金鱼Prox1基因cDNA的克隆及其在眼睛发育中的表达分析

脊椎动物的Prox1基因，与果蝇的转录因子prospero同源。为了探讨Prox1基因在金鱼眼睛发生过程中的表达图式，我们从金鱼眼睛SMART库中克隆了Prox1 cDNA。它全长共2 851bp，编码739个氨基酸。组织分布研究表明，Prox1主要分布于眼、脑、心、肝、脾和肾中。整体原位杂交显示，Prox1 mRNA首先是在晶体期的晶体原基中有转录，心跳期则在未成熟晶体的细胞中和视网膜的幼芽区可以检测到。晶体纤维形成后，它主要定位于视纤维层和内网织细胞层。免疫组化显示，心跳期Prox1蛋白的定位与mRNA相同，晶体纤维形成以后，Prox1蛋白主要定位在晶体上皮细胞内侧的晶体纤维上一个环状区域，与Prox1 mRNA的定位不同。这说明，Prox1 基因在晶体发生过程中有重要作用，且在晶体的不同发育时期起的作用可能有所不同。另外，Prox1在晶体发育过程中有一个从内向外的变化过程。     

PP-1与PP-2A在金鱼眼(球)不同组织/细胞中的表达模式

为确定蛋白磷酸酶-1和-2A(protein phosphatase-1 and phosphatase-2A,PP-1,PP-2A)在水生低等脊椎动物眼球不同组织/细胞中的表达模式,提取金鱼眼球中4种组织视网膜、晶体上皮细胞、晶体纤维和角膜的RNA和蛋白,利用RT-PCR和Western印迹技术进行检测.同时运用荧光免疫组织化学技术进行了定位表达研究.结果表明:1)在mRNA水平上,PP-1催化亚基α和PP-2A催化亚基β在金鱼视网膜、晶体上皮和角膜中表达强烈,与其在高等哺乳类脊椎动物如小鼠中的表达形成鲜明的对比;2)在蛋白水平上,PP-1和PP-2A催化亚基在金鱼视网膜和晶体上皮细胞中的表达较高,而在角膜中的表达最低.这与它们在小鼠眼球视网膜和角膜中同时具备高表达的模式形成对比;3)在金鱼发育48h的眼球中,PP-1和PP-2A催化亚基定位于视网膜的色素视网膜和神经视网膜细胞中,晶体上皮和纤维细胞中及角膜上皮细胞中,而在性成熟金鱼眼球中,此二酶催化亚基主要定位于视网膜的色素视网膜,神经视网膜及晶体上皮细胞中.这些结果为探讨PP-1和PP-2A在金鱼眼球不同组织中的功能打下了重要基础。     

棉花LIM结构域基因(GhLIM1)的克隆和表达分析

LIM结构域蛋白是一个重要的发育调控因子,参与基因转录,细胞骨架建成和信号传导等许多发育调控过程,胞质骨架是形成和稳定细胞形态以及传递物质,能量和信息的重要成分。为研究棉花纤维细胞发育过程中胞质骨架的形成和作用机理,通过棉花纤维EST序列整合,从陆地棉徐州142胚珠（含纤维）中扩增并克隆出棉花LIM结构域基因的编码区段。该棉花LIM结构域基因（GhL1M1)长848bp,包含一个570bp的开放阅读框,推导的氨基酸序列（189个氨基酸）与拟南芥,烟草和向日葵的LIM结构域蛋白有极高的同源性,而且两个LIM结构域完整,RT-PCR和Northerm杂交分析表明,该基因（GhL1M1）在陆地棉的根,茎尖,上胚轴,叶片,花蕾,花药,胚珠和不同发育时期的陆地棉纤维（4DPA、12DPA、18DPA）以及海岛棉纤维（18DPA）和中棉纤维（12DPA）中均有表达,但GhL1M1基因在茎尖,纤维和有纤维的胚珠中表达量更高,因此GhL1M1基因应与棉花纤维发育有密切关系。     

