MYP基因在中间球海胆及杂交海胆生殖腺不同发育时期的转录表达差异

摘要: 应用实时荧光定量PCR技术对主要卵黄蛋白(Major yolk protein, MYP)基因在中间球海胆和杂交海胆(中间球海胆♀×光棘球海胆♂)的生殖腺不同发育时期的转录表达差异进行了分析。结果表明, MYP基因在海胆雌雄个体生殖腺中转录水平上的表达差异不明显; MYP基因在中间球海胆生殖腺发育的4个时期的表达呈快速下降的趋势, 而在杂交海胆呈缓慢下降的趋势。在分析的生殖腺4个发育期中, 中间球海胆雌雄个体 MYP 基因的表达量(以18S rRNA为参照)分别从44.55%和41.17%下降到9.59%和1.83%; 杂交海胆的雌雄个体分别从37.66%和36.66%下降到19.22%和12.55%。MYP基因的转录表达量差异与杂交后生殖腺产生的变异密切相关。     

光棘球海胆(Mesocentrotus nudus)TGF-β基因克隆及其对海水酸化的响应

旨在明确光棘球海胆(Mesocentrotus nudus)转化生长因子-β(transforming growth factorβ,TGF-β)基因的序列及结构信息,探明该基因在海水酸化胁迫下的表达模式。利用同源克隆和cDNA末端快速扩增(RACE)技术首次获得光棘球海胆TGF-β基因(命名为MnTGF-β)的全长cDNA序列。结果显示:(1)光棘球海胆TGF-β基因的cDNA全长为2 299 bp,其中5'非编码区长度为745 bp,3'非编码区长度为261 bp;开放阅读框(ORF)长度为1 290 bp,编码430个氨基酸,相对分子量为48.31 kD,理论等电点为5.34。(2)同源性及系统进化分析显示,光棘球海胆MnTGF-β蛋白序列与紫球海胆(Strongylocentrotus purpuratus)SpTGF-β2蛋白序列高度保守(同源性97.69%)。(3)实时荧光定量PCR(qRT-PCR)检测发现,MnTGF-β基因在光棘球海胆性腺、体腔液、肠、围口膜、管足5种组织中均有表达,其相对表达量从高到低依次为:管足围口膜肠体腔液性腺。(4)与自然海水组(pH 8.06±0.01)相比,当海水△pH为-0.3时,随酸化时间延长,MnTGF-β基因在光棘球海胆性腺中的相对表达量呈先升高而后极显著降低(P0.01)再极显著升高(P0.01)趋势,在管足中的相对表达量呈现极显著上调趋势(P0.01),而在肠组织中的相对表达量则先极显著降低(P0.01)而后显著升高(P0.05);当海水△pH为-0.4和-0.5时,随酸化时间的延长,MnTGF-β基因在光棘球海胆性腺和管足中的相对表达量呈现先降低后升高的趋势,而在肠组织中的相对表达量则呈现极显著降低趋势(P0.01)。     

光棘球海胆seali基因克隆及表达特性分析

seali基因隶属于PIWI超家族, 其编码的RNA结合蛋白在生殖细胞发育过程中发挥重要作用。研究经同源比对从光棘球海胆(Mesocentrotus nudus)性腺转录组数据库中筛选得到seali基因片段, 随后通过cDNA末端快速扩增技术(Rapid amplification of cDNA ends, RACE), 获得其全长cDNA序列。光棘球海胆seali基因cDNA全长3462 bp, 其中3′UTR长度为416 bp, 5′UTR长度为180 bp, 其中3′UTR的加尾信号并非经典的AAUAAA或AUUAAA, 而是较少见的AAUACA。开放阅读框(Open Reading Frame, ORF)2862 bp, 编码954个氨基酸, 具有保守的PIWI和PAZ结构域, 多重序列比对和系统进化分析结果表明其属于Argonaute家族的PIWI亚家族成员。荧光定量PCR技术检测结果表明, Mnseali基因在光棘球海胆性腺、肠、管足和体腔细胞中均有表达, 在性腺中表达量最高。此外, Mnseali基因为母源因子, 在整个胚胎发育时期均有表达。在卵巢中, 随着卵母细胞的成熟, Mnseali的表达量逐渐升高, 而仅在成熟期的精巢中表达量显著上调。RNA原位杂交结果表明, Mnseali在光棘球海胆性腺的生殖细胞中特异表达, 是光棘球海胆生殖细胞标记基因。该研究为海胆生殖细胞发育相关的研究提供了支撑。     

