Construction of cytoplasmic molecular markers distinguishing Danio rerio from Gobiocypris rarus at high identity domains based on MP-PCR strategy and Sybr Green I detection

To distinguish the cytoplasm of Danio rerio from that of Gobiocypris rarus, we cloned G. rarus COXI and constructed cytoplasmic molecular markers at the high identity domains of COXI by mutated primer PCR (MP-PCR for short). Then Sybr Green I was used to detect the single amplicon. As a result, we succeeded in getting the cytoplasmic molecular markers, G.M COXI and Z.M COXI, by MP-PCR strategy. They were used to detect the sperm-derived mtDNA in the sexual hybrid embryos (D. rerio ♀ × G. rarus ♂) before the sphere stage. In the present study, all results demonstrate that MP-PCR approach and Sybr Green I detection are feasible to construct the molecular markers to identify genes that shared high identity.     

The complete mitochondrial genome of Chinese rare minnow, Gobiocypris rarus (Teleostei: Cypriniformes)

The Chinese rare minnow, Gobiocypris rarus, which is endemic to China, is an attractive aquatic laboratory animal in China. In the present study, the complete mitogenome sequence of G. rarus has been determined using long polymerase chain reaction (PCR) method. It was 16,601 bp in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region, the gene composition and order of which are similar to most other vertebrates. Except for eight tRNA and ND6 genes, all other mitochondrial genes are encoded on the heavy strand. The overall base composition of the heavy strand is 29.5% A, 27.6% T, 25.7% C, and 17.2% G, with a slight AT bias of 57.1%. There are 10 regions of gene overlap totaling 27 bp and 13 intergenic spacer regions totaling 63 bp. The mitogenome sequence of G. rarus could contribute to a better solution of its phylogenetic position within cyprinid fishes based on the whole mitogenomic data.     

稀有鮈鲫脑芳香化酶cDNA片段的克隆与表达分析

芳香化酶（P450arom）是雌激素合成过程中的关键酶,在性别分化中起重要作用。鱼类存在卵巢性和脑型两种芳香化酶,分别由Cyp19a和Cyp19b编码。稀有鮈鲫作为我国特有的实验动物,尚无其芳香化酶的有关资料,其性别分化机制亦不清楚。本研究采用RT-PCR的方法以简并引物扩增了稀有鮈鲫脑型芳香化酶基因Cyp19b的部分序列,其长度为1070 bp, 编码357个氨基酸残基,具有典型的芳香化酶结构域。RT-PCR分析发现该基因主要在稀有鮈鲫的脑中表达,在性腺、肠、肾中也有表达;其在雌、雄脑中的表达差异不显著。在胚胎发育阶段,Cyp19b的表达从囊胚期开始,至神经胚期达到较高水平,随后下降,孵化期又有所增强,孵化10天后维持在高水平。这些结果说明Cyp19b以脑中表达为主,可能在稀有鮈鲫神经系统发育和脑的性别分化中有重要作用。     

小鼠植入前胚胎紧密化相关基因Crg1的克隆

从已获得的运用抑制消减杂交技术(Suppression Subtractive Hybridization,SSH)分离、克隆和筛选代表8-细胞早期胚胎和紧密化8-细胞胚胎差别表达基因的ESTs片段(GenBank登录号：BQ740263、BQ740251)入手,经比较二者的同源性发现这两个EST末端反向互补,拼接成一个cDNA片段,经分析此序列包含一个完整的阅读框,提交给GenBank,登录号为AY134859。根据此序列设计引物从小鼠8-细胞紧密化胚胎cDNA中经PCR扩增出目的片段,克隆入pUCm—T载体后测序而获得全长cDNA,为小鼠植入前胚胎紧密化相关基因Crg1,分析比较证明Crg1基因与AY134859基本吻合。Crg1基因的cDNA全长为810bp,只有一个外显子,编码由150个氨基酸组成,分子量理论值为17．67kD的蛋白质。与最新的小鼠基因组工作草图进行电子杂交,该基因被定位在小鼠的14号染色体上。RT—PCR实验证明在小鼠植入前各个时期的胚胎、小鼠胚胎干细胞中均有表达,在小鼠胚胎成纤维细胞中没有表达。半定量RT—PCR实验证明Crg1基因在紧密化胚胎中表达较8—细胞胚胎高。采用Northern—blot手段分析Crg1基因在成年小鼠的8种组织中的表达情况,结果表明该基因只在小鼠卵巢中有微弱的表达,转录本大小为1．2kh,而在成年小鼠的脑、心脏、肾、睾丸、肝脏、肺、脾等中没有表达。研究表明,Crg1基因可能与小鼠胚胎紧密化及保持细胞的全能性相关。     

