Screening for reproductive biomarkers in Bactrian camel via iTRAQ analysis of proteomes

Bactrian camel is an ancient and precious species of livestock; that is, unique resources exist in the desert and have important economic and scientific value. In recent years, the number of Bactrian camels has declined sharply. Due to its long reproductive cycle and seasonal oestrus, the mechanism of oestrus is unknown. To identify candidate biomarkers of reproduction, we performed a comprehensive proteomic analysis of serum from Bactrian camel in oestrus and non-oestrus, using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with tandem mass spectrometry. We identified 359 proteins, of which 32 were differentially expressed: 11 were up-regulated and 21 were down-regulated in samples from camels in oestrus. We validated the differential expression of a subset of these proteins using qPCR and Western blot. Gene ontology annotation identified that the differentially expressed proteins function in cellular processes, metabolic processes and immune system processes. Notably, five of the differentially expressed proteins, PCGF5, histone H1.2, RBP4, FOLR1 and ANTXR2, are involved in reproductive regulatory processes in other animals. KEGG enrichment analysis demonstrated significant enrichment in several cardiac-related pathways, such as ‘dilated cardiomyopathy’, ‘hypertrophic cardiomyopathy’, ‘cardiac muscle contraction’ and ‘adrenergic signalling in cardiomyopathy’. Our results suggest that candidate biomarker (PCGF5, histone H1.2, RBP4, FOLR1 and ANTXR2) discovery can aid in understanding reproduction in Bactrian camels. We conclude that the profiling of serum proteomes, followed by the measurement of selected proteins using more targeted methods, offers a promising approach for studying mechanisms of oestrus.     

Comparative analysis of different methods revealing the involvement of proteins during ageing of rice seeds

Seed longevity is a very important characteristic controlling seed quality. However, the mechanism underlying this characteristic is poorly understood. In this study, 'FH7185', a storable rice variety, the germination rate of which was 66.63%, much higher than the control after artificial ageing under relative humidity 88% and 42°C for 21 days. Different methods were applied to reveal the involvement of proteins during ageing of rice seeds. In total, 35 differential proteins were identified from 2D‐PAGE, and 3,719 proteins from iTRAQ analysis. A comparison of these two methods showed that not all protein types could be detected on the 2D‐PAGE gels, and the dynamic range was somewhat limited. So the comprehensive proteome map from the iTRAQ analysis was used to identify the quantitatively regulated proteins that played roles in seed longevity, and found that the most marked change was the increased abundance of many metabolic enzymes, especially the ones involved in α‐linolenic acid metabolism in the embryos during ageing. OsLOX2 and OsLOX3 had a negative effect on seed longevity, which were lower in FH7185 than the control. The dehydrogenase (LOC_Os07g44430) which played a major catalytic role in lipid peroxidation, also changed significantly during ageing. Therefore, OsLOX2, OsLOX3 and Loc_OS 07G44430 may be involved in the regulation of seed longevity with a synergistic effect.     