LIM—only转录因子研究进展

LIM—only转录因子是LIM基因家族中的LIM—only基因编码的转录调节因子,其蛋白(LMO蛋白)结构中含1个或多个LIM结构域。LIM结构域由两个串联重复的种间高度保守的LIM基元组成。现已发现属于该家族的基因主要有LMO1、LMO2、LMO3、LMO4、dLMO、XLMO、PDLP、FHL等,并且还会有新的发现。迄今的研究结果表明,LMO蛋白是一种蛋白相互作用的适配器,它的LIM结构域可与核内广泛分布的LIM结构域结合蛋白或其他的转录调节因子结合形成转录复合体,启动特异基因的转录,调节生物的发育和分化,并与肿瘤的形成有关。     

菜粉蝶osiris基因家族鉴定与发育动态

osiris基因家族是昆虫特异性基因,迄今尚未在昆虫纲以外的物种中发现同源基因。本研究利用菜粉蝶转录组数据,鉴定了菜粉蝶16个osiris基因家族成员,分属11个亚家族。通过与菜粉蝶基因组比对,发现菜粉蝶osiris基因均为断裂基因,外显子数量为3-15个;通过结构域分析,发现菜粉蝶Osiris完整编码蛋白含有信号肽和一个未知功能结构域DUF1676,且多数Osiris蛋白含跨膜结构域。系统发育分析表明,osiris基因家族成员与其他昆虫种类相应成员更似直系同源,而非种内基因扩张,再次验证了osiris基因是在昆虫物种分化之前就已形成的多基因家族。发育转录组基因表达分析表明,osiris家族不同成员表达量在不同发育阶段趋势几乎完全一致,多在菜粉蝶1龄幼虫和5龄幼虫高表达,卵期、蛹期与成虫期低表达,预示着osiris基因家族不同成员转录调控机制的相似性与发育的相关性。     

PP2A-A/在金鱼及斑马鱼发育中的分化表达模式

以金鱼和斑马鱼为研究对象,运用RT-PCR和Western Blot技术分析蛋白磷酸酶2A(PP2A)结构亚基A(PP2A-A/)在金鱼、斑马鱼成体9种组织和12个发育时期胚胎中mRNA和蛋白水平的表达情况,得到其分化表达模式为:(1)在mRNA水平上,PP2A-A/在金鱼、斑马鱼9种组织中具有较强表达;种属差异性和组织差异性均较大;结构亚基A的两亚型A和A的表达存在差异。(2)在蛋白水平上,PP2A-A/在金鱼、斑马鱼9种组织中均有表达;种属差异性不大但出现明显的组织差异性。(3)PP2A-A/mRNA在金鱼和斑马鱼卵裂期到囊胚期胚胎中大量存在,PP2A-AmRNA在金鱼眼色素期量剧增推测其对金鱼眼色素的形成至关重要。(4)PP2A-A/基因在金鱼、斑马鱼12个发育时期胚胎中均有较高水平的蛋白存在,提示其为维持胚胎的正常发育发挥重要作用。       

棉花中一个新型转录因子GhWRI1的表达特征与功能分析

AP2(APETALA2)/EREBP(ethylene-responsive element binding protein)是一类特异性存在于植物中的转录因子,在植物中的许多代谢过程都起着十分重要的作用。从棉花中分离并获得了一个新的AP2基因GhWRI1,该基因长1 314 bp,所编码的蛋白含有437个氨基酸,分子量约为48 kD,pI为5.77。序列分析结果显示,GhWRI1蛋白含有2个AP2结构域,该蛋白属于AP2家族;与GFP蛋白的融合表达试验结果显示,在洋葱表皮细胞中,GhWRI1是特异性在细胞核中表达的。Real-time RT-PCR数据显示GhWRI1主要在真叶、根、纤维以及种子中表达,而且其在纤维和种子中的表达量是随着发育时间的推移而不断升高的。同时构建了GhWRI1与LUC共表达的载体,通过检测LUC的活性来反映GhWRI1的表达量,试验数据显示,当GhWRI1与LUC共表达后,LUC的活性提高了约20倍。试验结果都显示GhWRI1是一个很强的转录激活子。     

