两种泥鳅中Sox基因的检出

性别决定基因SRY都具有一个高度保守的基因序列——HMG-box,编码Sox蛋白,在胚胎发育过程中起重要作用。本文利用两种引物检测了泥鳅和大鳞副泥鳅的Sox基因。第一对引物特异扩增人类SRY基因的保守区。在扩增泥鳅的基因组DNA时可见长度分别为200、550、940和1000bp的四条扩增带。在大鳞副泥鳅中可以扩增出200、550、900bp三条带(Fig.1)。Southem杂交表明200和550bp两条带可以和SRY基因探针杂交(Fig．2)。第二对引物是兼并引物,可以特异扩增Sox基因的HMG-box区域。以基因组DNA为模板可以在大鳞副泥鳅中扩增出220、550、700和1500bp四条带(Fig．3);而在泥鳅中则为220、530、570乖1500bp四条带(Fig.4)。PCR产物的长度差异被认为是由于部分Sox基因的HMG-box区域中存在着内含子。没有发现性别特异扩增带。以上结果说明哺乳动物与性决定有关的组分在脊椎动物中是广泛存在的。但这些组分在鱼类中是否与性决定有关,或仅是哺乳动物性决定因子的进化前体尚不得而知。无论如何广泛深入研究鱼类的Sox基因对于揭示性决定系统和因子的进化方式是十分有意义的。     

两种泥鳅不同群体遗传变异的RAPD分析

应用RAPD技术,分析了采自于我国黄河、长江和珠江三大水系中游的大鳞副泥鳅和泥鳅不同群体间的遗传变异。结果表明：种内不同群体间带纹相似度在0．730－0．938之间;遗传距离为0．089－0．245;种间不同群体间的带纹相似度为0．392－0．505,遗传距离为0．620－0．800。两种泥鳞采自武汉的群体,其不同个体间的相似度较之其它群体为低,表明其群体内遗传变异程度较高。     

两种泥鳅中PdSox8和PdSox9基因的染色体定位

应用染色体原位杂交技术,以地高辛标记的大鳞副泥鳅ＰｄＳｏｘ８和ＰｄＳｏｘ９基因片段为探针,研究了二者在泥鳅和大鳞副泥鳅染色体组中的定位。结果表明,在大鳞副泥鳅中ＰｄＳｏｘ８、ＰｄＳｏｘ９分别于端部着丝粒染色体第４和第２号即１４和１２染色体上,信号位点距着丝粒的相对距离分别为４０．２％和６７．５％,在泥鳅中,则分别位于ｔ９、ｔ６上,信息位点距着丝粒的相对蹁分别为５８．３％和３０．８％。     

两种泥鳅RAPD标记遗传稳定性分析

用从20个随机引物中筛选出的7个引物对泥鳅（Misgurnus anguillicaudatus）和大鳞副泥鳅（Paramisgurnus dabryanus）及其自交与杂交子代进行了RAPD遗传稳定性分析。结果表明,RAPD谱带主要呈孟德尔式遗传,该类带纹明亮稳定,在泥鳅子代中占99．11％;在大鳞副泥鳅子代中占100％;在杂交子代中占99．36％。也存在非孟德尔式遗传谱带,该类带纹较暗、稳定性差,在泥鳅子代中为0．89％;在杂交子代中为0．64％;在大鳞副泥鳅中未见此类带纹。实验结果说明RAPD谱带具有很高的遗传稳定性。     

大鳞副泥鳅线粒体DNA九种限制性内切酶酶切图谱

大鳞副泥鳅ｍｔＤＮＡ经１１种限制性内切酶（ＢａｍＨＩ,ＢｇｌＩ,ＢｇｌⅡ,ＥｃｏＲＩ,ＨｐａＩ,ＫｐｎＩ,ＰｓｔＩ,ＳａｃＩ,ＳａｌＩ,ＸｂａＩ和ＸｈｏＩ）单酶完全酶解获得２３个酶切位点,这些酶酶切位点数依次分别是：２、７、０、３、３、０、３、１、１、２和１。通过琼脂糖凝胶电泳测定大鳞副泥鳅ｍｔＤＮＡ平均分子量为１０．３３±０．２２×１０６ｕ（原子质量）;其分子长约１６７２０±３５０碱基对（ｂｐ）。采用双酶完全酶解法构建了大鳞副泥鳅ｍｔＤＮＡ限制性内切酶酶切图谱。     

