Using Isolated Embryo Sacs and Early Proembryos for Localization of Calmodulin mRNA Before and After Fertilization in Nicotiana

An in situ hybridization technique for localization of calmodulin(CaM) mRNA in isolated entire embryo sacs and proembryos in Nicotiana tabacum L.cv.W38 has been developed. This technique can be applied to small amounts of materials in which a whole view of CaM mRNA distribution can be obtained. The authors revealed that CaM mRNA expression changes dramatically before and after fertilization. Especially interesting is that a prominent CaM mRNA band appears between the egg apparatus and polar nuclei temporarily during the period of pollination and fertilization. The band disappears just prior to fertilization and expands to a fan_shaped region that occupies the micropylar portion of the embryo sac. After fertilization, CaM mRNA accumulates in the elongated zygotes with higher concentration in their chalazal portion than in the micropylar portion. Such an asymmetrical pattern continues to manifest in the early proembryos. It is supposed that CaM mRNA may be involved in the early events and signaling steps associated with double fertilization and zygote polarization in higher plants.

Establishment of 6VS Telocentric Lines of Haynaldia villosa Resistant to Powdery Mildew Induced by Immature Embryo Culture

The line of T240-6 was selected from 32 SC 2 lines of immature embryo culture of T240 (common wheat (Triticum aestivum L.)× Wheat-Haynaldia villosa (L.) Schur. 6D/6V substitution line) through powdery mildew characterization, glutamate oxaloacetate transaminase (GOT) enzyme and gliadin (Gli) analyses and in situ hybridization. All of the individual plants resistant to powdery mildew lacked the locus of GOT at 6VL arm (GOT-V 2) and had gliadin locus at 6VS arm (Gli-V 2) of Haynaldia villosa. All the plants resistant to powdery mildew had one or two telocentric chromosomes that did not pair with wheat chromosomes but paired between themselves. T240-6 was identified as a telocentric line through in situ hybridization.

Expression pattern of a β_2 tubulin gene in zebrafish embryos identified by whole mount in situ hybridization

Zebrafish is a good vertebrate model for development studies. Research on gene expression and regulation during zebrafish embryonic development plays an important role in revealing the molecular mechanism of morphogenesis in vertebrate. Tubulin locates the cytoplasmic determinants in eggs, organizes the formation of basic cytoskeleton and controls various cell movements. There are several types of tubulin, each of which has specific expression regions and functions. In this study, the full_length cDNA of a Xenopus β 2 tubulin was used as template to generate Dig_labeled antisense RNA which was used as a probe to carry out in situ hybridization on whole mount zebrafish embryos. Under the experimental conditions, the tubulin transcript was first detected in a group of cells in embryos at the midblastula stage. With the development of the embryo, this transcript was gradually restricted to the nervous system. These results demonstrate that the expression of this β 2 tubulin gene in zebrafish embryos is neural specific.

Cloning and expression patterns of two Smad genes during embryonic development and shell formation of the Pacific oyster Crassostrea gigas

Increasing evidence indicates that transforming growth factor β （TGF-β） signaling pathways play many important roles in the early development of mollusks. However, limited information is known concerning their detailed mechanisms. Here, we describe the identification, cloning and characterization of two Smad genes, the key components of TGF-β signaling pathways, from the Pacific oyster Crassostrea gigas. Sequence analysis of the two genes, designated as cgi-smad1/5/8 and cgi-smad4, revealed conserved functional characteristics. The two genes were widely expressed in embryos and larvae, suggesting multiple roles in the early development of C. gigas. The mRNA of the two genes aggregated in the D quadrant and cgi-smad4 was highly expressed on the dorsal side of the gastrula, indicating that TGF-β signaling pathways may be involved in dorsoventral patterning in C. gigas. Furthermore, high expression levels of the two genes in the shell fields of embryos at different stages suggested important roles for TGF-β signaling pathways in particular phases of shell development, including the formation of the initial shell field and the biomineralization of larval shells. The results of this study provide fundamental support for elucidating how TGF-β signaling pathways participate in the early development of bivalve mollusks, and suggest that further work is warranted to this end.