虾夷马粪海胆(Strongylocentrotus intermedius)Vasa基因的克隆与表达

Vasa基因是DEAD-box家族成员中的一种ATP依赖的RNA解旋酶编码基因,在生殖系分化过程中发挥重要作用。采用同源克隆和cDNA末端快速扩增技术(RACE),从虾夷马粪海胆精巢中克隆得到Vasa基因3′端cDNA序列。该3′末端cDNA长1057bp,与太平洋牡蛎、紫球海胆、非洲爪蟾、小鼠及虹鳟经同源进行比对,其中与紫球海胆的同源率最高达到95%,并确定其为DEAD-box家族的Vasa亚家族成员。利用实时定量RT-PCR检测Vasa mRNA在虾夷马粪海胆组织和胚胎发育期的表达情况,研究结果表明Vasa基因在生殖腺表达量最高,雌、雄个体生殖腺中转录水平上的表达差异明显,雄性高于雌性,差异显著(P<0.05),在肠、体腔液、肌肉、围口膜、管足中表达量低,其中体腔液表达量最低。胚胎发育期检测发现Vasa基因在16细胞期时表达量最高,16细胞期后表达量呈下降趋势,囊胚期表达量最低。试验结果为虾夷马粪海胆生殖系统起源、分化研究提供科学依据。对于虾夷马粪海胆生殖系分化途径来讲,Vasa可作为一种有利的研究工具,追溯其生殖系的起源和分化。     

NPY和POMC基因在生长差异与饥饿再摄食鳙个体中的表达分析

为研究摄食促进基因和摄食抑制基因对鱼类生长调控和饥饿再摄食过程的影响,研究克隆了鳙神经肽Y(HynNPY)基因和前阿黑皮素原(HynPOMC)基因cDNA序列,采用qRT-PCR技术分析它们在生长显著差异鳙个体下丘脑、肠中的基因表达变化;设置对照组(连续投喂4周)、饥饿组、饥饿再摄食组,分析NPY和POMC在不同处理组鳙的下丘脑、肠中的基因表达变化。鳙NPY和POMC基因cDNA全长分别为839和799 bp,开放阅读框有291和657 bp,分别编码96和218个氨基酸。系统进化分析结果表明,鳙NPY和POCM基因具有高度保守性。鳙NPY在下丘脑的表达量最高,其次为肠和脑; POMC在肠道中的表达量最高,其次为下丘脑和肝脏。在相同环境下生长差异鳙个体的下丘脑和肠中, NPY在极大个体的表达量高于极小组个体, POMC在极小个体中的表达量高于极大个体。饥饿导致NPY在下丘脑表达上升,在肠表达量显著上升,恢复摄食后, NPY在下丘脑和肠中表达量下降; POMC在饥饿组下丘脑和肠中都表现为表达量呈显著上升,复投喂后POMC表达量逐步下降至接近对照组水平。肠组织学观察显示,极大个体的肠腔直径...     

饥饿及复投喂对大黄鱼肠型脂肪酸结合蛋白b基因表达的影响

为从分子水平研究肠型脂肪酸结合蛋白基因(intestines fatty acid-binding protein,IFABP)在鱼类脂代谢中的作用,该研究克隆并获得了1 362 bp大黄鱼I-FABPb基因序列,采用实时荧光定量PCR技术检测了I-FABPb基因在肌肉、肝、肠、胃、性腺、鳃、脑和心脏等8个不同组织中的表达情况,研究了饥饿和复投喂对大黄鱼I-FABPb基因在肠、肌肉和肝中表达的影响。结果显示,I-FABPb基因在8个被检测组织中均有表达,但在肠中表达量最高。饥饿对大黄鱼肠、肌肉和肝中I-FABPb基因表达影响显著,均呈现了先上升后下降的趋势,但在肠中变化最显著;长期饥饿后复投喂,I-FABPb基因在肠、肌肉和肝中的表达量均显著升高(P0.05),且显著高于饥饿0 d的水平(P0.05)。结果表明,饥饿及复投喂明显影响大黄鱼的脂肪代谢,I-FABPb在肠道脂肪代谢中起重要作用。     