叶花相关蛋白结构域对其转录辅激活及细胞核定位的影响

染色质相关蛋白在真核生物DNA复制、基因转录调控等过程中起着非常重要的作用．前期报道拟南芥叶花相关蛋白(leaf and flower related,LFR)蛋白定位于细胞核中, 其缺失突变体在叶、花发育及育性等方面存在着许多表型,但LFR蛋白的自身特征尚有待进一步探究．酵母单杂交实验表明,酵母转录因子GAL4 的DNA结合域与全长LFR的融合蛋白(GBD-LFR)具有转录辅激活活性,LFR的C端至少有2个犰狳蛋白(ARM)重复结构域及完整N端对于其转录辅激活活性是必需的．但在野生型拟南芥原生质体中,与典型的转录激活因子相比,GBD-LFR的转录辅激活活性并不明显．缺失或突变LFR与黄色荧光蛋白(YFP)的原生质体亚细胞定位的荧光显微观察表明,N端的1～25位氨基酸,特别是其中第22位的赖氨酸和第4、23以及25位精氨酸影响其核定位．利用激光共聚焦显微镜观察共表达黄色或青色荧光(CFP)融合蛋白的细胞核内分布,结果表明LFR与染色质结构蛋白组蛋白H4及染色质结合蛋白HMGA有一定的核内共定位．这些结果表明LFR可能作为一个染色质相关的蛋白质,在拟南芥的生长发育中发挥重要作用．     

棉花微管结合蛋白基因GhCLASP1的克隆与表达分析

CLASP蛋白(CLASPs)是一种能够特异地与微管结合,参与调节微管结构与功能的微管结合蛋白(MAPs),在植物细胞形成、细胞伸长等方面起着关键作用。该研究利用电子克隆结合RT-PCR技术从陆地棉(Gossypium hirsutum L.)中克隆获得1个CLASP基因,命名为GhCLASP1(NCBI登录号为KP742966)。序列分析显示,GhCLASP1属于CLASP基因家族,开放阅读框长度为4 188bp,编码含1 395个氨基酸残基的蛋白;GhCLASP1蛋白含有1个HEAT重复结构域和2个CLASP-N端结构域。进化分析表明,GhCLASP1与可可树(Theobroma cacao)CLASP蛋白的亲缘关系最近。qRT-PCR分析表明,GhCLASP1在棉花的根、茎、叶、花及纤维发育的不同时期均有表达,且GhCLASP1基因表达量最大值出现在纤维发育次生壁加厚期(开花后27d),推测GhCLASP1基因可能对棉花纤维次生壁的形成起着重要的作用。利用本氏烟草(Nicotiana benthamiana)叶片瞬时表达系统对GhCLASP1基因编码的蛋白进行亚细胞定位结果显示,GhCLASP1蛋白定位在细胞膜上。上述结果为进一步研究CLASP基因在棉花中的功能奠定了基础。     

唇?硬骨蛋白基因克隆及其在肌间刺发生过程中的表达

为探究硬化蛋白(Sclerostin, SOST)与肌间刺发生之间的关系, 研究通过转录组测序和RT-PCR获得了唇?(Hemibarbus labeo)硬化蛋白基因cDNA序列。序列分析表明, 硬化蛋白由信号肽和成熟肽两部分组成, 成熟肽包含一个胱氨酸结样结构域。系统进化树分析表明, 唇?硬化蛋白与金鱼(Carassius auratus)硬化蛋白最为接近。通过荧光定量PCR检测发现唇?硬化蛋白基因mRNA在所有被检测的组织中均有表达, 在鳃中的表达量最高。RNA原位杂交结果表明, 硬化蛋白mRNA分布于肌膈, 且随着肌间刺的发生信号逐步减弱。RT-qPCR显示, 在肌间刺4个发育阶段中, 硬化蛋白基因mRNA的变化显著, 在阶段Ⅰ表达量最高, 随后逐步降低。综上, 唇?硬化蛋白与肌间刺骨化存在一定的相关性。研究结果将为进一步调查鱼类硬化蛋白功能及硬化蛋白在肌间刺形成过程中的作用提供理论依据。     