鲤鱼(Cyprinuscarpio)外异蛋白A受体Edar基因的克隆及表达定位

外异蛋白A受体(Ectodysplasin-A receptor,Edar)基因最早是在哺乳动物中发现的,其在皮肤附属物的发育中具有重要的生物学功能。利用Genfishing差异筛选技术对建鲤和镜鲤的皮肤转录表达产物进行筛选,得到Edar基因的部分片段。通过克隆镜鲤mRNA全长发现,镜鲤该基因全长CDS为1 389 bp,编码一个含有462个氨基酸的蛋白质,包含一个信号肽位点、肿瘤坏死因子受体结合位点序列、跨膜区和死亡结构域。序列比对结果表明,镜鲤Edar蛋白与斑马鱼相似度最高,达88.31%,而与其亲缘关系较远的爪蟾,鸡,人类,小鼠相似度较低,分别是64.88%,63.79%,64.36%,63.50%。对5'-UTR和3'-UTR区进行分析表明,镜鲤与斑马鱼、青鳉5'-UTR的相似度只有23.78%,24.73%,而与非洲爪蟾、鸡、人和老鼠的相似度更低,仅为11.73%、18%、6.50%、10.04%;3'-UTR与青鳉的相似度为35.49%,与非洲爪蟾、人和小鼠的相似度分别为9.42%、8.05%、9.37%。整体原位杂交结果表明,该基因在建鲤和镜鲤鳞片发生时在鳞片着生的皮肤基质中特异表达,而在非鳞片发生区域表达较弱或者不表达,到所有鳞片发育形成后,该基因的表达消失,表明该基因可能参与鲤鱼鳞片的起始发育而不是后期的鳞片模式维持过程。

砷对大鼠胚胎p16和iNOS基因表达的影响

为进一步阐明三氧化二砷的发育毒性和致畸作用机制,本实验用非放射性原位杂交等技术研究了砷诱导畸胎发生过程中p16和iNOS基因表达的变化。腹腔注射砷剂量依次为0、1、4和10mg/kg。结果发现砷对SD大鼠胚胎的发育毒性与剂量正比关系,10mg/kg时,发育正常胚胎仅为16.7％。原位杂交结果显示在对照和1mg/kg砷组可见p16mRNA阳性表达信号,但iNOSmRNA表达阴性;在4和10mg/kg组p16mRNA阳性杂交信号未检出,而iNOSmRNA大量表达。p16蛋白缺失与胚胎细胞分裂周期有关;iNOS过表达可导致内源性NO产量增高,进而对胚胎造成氧化性损伤。扫描电镜下,可见≥4mg/kg砷对胚胎表皮细胞、卵黄囊胎盘间皮和内皮细胞表面的微绒毛及细胞膜均有明显的损害作用。

DIG标记DNA探针检测noggin在爪蟾胚胎中的表达

整体原位杂交 (Whole mountinsituhybridization)用于基因表达定位和表达分布模式的研究 ,已经成为一种非常重要的手段 .PCR方法进行探针标记 ,可以获得特异性高 ,片断大小可变的探针 .采用DIG标记 ,检测已知基因noggin在爪蟾胚胎时空分布 ,所得结果与文献报道一致 。

CLK2 mRNA在早期鸡胚中的表达及定位分析

为探究早期鸡胚(孵化后约48 h内)中蛋白激酶CLK2 mRNA的表达特点及部位,用qPCR法测定早期鸡胚中CLK2 mRNA的表达,利用分子克隆技术重组CLK2/pMD18-T质粒,制作CLK2 mRNA原位杂交探针并以地高辛标记,进而通过原位杂交技术测定不同时期鸡胚中CLK2 mRNA的表达和分布。结果表明,早期鸡胚中CLK2 mRNA在各时期均有表达但表达量有所不同;原位杂交结果显示,在鸡胚发育的各个阶段,CLK2 mRNA的表达有所差异,且在各个部位其表达的量也各不相同,头部和尾部的表达最为集中。综上,CLK2 mRNA在鸡胚中存在且表达,其表达分布提示CLK2可能与其头部和神经系统的发育密切相关。