结球甘蓝抗TuMV相关基因的克隆

以结球甘蓝高抗TuMV自交不亲和系84075为材料,构建了cDNA文库。根据抗病基因保守序列（NBS－LRR）设计一对简并引物,以84075的基因组DNA和cDNA为模板,进行PCR扩增,分别扩增出一条513bp片段,扩增片段进行克隆测序。选取两个与抗病基因同源性较高的克隆片段作探针（命名Borl,Bor2）,对构建的cDNA文库进行筛选,得到一个阳性克隆（命名TuR2）,测序及序列分析表明,该基因总长为762bp,编码226个氨基酸、包含681bp的开放阅读框。与已克隆的抗病基因有不同程度的同源性。利用TuR2作探针,进行了Southern杂交、Northern杂交以及抗病性的共分离检测分析。结果表明,TuR2可能吧单拷贝形式存在,其表达是组成成型的,且无组织特异性;初步确定是一个与结球甘蓝抗TuMV相关的基因。     

银鲫pou2基因在胚胎发育过程中的时空表达图式

用RACE-PCR方法从原肠期SMART文库中扩增到银鲫pou2基因的全长cDNA,其全长为2421bp,开放阅读框为1416bp,编码471个氨基酸,与斑马鱼pou2基因的氨基酸序列一致性高达91.0%。我们用RT-PCR和整体原位杂交的方法研究了银鲫pou2基因在胚胎发育过程中的时空表达图式。RT-PCR结果显示,银鲫pou2基因有母源转录本,其合子基因在高囊胚期强烈表达,在50%下包期和90%下包期也有高量的转录本,但在100%下包期表达量急剧降低,至体节期时已经完全检测不到其转录本。胚胎整体原位杂交结果显示其母源转录本在所有的胚盘细胞中。在高囊胚期和50%下包期,高度表达的合子转录本仍在所有的胚盘细胞中,但至90%下包期时,pou2的表达向胚胎背部的正中线汇聚,集中在神经板的两侧区域和脑部的两条横向条带。在100%下包期时,pou2的表达集中在神经板的中间区域以及预期形成的中后脑区域。至体节期时,转录本消失,这与RT-PCR结果高度一致。银鲫pou2基因的表达图式提示该基因在胚胎发育的早期具有重要作用,它可能参与调控神经板的形成和中后脑细胞的发育命运。     

雌核发育二倍体鲫鲤Dmc1基因的全长cDNA克隆及表达分析

Dmc1（disrupted meiotic cDNA）基因是一个在减数分裂前期Ⅰ表达的特异基因,其产物是减数分裂前期Ⅰ同源染色体配对所必需的。根据据酵母菌、小鼠以及人的DMC1中保守的氨基酸基序设计简并引物,PCR扩增克隆获得了第四代雌核发育二倍体鲫鲤（G4）Dmc1基因部分cDNA序列。在此基础上,通过RACE获得了G4Dmc1基因全长cDNA序列,长度为1369bp,其中开放阅读框为1029bp,编码含342个氨基酸的蛋白质。同时,系统进化分析表明,在进化过程中Dmc1基因在鱼类中保持着高度保守的进化特征。RT-PCR结果表明,Dmc1基因只在G4性腺中表达,在其他组织中不表达。通过实时荧光定量PCR,对Dmc1基因在G4和普通鲤鱼的早期卵巢的表达进行分析,发现G4表达比鲤鱼高。由此可见,雌核发育二倍体鲫鲤Dmc1基因也是减数分裂特异基因,而且其高表达暗示雌核发育二倍体鲫鲤具有正常的减数分裂过程并且其早期性腺存在着多倍体卵原细胞。     

