as a network of solubilized starch polymers with dispersed, uncooked and swollen starch granules, cell fragments, proteins and lipids. In this work, the influence of proteins on the masa viscoelastic behavior was studied in corn kernels under different nixtamalization conditions. Scanning electron microscopy, SDS-PAGE, differential scanning calorimetry and rheological analysis were used to characterize the corn samples. The micrographs showed that the nixtamalization modified the shape of the starch granules and protein bodies, but no changes in appearance were observed when protein was removed. SDS-PAGE showed that corn proteins polymerized during cooking. Lime promoted both calcium–protein and protein–calcium–protein interactions mainly by calcium bridges, which were difficult to disrupt and increased the protein thermo-resistance. In the absence of lime, corn proteins polymerized mainly by disulfide bond cross-linking. Thermal analysis (DSC) indicated that the gelatinization temperature increased in lime-treated samples compared to control samples. Rheological studies showed that the corn protein exhibited greater influence on gel strength by enhancing the elastic character of the samples (G′). These results suggested that polymerized corn proteins stabilized the gel structure, which in consequence influenced the viscoelastic behavior of masa.     

Combined effect of genetic and environmental factors on the accumulation of proteins in the wheat grain and their relationship to bread-making quality

Bread-making quality of wheat flour is largely determined by the accumulation, concentration and composition of the proteins in the grain, which are influenced by genetic (G) and environment (E) variation and their interactions. We have therefore evaluated the importance of G and E factors and their interactions in determining the accumulation and composition of the proteins in the wheat grain. The cultivar determined development time (CDDT), together with the amount and timing of N application, played a significant role in determining the accumulation and final composition of the wheat grain proteins, explaining 21–59% of the variation. At low temperature, N application both at spike formation and at anthesis explained the highest proportion of variation (36%) in the percentage of sodium dodecyl sulphate (SDS) unextractable polymers in the total amount of polymers (% UPP), while at high temperature CDDT contributed most to the variation in % UPP (20%). The largest contributor to variation in the amount of total SDS extractable proteins (TOTE) was N application at anthesis, both at low and high temperatures (12% and 36%, respectively). Thus, the climate should be considered in recommendations for improving the protein quality and thereby the bread-making quality of wheat.     

基于iTRAQ技术的干旱胁迫下玉米苗期蛋白质组学研究

采用同位素标记相对定量(iTRAQ)技术,对干旱胁迫条件下苗期玉米的蛋白质组学变化进行分析。结果表明,共检测到玉米幼苗中的207个蛋白在干旱胁迫后发生了显著的丰度变化。根据蛋白注释情况可将这些蛋白归入信号传导、渗透调节、蛋白合成与折叠、ROS清除、膜运输、转录相关、细胞结构与细胞周期、脂肪酸代谢、碳水化合物与能量代谢、光合作用与光呼吸等代谢途径。干旱胁迫后,涉及光反应和呼气作用的差异蛋白多表现为丰度上升;涉及碳水化合物及蛋白质合成差异蛋白多表现为丰度下降;与渗透调节相关的脱水蛋白、脯氨酸代谢和渗透胁迫相关的蛋白酶则显示为丰度上升。干旱胁迫还能导致植物体内活性氧大量产生,活性氧清除相关的酶类也会发生明显的丰度上升。根据研究结果推测,玉米苗期主要通过降低植株生长速率、减少水分散失、清除自由基等多种方式维持其在干旱胁迫条件下的生长发育过程。     

Rice Protein Aggregation During the Flaking Process

The effect of roasting of soaked paddy rice and its subsequent flaking during production of rice flakes on solubility of rice proteins in various solvents was investigated. The solvents were so chosen to provide an understanding of the type of protein–protein interactions occurring during processing. There was an increase in protein solubility upon roasting of soaked paddy in fine hot sand (175–250°C) – a prelude to the flaking process – however, after subsequent flaking solubility decreased. Gel filtration studies indicated that aggregates with molecular weights greater than 4×10⁷kDa formed during flaking as a result of disulphide bonding. The aggregates were composed of 68, 31–34 and 24 kDa proteins. Of these, the 31–34 kDa proteins were identified as «putative prolamins». These proteins require detergent for their initial solubilisation. The prolamins of 13–16 kDa were not involved in the formation of aggregates. Their solubility decreased after flaking. The higher molecular weight proteins involved in aggregate formation were susceptible to proteolysis, whereas the 13 kDa prolamin was resistant.     

A Therapeutic Potential for Marine Skeletal Proteins in Bone Regeneration

A vital ingredient for engineering bone tissue, in the culture dish, is the use of recombinant matrix and growth proteins to help accelerate the growth of cultivated tissues into clinically acceptable quantities. The skeletal organic matrices of calcifying marine invertebrates are an untouched potential source of such growth inducing proteins. They have the advantage of being ready-made and retain the native state of the original protein. Striking evidence shows that skeleton building bone morphogenic protein-2/4 (BMP) and transforming growth factor beta (TGF-β) exist within various marine invertebrates such as, corals. Best practice mariculture and the latest innovations in long-term marine invertebrate cell cultivation can be implemented to ensure that these proteins are produced sustainably and supplied continuously. This also guarantees that coral reef habitats are not damaged during the collection of specimens. Potential proteins for bone repair, either extracted from the skeleton or derived from cultivated tissues, can be identified, evaluated and retrieved using chromatography, cell assays and proteomic methods. Due to the current evidence for bone matrix protein analogues in marine invertebrates, together with the methods established for their production and retrieval there is a genuine prospect that they can be used to regenerate living bone for potential clinical use.