Borealin基因在小鼠中的表达

【目的】研究Borealin在小鼠早期胚胎及睾丸中的表达情况,初步探讨Borealin的生物学功能。【方法】利用体外合成RNA探针,采用全胚胎原位杂交检测Borealin基因在9.5～10.5日龄小鼠胚胎中的表达,免疫组化检测Borealin基因在成年小鼠睾丸中的表达,RT-PCR等方法检测Borealin基因在成年小鼠多组织中的表达。【结果】Borealin在小鼠9.5～10.5日龄胚胎的头部和成年小鼠生精小管基底部细胞的部位有较强的表达,在睾丸、卵巢、肠以及眼睛组织中的表达量较其他组织高。【结论】Borealin在小鼠9.5～10.5日龄胚胎头部及成年小鼠睾丸中均有特异性表达,可能参与了成体干细胞功能的维持。

Connexin43基因抑制对斑马鱼心血管系统发育的影响

为了研究cx43基因抑制对斑马鱼胚胎心血管系统发育的影响,针对cx43的翻译起始位点设计两个吗啉修饰的反义寡核苷酸抑制其表达,在斑马鱼受精卵一到两细胞期混合注射并且验证其有效性.注射后用原位杂交和原位免疫荧光检测心脏标志基因的表达以及心脏的表型,同时利用显微荧光造影和原位杂交检测血管的发育情况.用心室心房的标志基因vmhc和amhc反义RNA探针进行的原位杂交结果显示,vmhc表达抑制,而amhc表达上调.原位免疫荧光显示与原位杂交一致的结果表明:心房扩张心室缩小,并且心脏环化不全.用血管标志基因flk-1的RNA探针原位杂交和显微荧光造影表明,cx43基因抑制的斑马鱼胚胎血管无明显缺陷.此外,cx43基因抑制的斑马鱼胚胎心脏功能也有明显改变,包括心脏搏动无力,有血液回流现象.抑制cx43的表达可能通过影响两个细胞群的迁移导致斑马鱼胚胎心脏的发育缺陷,从而影响了心脏的功能,但是未发现胚胎血管系统发育的明显缺陷.

Expression analysis of eight amphioxus genes involved in the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway plays a crucial role in the embryonic development of metazoans. Although the pathway has been studied extensively in many model animals, its function in amphioxus, the most primitive chordate, remains largely uncharacterized. To obtain basic data for functional analysis, we identified and isolated seven genes （Lrp5/6, Dvl, APC, Ckla, CklS, Gsk3β, and Gro） of the Wnt/β-catenin signaling pathway from the amphioxus （Branchiostoma floridae） genome. Phylogenetic analysis revealed that amphioxus had fewer members of each gene family than that found in vertebrates. Whole-mount in situ hybridization showed that the genes were maternally expressed and broadly distributed throughout the whole embryo at the cleavage and blastula stages. Among them, Dvl was expressed asymmetrically towards the animal pole, while the others were evenly distributed in all blastomeres. At the mid-gastrula stage, the genes were specifically expressed in the primitive endomesoderm, but displayed different patterns. When the embryo developed into the neurula stage, the gene expressions were mainly detected in either paraxial somites or the tail bud. With the development of the embryo, the expression levels further decreased gradually and remained only in some pharyngeal regions or the tail bud at the larva stage. Our results suggest that the Wnt/β-catenin pathway might be involved in amphioxus somite formation and posterior growth, but not in endomesoderm specification.

Megasporogenesis and Megagametogenesis in Autotetraploid Indica/Japonica Rice Hybrid

Autotetraploid indic, a/japonica rice hybrid combines both the advantages of polyploidy and the heterosis between indica and japonica rice. Embryo sac abortion is an important factor influencing spikelet fertility in autotetraploid dce hybrid. To clarify the cytological mechanism of embryo sac abortion, the megasporogenesis and megagametogenesis in an autotetraploid japonicaAndica hybrid were examined by the whole-mount eosin B-staining confocal laser scanning microscopy （WE-CLSM） technique. Abnormalities were observed from the megasporocyte stage to the mature embryo sac stage. The degeneration of the tetrad cells and the functional megaspore was the characteristic of abnormalities during megasporogenesis. Abnormal small embryo sacs and disordered number of nuclei were frequently observed during embryo sac development. Some interesting phenomena, such as two functional megaspores, the diplospory-like megasporocyte, and five-nucleate embryo sac were found. The abnormalities that occurred during female gametophyte development resulted in more than five types of abnormal embryo sacs （i.e. embryo sac degeneration, embryo sac without female germ unit, embryo sac without egg apparatus, embryo sac with abnormal polar nuclei and abnormal small embryo sac） in autotetraploid japonica/ndica hybdd. Embryo sac fertility was lower in diploid japonica/ndica hybdd than in autotetraploid japonicaAndica hybrid although many abnormal phenomena were observed in autotetraploid hybrid.

