敲降VPS28基因对中国荷斯坦奶牛乳脂合成的调控

本课题组前期通过GWAS研究,发现VPS28基因在荷斯坦奶牛乳腺组织中特异性高表达,且其5′-UTR的突变位点-58C>T与乳脂性状关联,但其对乳脂性状的调控机理尚未明确。本研究为了明确VPS28基因及其突变位点-58C>T对乳脂的调控机理,首先利用启动子活性分析检测突变位点-58C>T对VPS28基因的影响,发现该突变位点显著降低VPS28基因启动子活性;然后利用RNA干扰技术敲降奶牛原代乳腺上皮细胞中VPS28基因表达量,检测VPS28通路和乳脂合成相关基因mRNA表达量以及细胞中脂肪滴形态,分析结果发现敲降VPS28基因可降低泛素化-溶酶体和泛素化-蛋白酶体通路基因和乳脂合成相关基因的表达量,并提高细胞中甘油三酯的合成,预示VPS28基因可能通过泛素化-溶酶体和泛素化-蛋白酶体途径调控乳脂生成。本研究结果在转录组水平揭示VPS28基因对乳脂合成的调控机制,为奶牛乳脂性状的分子育种研究提供参考依据。     

果生刺盘孢CfHAC1调控应答二硫苏糖醇胁迫的转录组分析

果生刺盘孢Colletotrichum fructicola是油茶炭疽病优势病原菌。前期研究发现bZIP转录因子CfHac1参与调控该菌的生长发育和致病性。为了揭示转录因子CfHac1调控果生刺盘孢响应内质网压力和致病机理,本研究测定了ΔCfhac1突变体对内质网压力胁迫剂的敏感性,发现突变体对二硫苏糖醇（dithiothreitol,DTT）的耐受性下降,说明CfHAC1基因可能参与调控果生刺盘孢响应内质网压力胁迫过程。进一步利用高通量RNA-seq技术对该病菌野生型菌株和CfHAC1敲除突变体菌株在DTT胁迫下的转录组进行了比较分析,结果表明差异表达基因共有2 680个,其中上调表达基因有1 181个,下调表达基因有1 499个。Gene Ontology 功能分析结果显示,差异表达基因主要参与催化活性、结合、代谢过程、细胞过程、细胞成分合成、生物过程调控和应激反应等生物学过程。KEGG功能富集分析表明,上调表达基因主要被富集到核糖体、真核细胞的核糖体生物合成、RNA转运和氰基氨基酸代谢通路中;下调表达基因显著富集在内质网蛋白质加工、N-聚糖生物合成、类固醇合成和蛋白质分泌等通路中。分析发现转录因子CfHac1调控内质网胁迫应答和致病相关基因的表达。本研究提供了在全基因组水平上对CfHAC1基因与内质网压力胁迫应答之间关联的新认识,为阐明果生刺盘孢响应内质网压力胁迫和致病机制奠定了基础。     

萱草叶片响应低温胁迫的转录组分析

低温胁迫是萱草（Hemerocallis fulva）生长过程中经常会遭遇的一种非生物胁迫。比较了萱草叶片在低温处理（10、5、0 ℃）下转录组与对照（15 ℃）数据的差异,共筛选出差异表达基因2 457个,其中上调基因1 253个,下调基因1 204个。差异表达基因主要富集在细胞过程、代谢过程和催化活性等49个GO过程,代谢途径、次生代谢产物的生物合成、植物激素信号转导等42条KEGG代谢途径中。其中参与植物激素信号转导通路的差异表达基因发生了不同程度的变化,GH3.10基因上调至对照组的13.624倍,IAA1基因下调0.120倍;参与可溶性糖合成通路的差异基因发生了0.076~28.114倍不同程度的变化。随后对3个低温处理组共有的29个差异表达基因进行热图和网络调控分析,基于基因在网络调控中的位置,对ABCF5、OFPs和SWEETs等基因在冷应答的作用进行了分析。本研究结果为进一步挖掘萱草低温响应的关键基因及耐寒萱草种质开发、分子育种提供了一定的理论支撑。     