团头鲂MSTN基因cDNA结构、表达及过表达对胚胎发育的影响

研究通过cDNA末端快速扩增法(RACE)克隆得到团头鲂生长抑制素(MSTN)基因的cDNA全长并分析了MSTN基因在团头鲂胚胎、成鱼组织中表达以及MSTN基因在胚胎中过表达情况。结果表明团头鲂MSTN基因的cDNA全长为2187 bp, ORF(开放阅读框)大小为1128 bp, 编码376个氨基酸。组织逆转录PCR (RT-PCR)结果显示, MSTN基因在肌肉、脑和精巢组织中大量表达, 肝脏、脾脏和卵巢组织中的少量表达, 肠、腮、心、眼和肾组织中的微量表达。胚胎逆转录PCR (RT-PCR)结果显示, 在0—44 hpf胚胎发育阶段, MSTN基因表达量较低; 而在48—52 hpf胚胎发育阶段, MSTN基因表达量逐渐升高。整胚原位杂交(WISH)结果显示, 胚胎发育的16 hpf时期MSTN基因主要在脊索中表达, 胚胎发育的28 hpf和55 hpf时期MSTN基因在脑中表达。MSTN基因过表达结果显示, 胚胎在体节发生期出现前-后轴拉长, 背-腹轴变短; 脊索发生扭曲, 强烈抑制体节发育而导致不分化等现象。研究为后续团头鲂MSTN基因的功能研究及团头鲂分子育种提供相关参考依据。     

青花菜雄性不育相关基因BoDHAR的克隆与表达分析

以一个与甘蓝显性核不育相关的差异表达片段的序列为信息探针,通过在NCBI与TAIR网站数据库中进行同源EST序列搜索,经人工拼接、RT-PCR、PCR克隆与序列分析,获得了青花菜脱氢抗坏血酸还原酶DHARdehydroascorbatereductase基因的cDNA与DNA全长序列,命名为BoDHAR。并利用双链接头介导PCR的染色体步行技术(genomewalking)克隆了其上游644bp的5′端序列。所获的BoDHAR基因全长1486bp,存在两个内含子,DNA编码区序列633bp,编码210个氨基酸;序列分析表明BoDHAR与同源基因AT1G19570.1cDNA序列有82.3%的一致性,推导的氨基酸序列有79.6%的一致性;编码的水溶性蛋白存在多个磷酸化位点;5′端上游区存在明显的转录调控序列。半定量RT-PCR结果表明BoDHAR在可育系花蕾中的表达量明显高于不育系花蕾,在花药中的表达明显高于其它部位。     

Sexually dimorphic and ontogenetic expression of dmrt1, cyp19a1a and cyp19a1b in Gobiocypris rarus

Fish have diverse sex determination and differentiation. DMRT1 and aromatase are conserved in the phyla and play pivotal roles in sex development. Gobiocypris rarus is a small fish used as a model in aquatic toxicology in China and has been used to study the effects of environmental endocrine disruptors on gene expression, but its sexual development remains elusive. Here, we report the full-length cDNA of G. rarus dmrt1 and its expression along with the expression of cyp19a1a and cyp19a1b, two genes encoding gonad and brain type aromatases, in adults and during ontogenesis. Both cyp19a1a and dmrt1 are expressed in the ovary and testis but show sexual dimorphism. Expression of cyp19a1a in the ovary is higher than in testes and dmrt1 follows the opposite pattern. Juvenile gonad histology changes at 15 days after hatching. The dimorphic expression of dmrt1 and cyp19a1a appears from 5 days after hatching, which is earlier than histological change. cyp19a1b is expressed coordinately with cyp19a1a until 15 days after hatching. These results show that dmrt1 and cyp19a1a play important roles in sex determination and sex differentiation in G. rarus.     