东亚飞蝗WntA在胚胎发育阶段的时空表达

【目的】探讨东亚飞蝗Locusta migratoria manilensis Wnt基因家族中WntA编码蛋白序列特征及其在胚胎发育阶段的时空表达谱,为进一步开展LmmWntA的功能研究及挖掘东亚飞蝗其他Wnt基因家族成员奠定基础。【方法】PCR克隆并利用邻接法(neighbor-joining,NJ)鉴定东亚飞蝗Wnt基因家族基因LmmWntA;通过同源序列多重比对分析LmmWntA氨基酸序列特征;利用整胚原位杂交技术对LmmWntA在东亚飞蝗产卵后(after egg laying,AEL)发育至12,24,35,46,56和65 h以及3,3.5,4,4.5,5,5.5,6.5,8,8.5,9.5和11 d共17个连续胚胎发育阶段进行转录信号筛查。【结果】克隆获得东亚飞蝗LmmWntA(GenBank登录号:MW052768),CDS全长1101 bp,编码336个氨基酸;LmmWtnA与头索动物、昆虫、有爪动物及环节动物WntA蛋白共同聚为WntA亚家族单系群;LmmWntA中段和C端与比对物种WntA蛋白序列保持了较高同源性,仅在N端信号肽区域出现差异,LmmWntA与膜翅目(Hymenoptera)西方蜜蜂Apis mellifera AmWntA蛋白聚为姊妹群,氨基酸序列一致性为59.05%。LmmWntA最先表达在东亚飞蝗35 h AEL胚胎期末端生长区并在该区域持续表达至4 d AEL胚胎期,同时在新生体节每节腹部形成条纹状表达;在46 h AEL胚胎期视叶后半区持续表达至8.5 d AEL胚胎期,该区域未来发育成复眼;在56 h AEL胚胎期脑持续表达至5.5 d AEL胚胎期;在65 h AEL胚胎期每节腹部的条纹状表达信号逐渐转移至腹中线两侧,在触角基部有明显的表达信号;在5.5 d AEL胚胎期表达信号进一步转移至腹神经;从3 d AEL胚胎期开始在腹侧体节远轴端表达,后期转移至上颚、足关节及末端;伴随4.5 d AEL胚胎末端开始内陷形成肛道,LmmWntA在内陷肛道的腹面及前端表达,最多内陷至腹部第7体节;至9.5 d AEL胚胎期时LmmWntA在翅芽盘处表达。【结论】LmmWntA在东亚飞蝗胚胎发育阶段动态表达,推测LmmWntA参与东亚飞蝗胚胎后端体节生长、神经系统(脑和腹神经)、复眼、触角、消化系统后端(肛道)、颚、胸部附肢(足和翅)等重要组织和器官的发生和形成。本研究结果为进一步开展LmmWntA功能缺失研究奠定发育生物学基础。