青壳鸡蛋与褐壳鸡蛋品质及营养成分比较分析

为了比较青壳鸡蛋和褐壳鸡蛋品质的差异,以苏禽青壳蛋鸡和海兰褐壳蛋鸡2个鸡种为试验素材,采用笼养饲养方式,测定苏禽青壳蛋鸡和海兰褐壳蛋鸡鸡蛋的常规蛋品质和营养成分含量。结果表明：在常规蛋品质方面,苏禽青壳蛋鸡鸡蛋的哈氏单位和蛋黄色泽极显著高于海兰褐蛋鸡鸡蛋（P＜0.01）,蛋黄比率显著高于海兰褐蛋鸡鸡蛋（P＜0.05）,蛋重极显著小于海兰褐蛋鸡鸡蛋（P＜0.01）,蛋形指数、蛋壳厚度和蛋壳强度差异不显著。在营养成分方面,苏禽青壳蛋鸡鸡蛋的干物质、脂肪、胆固醇和卵磷脂含量均极显著高于海兰褐蛋鸡鸡蛋（P＜0.01）,蛋白质和氨基酸含量差异不显著。钙、镁、铜含量两者差异不显著,但苏禽青壳蛋鸡鸡蛋的铁、锌、锰、硒、磷含量均极显著高于海兰褐蛋鸡鸡蛋（P＜0.01）,可见鸡种对鸡蛋常规品质和营养成分均具有一定的影响。     

氨基酸影响尖孢镰孢菌古巴专化型厚垣孢子的形成

香蕉枯萎病是由尖孢镰孢菌古巴专化型Fusarium oxysporum f. sp. cubense（Foc）侵染引起的一种土传真菌病害,已严重威胁香蕉产业的健康发展。该病菌产生的厚垣孢子可在土壤中存活多年,是香蕉枯萎病的初侵染源。本研究通过氨基酸添加试验,证明添加甘氨酸可抑制厚垣孢子的形成;通过对该病菌厚垣孢子形成前期、初期、中期和后期的转录组分析,发现氨基酸合成通路中有93个基因的表达水平在厚垣孢子形成过程中发生了显著变化;In silico 分析表明其中10个基因参与调控真菌的氨基酸合成,11个基因参与调控真菌种的生长发育和产孢,19个基因参与调控真菌种的致病性和毒素产生。由此推测,氨基酸合成通路不仅与尖孢镰孢菌古巴专化型厚垣孢子的形成相关,其有可能参与调控该病菌的致病性。     

麝鼠前列腺在繁殖期和非繁殖期转录组差异分析

麝鼠(Ondatra zibethicus L.)是季节性繁殖动物。成年雄性麝鼠在尿生殖孔上方肌肉与背皮之间有一对香腺,在繁殖期能分泌麝鼠香。与其他鼠类相比,麝鼠在繁殖期时前列腺—精囊腺组织极其发达。研究发现,经腹腔注射麝鼠香后,能够明显促进雄性小鼠的前列腺—精囊腺发育,表明两者之间可能存在一定的相关性。本研究利用RNA-seq技术对麝鼠繁殖期和非繁殖期的前列腺样品进行了转录组测序,对差异表达的基因进行GO分析和KEGG通路分析。结果显示,共筛选出1629个显著差异表达基因,涉及多种信号转导和能量代谢相关基因,其中OBP2、Bcl-2家族和肿瘤坏死因子受体超家族成员的差异表达提示麝鼠前列腺发育受多种机制调控,由香腺分泌的麝鼠香可能参与了前列腺的发育调节。     

过表达兴安落叶松TPP基因增强拟南芥对盐胁迫的耐受能力*

兴安落叶松(Larix gmelinii)是极为重要的造林针叶树种,具有早期速生、抗逆性强、生态效益好等特点。海藻糖参与调控干旱、寒冷、盐害等多种逆境胁迫,海藻糖-6-磷酸磷酸酶(TPP)是海藻糖合成通路的重要酶。从兴安落叶松逆境胁迫转录组中筛选到LgTPPI.1基因全长序列,克隆了其编码区(CDS),构建了重组载体并获得过表达LgTPPI.1拟南芥纯合株系。结果表明,LgTPPI.1 CDS全长1 236 bp,共编码411个氨基酸;LgTPPI.1基因的mRNA在根和茎中表达水平较低,在针叶中表达水平较高;过表达LgTPPI.1基因拟南芥在盐胁迫下海藻糖含量显著提高、脯氨酸和抗氧化酶类活性增加、胁迫响应标记基因表达上调、对盐胁迫的耐受性增强。这些结果表明,裸子植物利用与被子植物类似的海藻糖通路来耐受非生物胁迫。为后续进一步解析落叶松中海藻糖合成相关基因的功能,揭示裸子植物中针叶树对逆境的响应机制奠定基础。     