海胆主要卵黄蛋白研究进展

海胆的主要卵黄蛋白(major yolk protein,MYP)大量存在于海胆卵中,同时存在于海胆精巢和卵巢中,为配子发生及胚胎发育提供营养.经比对发现,海胆MYP cDNA序列与其他物种卵黄蛋白原并无明显相似性,而与铁传递蛋白具有更高的相似性,有研究认为MYP为铁传递蛋白.据其存在场所和合成时期的差异,MYP可分为为CFMYP(存在于体腔内,受精后合成)和EGMYP(存在于卵中,母源性),二者均由同一基因编码.就CFMYP和EGMYP合成时期、场所和功能作用等加以比较;并对海胆MYP与其他物种卵黄蛋白(原)发生、结构与分子量和功能等方面进行了综述.     

实时荧光定量PCR检测球孢白僵菌热休克蛋白基因hsp70在几种胁迫条件下的表达

本研究运用Realtime-PCR技术,对球孢白僵菌Beauveria bassiana hsp70基因在不同胁迫条件下的表达情况进行了检测。从mRNA转录水平探讨了不同胁迫条件对球孢白僵菌hsp70基因表达的影响。结果表明:38℃高温胁迫下,30min时表达量达到最高峰,为对照样品的10.18倍。随后表达量开始下降,至180min时,其表达量降为最低,为对照样品的2.85倍;4℃低温胁迫下,2h检测到hsp70的表达量下降至最低点,为对照样品的0.25倍。随后表达量开始回升,至10h表达量始终维持在对照样品的1.4-1.5倍左右;紫外胁迫下(波长253.7nm),3min后hsp70的表达量快速上升至最高峰,为对照样品的2.33倍。随后表达量迅速下降,至60min表达量始终维持在对照样品的0.2倍左右。因此推测,hsp70基因在球孢白僵菌抵抗高温、低温和紫外三种胁迫方面都可能具有重要作用。同时研究结果也表明,球孢白僵菌hsp70基因启动子在逆境下可引导基因高效表达,因而在抗逆工程菌株构建方面可能具有重要的应用价值。     

饥饿胁迫下家蚕5龄起蚕中的蛋白表达谱及 BmLp-c 6的转录分析

【目的】分析家蚕Bombyx mori受饥饿胁迫后的蛋白质谱变化,探索其耐受饥饿的机理。【方法】以家蚕品种932为实验材料,利用双向电泳和质谱技术检测5龄起蚕经过24 h饥饿胁迫的蛋白质谱差异变化,利用荧光定量PCR技术分析BmLp-c 6的转录表达。【结果】经比对饥饿蚕和正常取食蚕的血淋巴蛋白谱,饥饿蚕有62个特异蛋白点。蛋白点的等电点在4.22~6.98之间,分子量分布在20.81～144.69 kDa间。选取只在饥饿时出现的特异蛋白点No. 7111进行质谱鉴定,根据其氨基酸序列进行引物设计,获得了目的基因BmLp-c 6,经与载体pET-21d(+)连接重组后,成功获得诱导表达。经实时荧光定量PCR分析,当5龄起蚕受到饥饿胁迫影响时,BmLp-c 6基因在血淋巴中大量转录表达,但在中肠中的转录表达水平却极低。【结论】家蚕5龄起蚕在饥饿胁迫下,血淋巴中的蛋白质谱发生变化,BmLp-c 6会大量转录表达。     