黄鳝卵原细胞移植研究

【目的】明确黄鳝卵原细胞是否能迁移、整合到斑马鱼生殖嵴上,为建立黄鳝异体生殖途径打下研究基础。【方法】通过酶消化法制备黄鳝卵原细胞悬液,经PKH26标记卵原细胞后显微注射到受体斑马鱼胚胎中,在荧光显微镜下追踪受体斑马鱼胚胎中供体黄鳝卵原细胞的迁移、整合及增殖情况;采用组织形态学方法检测移植受体斑马鱼胚胎的发育情况,并以半定量PCR和荧光原位杂交检测受体斑马鱼胚胎中供体黄鳝卵原细胞标记基因nanos3的表达情况。【结果】黄鳝卵巢经蛋白酶消化后,分离得到的卵原细胞悬液浓度为2.4×10^(6)个/mL,以PKH26染色后在红色荧光下呈橙黄色。荧光追踪观察结果显示,在移植后0、12、24和48 hpt的受体斑马鱼胚胎中均能检测到供体黄鳝卵原细胞,且黄鳝卵原细胞迁移至受体斑马鱼胚胎的生殖嵴上进行整合及增殖,但72 hpt受体斑马鱼出膜后荧光信号丢失。组织形态学观察结果表明,黄鳝卵原细胞注入受体斑马鱼胚胎中48 hpt后其细胞质结构松散,生殖细胞由动物极向两端迁移而分化形成组织,72 hpt和30 dpt的移植受体斑马鱼与对照组斑马鱼无明显差别。半定量PCR检测结果显示,在48 hpt受体斑马鱼胚胎中能检测黄鳝nanos3基因表达,而在72 hpt和30 hpt受体斑马鱼中表达微弱。荧光原位杂交检测结果显示,48 hpt、72 hpt和30 dpt的移植受体斑马鱼均有黄鳝nanos3基因表达。【结论】黄鳝卵原细胞在48 hpt后仍存在于受体斑马鱼胚胎内,但72 hpt后在受体斑马鱼胚胎未检测到黄鳝卵原细胞,即黄鳝卵原细胞在斑马鱼受体中迁移并最终整合到生殖嵴上,但在斑马鱼出膜后无法继续存活。因此,为了提高黄鳝生殖细胞移植成功率应选择亲缘关系近的鱼种或构建性腺不育种质作为受体。

Development and characterization of interspecific hybrids between Oryza sativa and O. latifolia by in situ hybridization

Oryza sativa and O. latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, interspecific hybrids of these species were obtained using the embryo rescue technique. Hybrid panicle traits, such as long awns, small grain, exoteric large purple stigma, grain shattering and dispersed panicles, resemble that of the paternal parent, O. latifolia, whereas there is obvious heterosis in such respects as plant height, tillering ability and vegetative vigor. Chromosome pairing and the genomic components of the hybrid were subsequently investigated using genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) analysis. Based on the mitotic metaphase chromosome numbers of the root tips investigated, the hybrid is a triploid with 36 chromosomes. The genomic constitution of the hybrid is ACD. In the meiotic metaphase I of the hybrid pollen mother cell, poor chro- mosome pairing was identified and most of the chromosomes were univalent, which resulted in com- plete male sterility in the hybrid.

pcgf5a在斑马鱼胚胎早期发育中的作用

目的探讨pcgf5a基因在斑马鱼胚胎发育过程中的作用和机制。方法通过显微注射pcgf5a吗啉代寡核苷酸(MO)抑制pcgf5a基因mRNA翻译过程,观察斑马鱼胚胎发育过程中相关表型的变化;利用胚胎整封原位杂交和Real-time PCR等技术检测抑制pcgf5a对胚胎神经及眼睛发育相关标记基因的表达变化。结果抑制pcgf5a基因后,斑马鱼胚胎中外胚层发育受到影响,表现为脑区变小,眼睛发育迟缓,尾部卷曲及体轴缩短(548/891),同时神经发育(sox2:82/98、sox3:73/84、foxg1:70/88、pax6a:36/45)和眼睛发育(pax2a、vsx2和rx1,n=5)相关基因表达明显降低。结论pcgf5a可能通过调节神经与眼睛发育相关转录因子的表达,进而影响细胞增殖和凋亡而参与胚层分化以及后续的神经系统与眼睛的发育。