RNA-seq用于获得共轭亚油酸对贵妃鸡肌内脂肪代谢调控的关键基因

通过转录物组测序获得在贵妃鸡基础日粮中添加共轭亚油酸(CLA)对肌内脂肪代谢的差异表达基因,经生物信息学分析获得相关的信号通路及可能发挥重要作用的候选基因,为CLA对肌内脂肪沉积的分子机制奠定基础。本研究选用55日龄健康的贵妃鸡为试验动物,在基础日粮中添加CLA 0%、1%和2%,预饲期1周,正饲期6周。屠宰采集胸肌组织进行转录物组测序,对测序数据进行差异表达分析,差异表达基因GO功能和差异表达基因KEGG通路富集分析,筛选出与胸肌脂类代谢相关的差异表达基因,利用qRT-PCR对差异表达基因进行验证。结果显示,共获得1 065个差异表达基因,其中上调基因703个,下调基因362个。GO富集结果显示,差异表达基因主要富集在生物过程的细胞过程、单一生物过程、生物调节和代谢过程。KEGG信号通路富集显示,差异表达基因显著富集在黏着斑、不饱和脂肪酸生物合成、脂肪酸生物合成和类固醇生物合成等信号通路中,发现11个主要与肌内脂肪代谢相关的候选基因,分别是FADS1、FADS2、ELOVL5、ACOX2、SLC27A1、FABP5、LPL、LOC107050163、ENSGALG00000030996、ENSGALG00000005043和ENSGALG00000048882。并随机选取6个基因进行qRT-PCR验证,其相对表达量变化趋势与测序结果一致。本研究筛选到CLA影响贵妃鸡胸肌脂类代谢相关的差异表达基因,并对11个主要参与脂肪代谢相关的基因进行分析,为揭示CLA调控肌内脂肪沉积的分子机制奠定基础。     

基于RNA-Seq分析Ras1~(CA)在家蚕后部丝腺过表达对细胞周期通路基因的影响

【目的】本文旨在挖掘野生型家蚕Bombyx mori和Ras1CA过表达转基因家蚕后部丝腺中的转录本差异,分析和验证细胞周期通路中的差异表达基因,从而探讨Ras1CA过表达转基因家蚕提高蚕丝产量的分子机制。【方法】利用转录组学比较野生型和Ras1CA过表达转基因家蚕后部丝腺中的基因转录本差异,经实时荧光定量PCR(q RT-PCR)验证。【结果】野生型家蚕和Ras1CA过表达转基因家蚕后部丝腺组织中有2 636个差异表达基因,其中细胞周期信号通路中有42个差异表达基因,包括细胞周期依赖性激酶(CDK)、细胞周期素(Cyclin)以及转录因子等。通过q RT-PCR检测cdk1、cyclin D1、cyclin D2、cdc7、cdh1、dp-1,2等6个基因在野生型和Ras1CA过表达转基因家蚕后部丝腺组织中的相对表达量,发现转基因家蚕中的表达量均显著高于野生型(P<0.05),其中cyclin D2的表达差异极显著(P<0.01)。q RT-PCR结果与转录本差异一致,表明Ras1CA过表达后,能够促进细胞周期通路基因的表达。【结论】野生型家蚕和Ras1CA过表达转基因家蚕后部丝腺中有大量差异表达基因,且Ras1CA能够在转录水平上调控细胞周期通路,影响后部丝腺组织的细胞分裂和器官生长,从而促进蚕丝蛋白的合成。     

苗期水稻响应褐飞虱取食的基因差异表达分析

【目的】褐飞虱Nilaparvata lugens (St?l)是水稻的重要害虫之一,主要刺吸水稻韧皮部汁液为害,对水稻生产造成严重的产量和经济损失。为研究水稻响应褐飞虱取食的分子机制,对褐飞虱取食6 h后的苗期水稻进行转录组测序及基因差异表达分析。【方法】采用illumina二代测序技术获得褐飞虱取食前后水稻组织的转录组数据,利用RSEM软件进行基因表达定量和DEseq2进行差异表达分析;从差异表达基因中随机选取20个基因采用荧光定量PCR技术进行验证;采用GeneMerge软件对差异表达基因进行KEGG和GO富集分析。【结果】褐飞虱取食后,水稻转录组中的1 104个基因出现了差异表达,其中435个基因表达上调,669个基因表达下调。荧光定量PCR结果显示,20个差异表达基因中18个基因的表达变化趋势和测序结果一致,证明了转录组分析结果可靠。GO和KEGG富集分析表明,表达上调基因主要与水稻氧化应激、海藻糖合成及次生化合物代谢有关,显著富集在14个KEGG通路和30个GO功能分类中;而表达下调基因主要参与水稻纤维素、蛋白质及脂肪酸合成过程,显著富集在29个KEGG通路和26个GO功能分类。在差异表达基因中,分别有61个转录因子和13个水杨酸和茉莉酸信号通路相关基因。【结论】褐飞虱取食激发了水稻的应激反应和保护机制,同时还降低了营养合成的过程,是飞虱为害造成水稻减产的原因之一。本研究初步揭示了苗期水稻响应褐飞虱取食的差异表达基因,为研究水稻-褐飞虱互作机制以及褐飞虱抗性水稻品种培育提供了参考和依据。     