Developmental expression of CagMdkb during gibel carp embryogenesis

Midkine (Mdk) genes have been revealed to have different expression patterns in vertebrates and therefore, additional studies on Mdk expression patterns are required in more species. In this study, CagMdkb has been cloned and characterized from a SMART cDNA library of 10-somite stage embryos of Carassius auratus gibelio. Its full length cDNA is 1091 bp and encodes a sequence of 147 amino acids, which shows 97.3% identity to zebrafish Mdkb on the amino acid level. RT-PCR analysis reveals that CagMdkb is first transcribed in gastrula embryos and maintains a relatively stable expression level during subsequent embryogenesis. Western blot analysis reveals a 19 kDa maternal CagMdkb protein band and the zygotic CagMdkb protein is expressed from gastrula stage. At around 10 somite stage, the 19 kDa CagMdkb is processed to another protein band of about 17 kDa, which might be the secreted form with the 21-residue signal peptide removed. With immunofluorescence analysis, maternal CagMdkb protein was found to be localized in each blastamere cell of early embryos. The zygotic CagMdkb positive fluorescence signal was detected from a pair of large neurons at 18-somite stage. At the later stages, CagMdkb protein was also extended to numerous small neurons in the forebrain, midbrain and hindbrain, as well as to nerve fibers in the spinal cord. Co-localization with 3A10 antibody revealed CagMdkb immunoreactivity on developing Mauthner neurons, a member of reticulospinal neurons. In addition, ectopic expression of CagMdkb in early embryos of gibel carp and zebrafish suppressed head formation and CagMdkb function was found to depend on secretory activity. All these findings indicate that CagMdkb plays an important role in neural development during gibel carp embryogenesis and there is functional conservation of Mdkb in fish head formation.     

一个新的二色补血草金属硫蛋白基因LbMT2的克隆及其表达分析

从二色补血草cDNA文库中分离出一个新的金属硫蛋白基因LbMT2全长cDNA序列。该基因全长518 bp, 其中5′非翻译区(UTR)64 bp, 3′非翻译区205 bp, 开放阅读框(ORF)249 bp, 编码由82个氨基酸组成的蛋白质, 编码蛋白的分子量为8.1 kDa, 理论等电点(pI)为4.72。利用实时定量PCR方法研究了二色补血草在CuSO4、CdCl2、NaCl、低温和PEG胁迫下不同时间该基因的表达模式。结果表明, CuSO4、CdCl2、NaCl和低温处理均能诱导LbMT2基因在二色补血草的根和叶中的表达, 而PEG处理则抑制了LbMT2在根和叶中的表达。构建LbMT2基因的原核表达载体pGEX-LbMT2, 通过IPTG诱导在大肠杆菌(Escherichia coli) BL21中融合表达, SDS-PAGE 电泳获得35 kDa 的蛋白条带, 大小与预期相符。     

水稻EPSP合酶cDNA克隆、序列分析及其拷贝数测定

根据本室分离的水稻EPSP合酶基因的基因组序列设计一对引物,利用RT-PCR方法首次从水稻(Oryza sativa L. subsp. indica)叶片的RNA中扩增获得了水稻编码EPSP合酶的全长为1 585 bp的cDNA片段,它含有一个完整的开放读码框,编码511个氨基酸,包括444个氨基酸组成的成熟肽序列以及N端的67个氨基酸组成的叶绿体转运肽序列.成熟肽氨基酸序列对比表明,除真菌来源的EPSP合酶变异较大外,其他来源的EPSP合酶同源性较高,均在51%以上.而叶绿体转运肽氨基酸序列同源性较低.Southern杂交表明水稻EPSP合酶基因在水稻基因组中以单拷贝形式存在.RT-PCR分析表明,水稻EPSP合酶基因在根、未成熟种子和叶片中均有转录表达,在叶片中表达量最高.     

Overexpression of the cytochrome c oxidase subunit I gene associated with a pyrethroid resistant strain of German cockroaches, Blattella germanica (L.)