牦牛不同组织TLR3、TLR5 mRNA转录水平相对定量研究

参考牦牛TLRs基因序列设计荧光定量PCR特异性引物,建立检测牦牛TLRs相对表达量的荧光定量PCR方法,分析TLR3及TLR5基因在牦牛不同器官组织中的转录水平。结果显示两基因具有不同的表达谱及表达量,其中TLR3基因除在乳腺组织外,在其它组织器官中均有转录,其中在心、大肠、胃和肌肉组织中表达量较高;TLR5基因则在心、肺、大肠、小肠、胃、肌肉、卵巢组织中具有较高的表达量。该试验结果表明,TLRs在不同组织器官转录水平差异较大,可能与其对病原体的识别有关;同一个基因在不同组织中存在差异性,这可能与基因作用机理相关。     

The sea urchin major yolk protein is synthesized mainly in the gut inner epithelium and the gonadal nutritive phagocytes before and during gametogenesis

Major yolk protein (MYP), the predominant component of yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP is stored in ovarian and testicular nutritive phagocytes prior to gametogenesis and is used during gametogenesis as material for synthesizing proteins and other components necessary for eggs and sperm. To reveal the expression profile and the main production site of MYP, we analyzed MYP mRNA expression in immature and maturing Pseudocentrotus depressus. Real‐time reverse‐transcribed polymerase chain reaction analysis showed that MYP mRNA was expressed predominantly in the digestive tract (stomach, intestine and rectum) and the gonad of both sexes. The total amounts of MYP mRNA in the whole digestive tract and in the whole gonad were at similar levels in both immature and maturing sea urchins. MYP mRNA was also detected in white morula cells and vibratile cells separated from the coelomic fluid by density gradient centrifugation, but the expression levels in these cells were very low compared with those in the digestive tract and the gonad. Using in situ hybridization analysis, MYP mRNA was detected in the inner epithelium of the digestive tract and in nutritive phagocytes of the ovary and testis, but was not detected in the germ cells. We conclude that the adult sea urchin has two predominant production sites for MYP regardless of sex and reproductive stage: the inner epithelium of the digestive tract and the nutritive phagocytes of the gonad. Mol. Reprod. Dev. 77: 59–68, 2010. © 2009 Wiley‐Liss, Inc.     

Accumulation of the major yolk protein and zinc in the agametogenic sea urchin gonad

Sea urchins of both sexes store the nutrients necessary for gametogenesis in nutritive phagocytes of the agametogenic gonad. A zinc-binding protein termed the major yolk protein (MYP) is stored here as two isoforms: the egg-type (predominant in egg yolk granules) and the coelomic fluid-type (a precursor with greater zinc-binding capacity). MYP is used during gametogenesis as material for synthesizing gametic proteins and other components. We investigated its accumulation and relationship to zinc contents in gonads during the non-reproductive season in Pseudocentrotus depressus. MYP constituted most of the protein in coelomic fluid and gonads. Both ovaries and testes grew gradually, accumulating MYP and zinc during the year. Total zinc contents and the ratio of coelomic fluid-type to egg-type protein were higher in ovaries than in testes as gametogenesis approached. Most of the zinc in the coelomic fluid was bound to MYP, and the concentrations of MYP and zinc were elevated toward the onset of oogenesis in the female coelomic fluid. Thus, MYP accumulates in the agametogenic ovaries and testes during the non-reproductive season, playing a role as a carrier to transport zinc to the gonad. Transportation of zinc by MYP is more active in females than in males.     

Changes of major carotenoids in gonads of sea urchins (Strongylocentrotus intermedius and S. nudus) at maturation

Changes in the carotenoid content in gonads of two sea urchins species were investigated during maturation. The content of echinenones and carotenes, the two major carotenoid fractions in gonads, is highest for Strongylocentrotus intermedius at the spawning gametogenic stage of gonad maturation for both sexes. For S. nudus, the content of these pigments is highest at stages of active gametogenesis and spawning for males and at the growth stage for females. A comparison of the carotenoid content dynamics during maturation of gonads for males, females and animals at the resting (sexual inactivity) stage was also carried out.     