An EAV-HP Insertion in 5′ Flanking Region of SLCO1B3 Causes Blue Eggshell in the Chicken

The genetic determination of eggshell coloration has not been determined in birds. Here we report that the blue eggshell is caused by an EAV-HP insertion that promotes the expression of SLCO1B3 gene in the uterus (shell gland) of the oviduct in chicken. In this study, the genetic map location of the blue eggshell gene was refined by linkage analysis in an F₂ chicken population, and four candidate genes within the refined interval were subsequently tested for their expression levels in the shell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was found to be the only one expressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencing analysis showed that only the allele of SLCO1B3 from blue-shelled chickens was expressed in the uterus of heterozygous hens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion transporting polypeptide (OATP) family; and the OATPs, functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds, including bile salt in mammals. We subsequently resequenced the whole genomic region of SLCO1B3 and discovered an EAV-HP insertion in the 5′ flanking region of SLCO1B3. The EAV-HP insertion was found closely associated with blue eggshell phenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell is associated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the EAV-HP insertion is the causative mutation for the blue eggshell phenotype. The insertion was also found in another Chinese blue-shelled breed and an American blue-shelled breed. In addition, we found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which implied independent integration events in the blue-shelled chickens from the two continents, providing a parallel evolutionary example at the molecular level.     

Polymorphisms in eggshell organic matrix genes are associated with eggshell quality measurements in pedigree Rhode Island Red hens

Novel and traditional eggshell quality measurements were made from up to 2000 commercial pedigree hens for a candidate gene association analysis with organic eggshell matrix genes: ovocleidin-116 , osteopontin ( SPP1 ), ovocalyxin-32 ( RARRES1 ), ovotransferrin ( LTF ), ovalbumin and ovocalyxin-36 , as well as key genes in the maintenance and function of the shell gland [ estrogen receptor ( ESR1 ) and carbonic anhydrase II ( CAII )]. Associations were found for (i) ovalbumin with breaking strength and shell thickness; (ii) ovocleidin-116 with elastic modulus, shell thickness and egg shape; (iii) RARRES1 with mammillary layer thickness; (iv) ESR1 with dynamic stiffness; (v) SPP1 with fracture toughness and (vi) CAII with egg shape. The marker effects are as large as 17% of trait standard deviations and could be used to improve eggshell quality.     

Selenium distribution in eggs of avian species

We studied the effect of egg mass of eight different avian species on Se distribution between egg components and the effect of incubation on Se accumulation by chicken eggshell and shell membrane. Eight groups of birds received a diet without Se supplementation. Unfertile eggs were collected after 35 days of feeding; yolk, albumen, shell and shell membrane were assayed separately for Se. All avian species studied showed identical Se concentration in yolk–albumen complex equal to 38.7 μg Se/100 g, reflecting a linear correlation between yolk–albumen mass and Se content. Shells and shell membrane Se accumulation showed quadratic correlation with the appropriate mass thus explaining unusually high Se concentration in ostrich shell and shell membrane, that reached values 1785 and 1904 μg Se/kg respectively. Incubation of fertile eggs decreased eggshell Se content, the effect being more expressed in eggs from hens fed sodium selenite compared to organic Se utilization (Sel-Plex). It was concluded that shell might be an additional Se source for an embryo.     

Ovocalyxin-36 Is a Pattern Recognition Protein in Chicken Eggshell Membranes

The avian eggshell membranes are essential elements in the fabrication of the calcified shell as a defense against bacterial penetration. Ovocalyxin-36 (OCX-36) is an abundant avian eggshell membrane protein, which shares protein sequence homology to bactericidal permeability-increasing protein (BPI), lipopolysaccharide-binding protein (LBP) and palate, lung and nasal epithelium clone (PLUNC) proteins. We have developed an efficient method to extract OCX-36 from chicken eggshell membranes for purification with cation and anion exchange chromatographies. Purified OCX-36 protein exhibited lipopolysaccharide (LPS) binding activity and bound lipopolysaccharide (LPS) from Escherichia coli O111:B4 in a dose-dependent manner. OCX-36 showed inhibitory activity against growth of Staphylococcus aureus ATCC 6538. OCX-36 single nucleotide polymorphisms (SNPs) were verified at cDNA 211 position and the corresponding proteins proline-71 (Pro-71) or serine-71 (Ser-71) were purified from eggs collected from genotyped hens. A significant difference between Pro-71 and Ser-71 OCX-36 for S. aureus lipoteichoic acid (LTA) binding activity was detected. The current study is a starting point to understand the innate immune role that OCX-36 may play in protection against bacterial invasion of both embryonated eggs (relevant to avian reproductive success) and unfertilized table eggs (relevant to food safety).     