利用iTRAQ技术和转录组筛选芍药属远缘杂交不亲和基因

【目的】远缘杂交育种是目前牡丹、芍药品种改良和育种的主要方法,而远缘杂交不亲和一直是制约其快速发展的主要因素。本研究从牡丹、芍药远缘杂交授粉后不亲和应答相关的柱头差异蛋白与转录组方面深入研究,揭示牡丹、芍药远缘杂交不亲和的分子机理,为杂交育种提供理论依据。【方法】以芍药‘粉玉奴’自交、芍药‘粉玉奴’与牡丹‘凤丹白’杂交为供试材料,在授粉后24 h采取柱头,分别进行同位素标记相对定量（iTRAQ）和转录组技术分析。对所获得的蛋白和转录组数据进行生物信息学分析,并对其中可能与远缘杂交不亲和相关的基因进行定量PCR验证。【结果】利用iTRAQ技术分析牡丹、芍药远缘杂交后柱头中蛋白质的表达差异,共鉴定到685个差异蛋白,富集到了188条通路,其中显著富集的Pathway有18条。与不亲和授粉相关代谢通路有RNA降解、钙信号途径、丝裂原活化蛋白激酶（mitogen-activated protein kinase signaling pathway,MAPK）信号途径、磷脂酰肌醇信号系统。在RNA降解代谢通路中,烯醇酶（Enolase）、热休克蛋白DnaK（HSP70）及病菌抗原（GroEL）均表达下调。在钙信号途径中,钙调蛋白（CALM）表达下调,腺苷酸转运酶（adenine nucleotide translocase,ANT）表达量增加,表达上调。MAPK信号途径中,乙二醛酶Ⅰ（GloI）表达下调。磷脂酰肌醇信号系统中的钙调蛋白（CALM）表达下调。随机选取与差异蛋白相关的6个基因进行qRT-PCR验证,结果显示,6个基因的表达与蛋白质水平趋势相一致,均表达下调。通过转录组测序,共获得了52 998个有注释信息的Unigene,占所有Unigene的40.37%。基于6组样品的RPKM（Reads Per Kilobase per Million）值,共筛选到16 224个差异基因。其中上调基因13 361,下调基因2 863个。对差异基因进行Pathway显著富集分析,杂交与自交相比,不亲和差异表达的基因主要富集在氧化磷酸化代谢、ABC转运蛋白、次级代谢产物等通路。与远缘杂交不亲和相关且发生显著变化的基因有CalS-5、CalS-12（胼胝质酶）和SPL（squamosa promoter binding protein-like）表达上调,ABCF（ABC transporter family protein）表达下调。【结论】在转录组和蛋白数据共注释到6个蛋白、4个基因与植物不亲和性密切相关,这些蛋白与基因可能在远缘杂交不亲和方面发挥着重要作用。     

玉米籽粒早期发育相关蛋白的差异表达特性

玉米籽粒发育早期,代谢活动旺盛,细胞分裂与增大活跃,为后续贮藏物质的合成形成充足库容。为阐明籽粒早期发育的蛋白合成、积累与调控过程,本研究以夏玉米品种登海661为试验材料,在开花期人工饱和授粉后第3、第5、第10天取果穗中部籽粒,利用同位素标记相对定量(iTRAQ)技术分析其蛋白差异表达特性。玉米籽粒早期发育阶段总计鉴定及定量2639种蛋白,这些蛋白涉及多种生物过程与分子功能,其中代谢过程和分子过程是最主要的2个生物过程;催化活性和绑定功能是最主要的2个分子功能,这些生物过程与分子功能对籽粒早期发育具有重要作用。定量分析结果表明137种蛋白在籽粒发育早期显著差异表达,其功能涉及蛋白代谢、胁迫响应、细胞生长与分裂、碳水化合物与能量代谢、转运、次生物质代谢、淀粉合成、转录、油脂代谢、信号转导、氨基酸代谢等。其中,表达差异较大的是与蛋白代谢、胁迫响应、细胞生长与分裂以及碳水化合物与能量代谢相关的蛋白。表达模式聚类结果显示这些不同功能类别的蛋白协同表达,共同调控玉米籽粒的早期发育。     

弓形虫卵囊感染小鼠的急性期与慢性期的脑组织蛋白质组变化

弓形虫是一种呈世界性分布的机会性致病原虫,可引起致命性脑炎。人弓形虫病急性感染往往与弓形虫卵囊污染密切相关。探究弓形虫卵囊感染对宿主的致病机制和防控弓形虫病具有重要意义。因此,本研究利用iTRAQ技术,结合2D-LC-MS/MS分析弓形虫PRU虫株卵囊急、慢性感染小鼠后,其脑组织蛋白质的差异表达情况。结果表明,在急性感染期和慢性感染期小鼠脑组织中分别鉴定到85和51个差异表达的蛋白,而慢性感染期与急性感染期相比有114个蛋白差异表达。对差异表达蛋白进行功能分析发现其主要涉及到免疫、代谢过程等通路。本研究结果为进一步阐明弓形虫卵囊急慢性感染引起宿主脑组织损伤及致病机制提供了重要的数据。     