Prox 1 in eye degeneration and sensory organ compensation during development and evolution of the cavefish Astyanax

We have investigated expression of the homeobox gene Prox 1 during eye degeneration and sensory organ compensation in cavefish embryos. The teleost Astyanax mexicanus consists of sighted surface-dwelling forms (surface fish) and several populations of blind cave-dwelling forms (cavefish), which have evolved independently. Eye formation is initiated during cavefish development, but the lens vesicle undergoes apoptosis, and the eye subsequently arrests and degenerates. The requirement of Prox 1 for lens fiber differentiation and γ-crystallin expression in the mouse suggests that changes in the expression of this gene could be involved in cavefish eye degeneration. Surface fish and cavefish embryos stained with a Prox 1 antibody showed Prox 1 expression in the lens, neuroretina, myotomes, heart, hindbrain, and gut, as reported in other vertebrates. We found that Prox 1 expression is not altered during cavefish lens development. Prox 1 protein was detected in the lens vesicle as soon as it formed and persisted until the time of lens degeneration in each cavefish population. The cavefish lens vesicle was also shown to express a γ-crystallin gene, suggesting that Prox 1 is functional in cavefish lens development. In addition to the tissues described above, Prox 1 is expressed in developing taste buds and neuromasts in cavefish, which are enhanced to compensate for blindness. It is concluded that the Prox 1 gene is not involved in lens degeneration, but that expansion of the Prox 1 expression domain occurs during taste bud and neuromast development in cavefish. Received: 31 July 1999 / Accepted: 8 November 1999     

Analysis of the Expression Pattern of Regulatory Genes Pax6, Prox1, and Six3 during Regeneration of Eye Structures in the Newt

We studied tissue-specific expression of homeobox genes Pax6, Prox1, and Six3 during regeneration of the retina and lens. In the native retina, mRNA of Pax6, Prox1, and Six3 was predominantly localized in ganglion cells and in the inner nuclear layer of the retina. Active Pax6, Prox1, and Six3 expression was detected at early stages of regeneration in all proliferating neuroblasts forming the retinal primordium. Low levels of Pax6, Prox1, and Six3 mRNA were revealed in depigmented cells of the pigment epithelium as compared to the proliferating neuroblasts. At the intermediate stage of retinal regeneration, the distribution of Pax6, Prox1, and Six3 mRNA was diffuse and even all over the primordium. During differentiation of the cellular layers in the course of retinal regeneration, Pax6, Prox1, and Six3 mRNA was predominantly localized in ganglion cells and in the inner part of the inner nuclear layer, which was similar to the native retina. An increased expression was revealed in the peripheral regenerated retina where multipotent cells were localized. The dual role of regulatory genes Pax6, Prox1, and Six3 during regeneration of eye structures has been revealed; these genes controlled cell proliferation and subsequent differentiation of ganglion, amacrine, and horizontal cells. High hybridization signal of all studied genes was revealed in actively proliferating epithelial cells of the native and regenerating lens, while the corneal epithelium demonstrated a lower signal. Pax6 and Prox1 expression was also revealed in single choroid cells of the regenerating eye.     

Expression of βA3/A1-crystallin in the developing and adult rat eye

Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat βA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC). βA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life. After birth, βA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE). This suggested that βA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues. In summary, expression of βA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression. Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression, likely account for developmental abnormalities in Nuc1.     