Ege A,a novel C2 domain containing protein,is essential for GPCR-mediated gene expression in dictyostelium

The major yolk protein (MYP) in sea urchins has historically been classified as a vitellogenin based on its abundance in the yolk platelets. Curiously, it is found in both sexes of sea urchins where it is presumed to play a physiological role in gametogenesis, embryogenesis, or both. Here we present the primary structure of MYP as predicted from cDNAs of two sea urchins species, Strongylocentrotus purpuratus and Lytechinus variegatus. The sequence from these two species share identity to one another, but bear no resemblance to other known vitellogenins. Instead the sequence shares identity to members of the transferrin superfamily of proteins. In vitro iron binding assays, including both (59)Fe overlay assays of MYP enriched coelomic fluid and immunoprecipitation of native iron-bound MYP from coelomic fluid, support this classification. We suggest that one of MYP's transferrin-like properties is to shuttle iron to developing germ cells.     

The major yolk protein is synthesized in the digestive tract and secreted into the body cavities in sea urchin larvae

Major yolk protein (MYP), a transferrin superfamily protein contained in yolk granules of sea urchin eggs, also occurs in the coelomic fluid of male and female adult sea urchins regardless of their reproductive cycle. MYP in the coelomic fluid (CFMYP; 180 kDa) has a zinc-binding capacity and has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). CFMYP is thought to be synthesized in the digestive tract and secreted into the coelomic fluid where it is involved in the transport of zinc derived from food. To clarify when and where MYP synthesis starts, we investigated the expression of MYP during larval development and growth in Pseudocentrotus depressus. MYP mRNA was detected using RT-PCR in the early 8-arm pluteus stage and its expression persisted until after metamorphosis. Real-time RT-PCR revealed that MYP mRNA increased exponentially from the early 8-arm stage to metamorphosis. Western blotting showed that maternal EGMYP disappeared by the 4-arm stage and that newly synthesized CFMYP was present at and after the mid 8-arm stage. In the late 8-arm larvae, MYP mRNA was detected in the digestive tract using in situ hybridization, and the protein was found in the somatocoel and the blastocoel-derived space between the somatocoel and epidermis using immunohistochemistry. These results suggest that CFMYP is synthesized in the digestive tract and secreted into the body cavities at and after the early 8-arm stage. We assume that in larvae, CFMYP transports zinc derived from food via the body cavities to various tissues, as suggested for adults.     

Long-Term Changes in the Gonad Condition of the Sea Urchin Strongylocentrotus intermediusfrom Alekseev Bight (Amurskii Bay) under Polluted Conditions

The gonad condition of the sea urchin Strongylocentrotus intermediuscollected in August 1997 at two stations in Peter the Great Bay was examined. One of the stations was located in a polluted area (Alekseev Bight, Popov Island) and the other, in a relatively clean area (the Verkhovskii Islands). The results were compared with analogous data for 1984, 1985, and 1989. In 1997, the gonad condition of sea urchins inhabiting the two areas differed significantly. The mean value of the gonad index (GI) for sea urchins from Alekseev Bight was less than half and the maturity index was about twice that of sea urchins from the Verkhovskii Islands. The GI of sea urchins from Alekseev Bight decreased by a factor of 1.5 between 1984 and 1997. Pronounced histopathological changes were found in sea urchin gonads at this station: granular and hydropic dystrophy of oocytes, resorption and a sharp decrease in the number of gametes (in about 20% of the sea urchins, hardly any gametes were found in the gonads), changes in the morphology of accessory cells (hypertrophy, atrophy, and necrosis), and accumulation of lipofuscin in the cytoplasm of accessory cells and oocytes, in the hemal sinuses and mesentery. The suppressed gonad condition of the sea urchin S. intermediusin Alekseev Bight may be a consequence of the unfavorable environmental situation that formed in the bight in the 1980s–1990s. The main negative factor is anthropogenic pollution of Amurskii Bay.     