广叶绣球菌子实体不同发育阶段的比较蛋白质组学分析

利用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification,iTRAQ)标记结合二维液相色谱串联质谱(two-dimensional liquid chromatography tandem mass spectrometry,2D LC-MS/MS)技术,研究广叶绣球菌(Sparassis latifolia)原基期、幼菇期和成熟子实体阶段的差异表达蛋白质组.采用Q-Exactive质谱鉴定并经ProteinPilot软件搜库,对所获得的差异蛋白进行GO (gene ontology)、KEGG(kyoto encyclopedia of genes and genomes)和转录因子注释分析.结果共鉴定到可信蛋白2305个,其中2219个蛋白具有相对定量信息.与原基期相比,幼菇期显著上调蛋白104个,下调蛋白142个,子实体阶段显著上调蛋白155个,下调蛋白460个.GO分子功能提示这些差异性蛋白主要参与催化活性、蛋白结合和水解酶活性.KEGG代谢通路分析结果显示,差异蛋白主要涉及碳代谢、氨基酸合成、核糖体、糖酵解/糖衍生等代谢过程.差异蛋白中与信号传导和转录因子相关的蛋白数量分别为27个和7个.     

TMV侵染烟草的早期响应蛋白分析

为了完善烟草花叶病的预警防控体系,分析烟草感染烟草普通花叶病毒后的蛋白质组早期变化,筛选烟草的早期响应蛋白。[方法]采用iTRAQ技术分析了蛋白质组变化,采用qPCR技术检测基因的表达。[结果]蛋白质组分析发现160个差异表达蛋白质,包括有64个上调表达蛋白和96个下调表达蛋白,这些差异表达蛋白质主要与氧化还原平衡调节、RNA降解、叶绿体组成及光合作用、逆境胁迫响应有关;qPCR结果显示这些差异蛋白质的mRNA表达变化趋势与iTRAQ数据基本一致;及时施用抗病毒药剂(氨基寡糖素和吗胍.乙酸铜)和植物诱抗剂(盐酸吗啉胍和氮苷吗啉胍)能够预防和控制烟草花叶病的发生,但同时发现PR10、LHCB、POD、HSP90、14-3-3和AGO1等蛋白的表达也受到了影响。[结论]试验筛选出一些TMV感染相关的烟草早期响应蛋白,不仅有助于阐述烟草花叶病发生的机制,也可用于筛选新的抗病毒药剂和植物诱抗剂。     

应用iTRAQ定量蛋白质组学技术筛选无乳链球菌鱼源株与人源株差异表达蛋白

以斑马鱼和巨噬细胞作为体内、外感染模型,比较无乳链球菌鱼源株GD201008-001与人源株A909的致病性差异,利用iTRAQ技术和质谱分析技术进行差异表达蛋白鉴定,以期为揭示无乳链球菌不同宿主来源株致病机制提供新思路。实验通过测定菌株GD201008-001、A909的斑马鱼半数致死量和巨噬细胞吞噬率,比较二者致病性差异;提取全菌蛋白,经iTRAQ试剂标记后进行质谱鉴定,质谱数据用软件Mascot 2.2和Proteome Discoverer 1.4进行查库(Uni Prot数据库)鉴定及定量分析,并对差异蛋白进行GO功能注释和KEGG通路分析。结果显示,GD201008-001毒力显著高于A909;通过iTRAQ分析两株菌差异表达蛋白,发现差异蛋白涉及的生物学功能较为广泛,在鉴定出的368个差异表达蛋白中,GD201008-001中上调表达蛋白193个(比值1.5),下调表达蛋白175个(比值0.667)。生物信息学分析预测这些蛋白主要涉及26个生物学功能,14个通路,推测Clp X、Glm S和Cps IVK可能在两株菌致病性差异中发挥重要作用。本研究为阐明无乳链球菌不同宿主来源株致病性差异奠定了基础。     

应用iTRAQ结合2D LC-MS/MS技术分析草菇同核和异核菌丝蛋白质组

首次利用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification,iTRAQ)技术结合二维液相色谱串联质谱对草菇(Volvariella volvacea)同核和异核菌丝蛋白质组进行研究。采用QExactive质谱鉴定并经MASCOT软件搜库,对所有鉴定蛋白进行主成分分析(principal component analysis,PCA)、层次聚类(hierarchical clustering)分析和GO(gene ontology)注释,并进行生物信息学分析。结果共获得2335个不同肽段,鉴定到1039个蛋白,其中1030个蛋白具有定量信息。在同核菌丝中显著上调蛋白83个,下调蛋白97个,表明iTRAQ标记技术结合二维液相色谱串联质谱可有效地分离和鉴定草菇菌丝蛋白质组。该研究结果为更深入全面地研究草菇菌丝蛋白质组学奠定基础。