An evaluation of different methods for assessing eggshell pigmentation and pigment concentration using great tit eggs

The striking variation in colour and maculation of bird eggs has fascinated biologists since centuries, and many hypotheses based on mechanical, physiological or signalling functions have been proposed for its evolution by natural and sexual selection. Protoporphyrin is the main eggshell pigment found in brown maculated eggs, and is assumed to function as a mediator of these selection processes. It is a precursor of heme with pro‐oxidant properties, and hence a link between brown maculation and female condition has been proposed and tested in a number of studies, albeit with contrasting results. A variety of different visual methods have been used to quantify outer eggshell pigmentation, which has been assumed to correspond to overall quantity of protoporphyrin in the shell. Yet, this relationship has rarely been tested. The aim of this study was to apply four commonly used methods to assess pigmentation in great tit eggs with protoporphyrin as the predominant eggshell pigment, and to compare the results of these methods. Specifically, we 1) ranked eggshell pigmentation by human naked eye, 2) applied a granularity approach and 3) measured spectrophotometric reflectance of eggshell pigments. Second, we estimated the relationship between outer eggshell pigmentation (i.e. estimated by three different methods above) and true protoporphyrin concentration deposited in the entire shell measured by HPLC. Among‐method estimates were significantly correlated for the traits describing pigment ‘darkness’ only. While the model including scores based on human naked eyes explained 16% of the variance of pigment concentration in the entire shell, spectrometry explained 27%, and the granularity approach explained 40%. Thus, the estimation of true pigment concentration in the entire shell from the visible outer side of the shell is most reliable with the granularity approach. It is relevant for studies where the maintenance of the integrity of the eggs is essential.     

Effects of phytoestrogen supplementation in the feed on the shell gland of laying hens at the end of the laying period

Shell quality decreases as laying hens age and the aim of present study was to investigate how a supplement of daidzein, a natural phytoestrogen in soya, affects key factors in the shell gland and eggshell quality in late-stage laying hens. Hybrids of Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB), received either a daidzein diet (50mg/kg feed) or a control diet from 60 to 72 weeks of age. Both the total number of capillaries and capillaries with carbonic anhydrase (CA) activity were higher in the LSL hybrid than in the LB. After daidzein supplementation the number of CA positive capillaries was unaffected in the LSL but increased in the LB hybrid indicating a higher sensitivity to daidzein in this hybrid. Estrogen receptor alpha and beta (ERα, ERβ) were localized and the complete picture of the two ERs can now be described in shell gland of domestic hens. Nuclear and cytoplasmic staining was generally stronger for ERβ, while membrane associated staining was present only for ERα. Interestingly, capillary endothelium contained only ERβ and since estrogen regulation of CA is well documented, the presence of an endothelial ER provides one possible route for the increase in CA positive capillaries found in LB hybrids. Eggshell quality or egg production was not affected by daidzein supplementation. The hybrids used in this study showed anatomical differences and reacted differently to daidzein supplementation, but if this can be explained by the divergences in ERβ localization noted between the hybrids remains to be clarified.     

Presence of histamine and mast cells in chicken oviduct

The purpose of the present study was: (1) to demonstrate immunocytochemically the localization of histamine in the wall of four chicken oviductal parts, i.e. infundibulum, magnum, isthmus, and shell gland, (2) to identify the presence of mast cells in chicken oviduct, and (3) to determine histamine concentration in oviductal tissue by the spectrofluorometric method. Experiments were carried out on Isa Brown laying hens decapitated just after oviposition. The specific immuno-reactivity for histamine and the presence of mast cells were found in the wall of all the examined oviductal parts. The immuno-reactive histamine was localized in epithelium, tubular glands, connective tissue layer, circular and longitudinal muscles, and endothelium and muscles of blood vessels. The intensity of immuno-positive reaction was as follows: infundibulum > shell gland > magnum = isthmus and correlated with quantitatively determined histamine level and tissue density of mast cells. It is suggested that mast cells are the main source of histamine in the chicken oviduct.     

Differential label‐free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property

Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label‐free MS‐based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.