Stereological analysis of nutritive phagocytes and gametogenic cells during the annual reproductive cycle of the green sea urchin, Strongylocentrotus droebachiensis

Abstract. The germinal epithelium of sea urchin gonads contains two interdependent populations of cells: somatic cells called nutritive phagocytes (NPs) and germ cells—ovarian and testicular gonial cells and their derivatives, gametocytes and gametes. Annually, NPs vary their structure and function to produce a changing microenvironment for germ cells during gametogenesis and after spawning. Here, we describe seasonal changes in the NPs as they interact with germ cells during successive gametogenic stages in both sexes of the green sea urchin, Strongylocentrotus droebachiensis. Monthly samples were collected from the Gulf of Maine at a depth of 10 m and analyzed with light microscopy and stereology. Shorter day length and falling seawater temperatures were correlated with nutrient mobilization from NPs, initiation of gonial cell mitosis, and subsequent gametogenesis. During gametogenesis, NPs in both sexes were depleted of nutrients and eventually phagocytize residual ova or spermatozoa. Observations from this study are important for understanding both the cellular aspects of the reproductive biology of sea urchins and those environmental factors that affect the onset and progress of gametogenesis.     

Selective transport and packaging of the major yolk protein in the sea urchin

The major yolk protein of sea urchins is an iron-binding, transferrin-like molecule that is made in the adult gut. Its final destination though is the developing oocytes that are embedded in somatic accessory cells and encompassed by two epithelial layers of the ovary. In this study, we address the dynamics of yolk transport, endocytosis, and packaging during the vitellogenic phase of oogenesis in the sea urchin by use of fluorescently labeled major yolk protein (MYP). Incorporation of MYP into the accessory cells of the ovary and its packaging into yolk platelets of developing oocytes is visualized in isolated oocytes, ovary explants, and in whole animals. When MYP is introduced into the coelom of adult females, it is first accumulated by the somatic cells of the ovarian capsule and is then transported to the oocytes and packaged into yolk platelets. This phenomenon is specific for MYP and accurately reflects the endogenous MYP packaging. We find that oocytes cultured in isolation are endocytically active and capable of selectively packaging MYP into yolk platelets. Furthermore, oocytes that packaged exogenous MYP are capable of in vitro maturation, fertilization, and early development, enabling an in vivo documentation of MYP utilization and yolk platelet dynamics. These results demonstrate that the endocytic uptake of yolk proteins in sea urchins does not require a signal from their surrounding epithelial cells and can occur autonomous of the ovary. In addition, these results demonstrate that the entire population of yolk platelets is competent to receive new yolk protein input, suggesting that they are all made simultaneously during oogenesis.     

Prenatal and postnatal expression of mRNA coding for rat prothrombin.

The levels of prothrombin mRNA in prenatal and postnatal rat tissues were analyzed in order to determine tissue distribution of prothrombin expression and to determine if increases in liver prothrombin mRNA during development correlated with previously documented developmental increases in plasma prothrombin levels. Maternal tissues were also analyzed in order to determine if prothrombin mRNA levels varied due to gestational or postpartum influences. Northern analysis demonstrated that rat liver prothrombin mRNA levels increased several-fold late in gestation and reached maximal levels by 13 days after birth. Prothrombin mRNA was also expressed in diaphragm, stomach, intestine, kidney, spleen and adrenal tissues during development. In maternal tissues during pregnancy, prothrombin mRNA was expressed in liver, diaphragm, stomach, uterus and placenta. Prothrombin mRNA levels in each of these tissues that were positive by Northern analysis were quantitated by solution hybridization analysis. Between gestational day 18 and postnatal day 13, liver prothrombin mRNA levels increased from approx. 600 to 2100 molecules per cell (a 3.5-fold increase). In maternal liver during pregnancy, between day 18 and day 22, prothrombin mRNA levels increased from approx. 1800 to 2100 molecules per cell. Immediately after delivery, maternal liver prothrombin mRNA levels decreased to approx. 50% of preparturition levels. Prothrombin mRNA levels in placental tissue ranged from approx. 100 to 250 molecules per cell. In other fetal, postnatal and maternal tissues, prothrombin mRNA expression was less than 100 molecules per cell. These results demonstrate that the level and tissue-type expression of prothrombin mRNA varies in response to prenatal and postnatal influences.