Developmental and physiological pattern of aldose reductase mRNA expression in lens and retina

Aldose reductase (AR), an enzyme which converts glucose to sorbitol, has been implicated in the pathogenesis of diabetic cataracts and retinopathy. The normal physiological role of this enzyme in ocular tissue, however, remains unclear. In a developmental study in the rat using in situ and Northern hybridization analyses, we have found that there is a high level of AR mRNA expression in optic cup and lens as early as embryonic day 13. Serial sections through whole embryos at this stage showed that the eye was the only site of AR mRNA hybridization. Levels of AR mRNA declined in the retina as differentiation proceeded and were very sparse there postnatally. As lens development progressed, epithelial AR mRNA levels remained high, especially in the germinative zone, which is the source of the cells that will become lens fibers, and in the bow region, where these cells undergo a dramatic morphogenetic differentiation into lens fibers. AR mRNA was undetectable in terminally differentiated lens fibers. Since it has been suggested that AR-catalyzed sorbitol production could be an osmoprotective device of lens epithelium during systemic hyperosmolar stress, AR mRNA levels from dehydrated hyperosmolar rats were compared with euvolemic control values, and no difference was found. In summary, AR appears to be of particular importance in the development of the eye, with its retinal role receding relative to lens as differentiation is completed. A continued high level of expression in lens epithelium in adulthood may be explained by the fact that lens tissue, unlike retina, normally continues to proliferate and differentiate after birth. The temporal and spatial pattern of distribution of AR mRNA is strongly suggestive of a role for this enzyme in lens fiber morphogenesis.     

Structure and Chromosomal Localization of the Human Homeobox Gene Prox 1

The genomic organization and nucleotide sequence of the human homeobox gene Prox 1 as well as its chromosomal localization have been determined. This gene spans more than 40 kb, consists of at least 5 exons, and encodes an 83-kDa protein. It shows 89% identity with the chicken sequence at the nucleotide level in the coding region, while the human and chicken proteins are 94% identical. Among the embryonic tissues analyzed (lens, brain, lung, liver, and kidney), the human Prox 1 gene is most actively expressed in the developing lens, similar to the expression pattern of the chicken Prox 1 gene. The Prox 1 gene was mapped to human chromosome 1q32.2–q32.3.     

Differential expression of the HMGN family of chromatin proteins during ocular development

The HMGN proteins are a group of non-histone nuclear proteins that associate with the core nucleosome and alter the structure of the chromatin fiber. We investigated the distribution of the three best characterized HMGN family members, HMGN1, HMGN2 and HMGN3 during mouse eye development. HMGN1 protein is evenly distributed in all ocular structures of 10.5 days post-coitum (dpc) mouse embryos however, by 13.5dpc, relatively less HMGN1 is detected in the newly formed lens fiber cells compared to other cell types. In the adult, HMGN1 is detected throughout the retina and lens, although in the cornea, HMGN1 protein is predominately located in the epithelium. HMGN2 is also abundant in all ocular structures of mouse embryos, however, unlike HMGN1, intense immunolabeling is maintained in the lens fiber cells at 13.5dpc. In the adult eye, HMGN2 protein is still found in all lens nuclei while in the cornea, HMGN2 protein is mostly restricted to the epithelium. In contrast, the first detection of HMGN3 in the eye is in the presumptive corneal epithelium and lens fiber cells at 13.5dpc. In the lens, HMGN3 remained lens fiber cell preferred into adulthood. In the cornea, HMGN3 is transiently upregulated in the stroma and endothelium at birth while its expression is restricted to the corneal epithelium in adulthood. In the retina, HMGN3 upregulates around 2 weeks of age and is found at relatively high levels in the inner nuclear and ganglion cell layers of the adult retina. RT-PCR analysis determined that the predominant HMGN3 splice form found in ocular tissues is HMGN3b which lacks the chromatin unfolding domain although HMGN3a mRNA is also detected. These results demonstrate that the HMGN class of chromatin proteins has a dynamic expression pattern in the developing eye.