小鼠胚胎整体原位杂交实用技术的建立

目的 优化小鼠胚胎整体原位杂交及数据分析方法。 方法 小鼠胚胎用 4 %多聚甲醛 4℃固定过夜 ,10mg L蛋白酶K(PK)室温消化 30min ,预杂交 2h后 ,加入探针 6 3℃反应 16～ 18h ,用盐溶液高温洗涤多次 ,RNA酶 37℃消化 1h ,封闭 4h后加入地高辛抗体 4℃过夜。抗体反应后反复洗涤并在水平摇床上振摇 ,NBT和BCIP显色系统避光显色。提取图像灰度值 ,扣除背景、对照等假阳性信号。 结果 KIAA170 8探针在脑、脊髓等 8个区域有杂交信号 ,对图像数据分析结果表明 ,只有 1个区域的信号在 95 %可信限范围内 ,为阳性信号。 结论 通过控制蛋白酶K消化时间、探针和地高辛抗体浓度 ,以及洗涤、显色过程 ,得到了低本底、着色清晰的杂交结果 ,结合图像的数据分析步骤 ,消除假阳性结果。建立了一套实用的小鼠胚胎整体原位杂交方法。

百合杂种系间杂交障碍的克服及3种幼胚离体培养方法的比较研究

分别以麝香百合（Longiflorum,L）White Fox、亚洲百合（Asiatic,A）Conneticut King、东方百合（Oriental,O）Sorbonne及喇叭百合（Trumpet,T）Royal Goden为亲本,用切割柱头法进行杂交,采用离体培养技术对杂种幼胚进行抢救,并运用基因组原位杂交技术对所得杂种进行鉴定.结果显示,在所选的6个杂交组合中,切割柱头授粉能不同程度地克服除LL×TT之外的5个杂交组合的受精前障碍;胚、胚乳及胚珠3种外植体的离体培养萌发率分别为100%、21.5%和1%,所得植株占所得杂种的比例分别为9.6%、89.9%和0.5%;基因组原位杂交分析显示,所获植株均为真杂种.结果证明,胚、胚乳及胚珠培养是从幼胚获得百合杂种植株的高效途径,且能有效防止伪杂种混杂,胚乳培养是获得百合杂种一代的主要方式.

砷对大鼠胚胎神经系统发育和HSP70mRNA表达的影响

应用显微解剖、原位杂交组织化学、扫描电镜等技术深入研究了三氧化二砷对大鼠胚胎的发育毒性和神经毒性及ＨＳＰ７０ｍＲＮＡ表达与胚胎神经系统发育的关系。结果表明反映胚胎神经系统发育的头长，神经管未闭、体位异常和脑部形态异常发生率均与染砷剂量和作用时间呈正比关系（Ｐ＜００５）。扫描电镜观察发现大鼠胚胎脑部表皮细胞的微绒毛数量减少，回缩变短，细胞膜表面出现许多小的空洞性病理改变。原位杂交结果表明砷能激发大鼠胚胎产生应激反应，１０ｍｇ／ｋｇ砷诱导的神经管未闭发生率（３５７％）和ＨＳＰ７０ｍＲＮＡ原位表达强度均明显高于４ｍｇ／ｋｇ砷的作用，证明热休克反应具有双重作用，揭示了砷的发育毒性与其剂量和作用时间是否大于细胞内ＨＳＰ７０合成量的自我调控水平有关，同时亦与损害时间发生于胚胎器官形成期的那一时段密切关联。

视黄酸影响斑马鱼胚胎心脏房室分化

目的观察外源性视黄酸对斑马鱼心脏前后轴发育即心脏房室分化的影响。方法采用化学遗传学方法在斑马鱼胚胎发育至12.5hpf(hours post fertilization)给予5×10-8mol/L和10-7mol/L外源性视黄酸处理1.5h。应用胚胎整体原位杂交观察视黄酸对心脏特异基因vmhc,amhc,nkx2.5和nppa表达的影响。结果vmhc,amhc探针的整体原位杂交结果显示视黄酸处理会导致斑马鱼胚胎心脏的前后轴发育异常,表现为vmhc表达细胞的范围缩小,amhc表达细胞的范围明显增大。视黄酸可以明显增强nppa在心脏的表达。视黄酸对心脏房室分化的影响在48hpf后表现得明显。结论5×10-8mol/L和10-7mol/L视黄酸在12.5hpf处理斑马鱼胚胎对斑马鱼心脏早期发生没有影响。斑马鱼发育过程中,视黄酸对心脏的房室分化过程起着重要的调控作用,视黄酸支持心房的分化和发育,同时抑制心室发育。