A cytochrome c oxidase subunit I gene (COXI) was identified and isolated as a differentially expressed gene between insecticide susceptible ACY and resistant Apyr-R German cockroach strains using PCR-selected subtractive hybridization and cDNA array techniques. The cDNA sequence of COXI has an open reading frame of 1533 nucleotides encoding a putative protein of 511 amino acid residues. Northern blot analysis indicated that levels of COXI expression were similar in three life stages (eggs, nymphs, and adults) of the susceptible ACY strain. The expression of COXI in the resistant Apyr-R strain was developmentally regulated, with low expression in eggs, an increase (approximately 1.4-fold) in nymphs, and rose to a maximum (approximately 3-fold) in both adult females and males. Comparison of COXI expression between ACY and Apyr-R strains indicated that there was no difference in the eggs of the two strains, but expression was higher (approximately 1.5-fold) in nymphs and much higher (approximately 3- to 4-fold) in adult males and females of the Apyr-R strain. The levels of COXI mRNA showed about 1.4- and 1.7-fold increase in the abdomen tissues compared with the head+thorax tissues of ACY and Apyr-R strains, respectively. Although expression patterns of COXI in head+thorax and abdomen tissues were similar (i.e. lower in the head+thorax tissues and higher in the abdomen tissues) in both the ACY and Apyr-R strains, the expression of COXI was about 2.5-fold higher in the head+thorax and approximately 3-fold higher in the abdomen tissues of the Apyr-R strain compared with the corresponding ACY samples. The overexpression of COXI in resistant German cockroaches merits the investigation of the importance of the gene in insecticide resistant German cockroaches.     

Molecular cloning,sequence and expression analysis of <Emphasis Type="Italic">ZmArf2</Emphasis>, a maize ADP-ribosylation factor

A full-length cDNA encoding a maize GTP-binding protein of the ADP-ribosylation factor family was cloned by suppression subtractive hybridization and an in silico cloning approach. The cDNA was 938 bp in length and contained a complete ORF of 612 bp, which encodes a protein of 203 amino acid residues. Its deduced amino acids sequence had an 83% identity with that of a GTP-binding protein in rice. The gene was designated ZmArf2. The ZmArf2 gene consists of G1, G2, G3, G4 and G5 boxes, and Switch I and Switch II regions. Eight nucleotides differed and five amino acids changed between the popcorn inbred N04 and the dent corn inbred Dan232. One changed amino acid was in the G1 box. RT-PCR analysis showed that ZmArf2 expression increased in the early stages of endosperm development and was not tissue-specific.     

人BAFF转基因斑马鱼的构建及早期免疫基因表达检测

目的建立人BAFF转基因斑马鱼模型,探讨其在自身免疫性疾病发病中的作用。方法RT-PCR法由人淋巴瘤细胞克隆了人BAFF基因全长855bp蛋白编码区域,构建表达人BAFF重组质粒Tol2-hBAFF,体外细胞转染并通过免疫印迹法验证蛋白表达。重组载体经显微注射斑马鱼受精卵后,GFP荧光跟踪并筛选阳性鱼。qPCR法检测早期免疫相关基因表达情况。结果人BAFF-GFP融合蛋白可成功表达,利用Tol2-hBAFF重组质粒显微注射斑马鱼受精卵可获得表达人BAFF的转基因斑马鱼,且表达人BAFF斑马鱼1dpf胚胎中TCRAC明显高表达,而Ikaros则表达量显著降低,表明在斑马鱼胚胎中表达人BAFF蛋白会造成早期淋巴系统中基因的过早表达。结论建立的表达人BAFF的转基因斑马鱼,可为系统性红斑狼疮等与BAFF功能亢进密切相关的自身免疫性疾病的治疗,及相关机制研究提供一种具有诸多优点的新型工具。     

Germ cell specific expression of Vasa in rare minnow, Gobiocypris rarus

Germ cells are set aside early with somatic cells and take roles for reproduction of species from one generation to the next generation. Vasa, a member of DEAD family is well documented as germ cell marker in the animal kingdom. Rare minnow, Gobiocypris rarus, is an emerging model fish in China to study development and toxicology, etc. A suitable germ cell marker will benefit the studies of the factors that may influence germ cell development. Here, we report the cloning and characterization of G. rarus vasa named Grvas whose protein product has the typical characteristics of Vasa proteins. RT-PCR results showed that Grvas is expressed specifically in the gonads of male and female, it is maternally deposited into the eggs for embryos and is continuously expressed in the embryos from the zygote to larvae and adult. Grvas mRNA and/or protein is restricted to the germ cells of ovary and testis. Temporal expression of Grvas mRNA is similar to that of zebrafish vasa during embryogenesis. Grvas signals are coincident with primordial germ cells. These results mean that a germ cell marker, Grvas is isolated from rare minnow and its expression is exclusively in germ cells.