两种泥鳅不同核质关系下LDH同工酶基因表达的研究

采用聚丙烯酰胺不连续系统凝胶电泳方法分析了泥鳅和大鳞副泥鳅不同杂交组合不同核质关系下胚发育阶段（0－145h）中乳酸脱氢酶（LDH）同工酶的分化表达谱式,ＬＤＨ同工酶的基因表达随细胞质、细胞核不同及胚胎发育各个时期具有不同的个体发育谱式,以泥鳅卵子为细胞质的Ⅰ、Ⅱ两组,杂交组（Ⅱ）ＬＤＨ同工酶基因在受精后３min至原肠中期雄核基因参与表达与调控,部分基因位点比本交组（Ⅰ）启动与表达的时间要早,两组的管家酶主要来自细胞质。以大鳞副泥鳅为细胞质的Ⅲ、Ⅳ两组,各酶均以细胞质调控为主,其管家酶与泥鳅有很大不同。并具体分析和讨论了不同核质关系ＬＤＨ同工酶基因的表达和调控的时空顺序。     

黄鳝P-450芳香化酶基因的克隆及序列分析

P-450芳香化酶(P450arom)是催化雄激素生物合成雌激素的关键酶。本文采用RT-PCR和RACE(Rapid amplifi- cation of cDNA ends)法,首次分离和克隆了雌雄同体鱼黄鳝卵巢中P450 arom基因。该基因cDNA全长1802bp(不包 括poly(A)),5'端非翻译区有49bp,3'端202bp(不包含poly(A)),阅读框(Open reading frame,ORF)1551bp,翻译成517 个氨基酸,计算的蛋白质分子量58．2kDa。同源性分析显示,黄鳝卵巢P450arom的氨基酸序列与其他鱼卵巢 P450arom具有63％-80％同源性,与其他鱼脑P450arom为58％-60％同源,与人胚盘和鸡卵巢P450arom则为 50％-52％同源;但在芳香化酶高保守区(包括1-螺旋区,芳香化酶特异保守区和血红素结合区)的同源性高达 76％-92％。系统发育分析表明芳香化酶基因是单起源,黄鳝卵巢芳香化酶基因与鳉鱼卵巢的关系最近,与鱼类卵 巢P450arom属于同一分支的,与鱼类脑及鸡和人的属于不同分支。     

赤点石斑鱼两种芳香化酶cDNA的克隆及其表达的组织特异性

以赤点石斑鱼 (Epinephelusakaara)脑垂体中提取的RNA为模板 ,根据芳香化酶的保守序列设计引物 ,利用GeneRacerTM 技术 ,克隆出两种芳香化酶即脑芳香化酶 (P4 5 0aromB)和性腺芳香化酶 (P4 5 0aromA)的cDNA ,其全长分别为 190 1bp (编码 5 0 9aa)和 1833bp (编码 5 18aa)。序列分析结果表明 ,赤点石斑鱼两种芳香化酶cDNA序列的同源性为 5 1 6 % ,氨基酸序列之间同源性为 6 2 5 % ,与斜带石斑鱼两种芳香化酶氨基酸同源性分别为 94 7%和 97 9%。对 8个科的 10种鱼进行了分子系统进化树分析 ,结果与根据传统的形态学和生化特征分类进化地位基本一致。以特异性引物扩增雌、雄赤点石斑鱼各种组织 (垂体、嗅球、端脑、下丘脑、中脑、后脑、延脑、心脏、肾脏、肝脏、脾脏、性腺、鳃、胃、肠、皮肤、脂肪、肌肉、头肾、胸腺、鳔 ) ,以β actin作内标比较各组织芳香化酶基因表达量的差异 ,结果表明 ,赤点石斑鱼脑芳香化酶 (P4 5 0aromB)有广泛的组织分布 ,脑和垂体的表达量很高 ,各组织表达量有明显的雌、雄差异 ;而性腺芳香化酶 (P4 5 0aromA)表达主要集中于垂体和性腺 ,且不论雌雄 ,其性腺表达量均高于脑垂体 ,和P4 5 0aromB的表达模式明显不同 ,表现为在脑部 ,P4 5 0aromB表达量高于P4 5 0aromA ,而在性腺 ,     

北柴胡细胞色素P450酶基因的克隆及其植物过量表达载体的构建

目的:克隆北柴胡中可能参与柴胡皂苷生物合成的细胞色素P450酶基因,构建其过量表达载体,为通过转基因验证其功能奠定基础。方法:在454高通量测序获得5'和3'端部分cDNA序列的基础上,利用LD-PCR方法获得全长cDNA,根据全长cDNA序列设计含有酶切位点的PCR引物,利用高保真酶,以RNA反转录产物为模板PCR扩增细胞色素P450酶基因的开放读框,扩增产物与pEASY-T1 Simple载体连接,转化大肠杆菌DH5α;重组质粒pT1-P450经菌液PCR和酶切方法验证后测序,采用NCBI在线Blastx、DNAman和MEGA4软件对序列进行生物信息学分析,随后将pT1-P450的酶切产物插入双元载体pCAMBIA-SUPER 1300,菌液PCR和酶切验证重组质粒p1300-P450。结果:扩增到了北柴胡细胞色素P450酶基因BcCYP87E,构建了这一基因的过量表达载体。结论:细胞色素P450酶基因的克隆和转基因载体的构建,为后续开展转基因研究,验证其生物功能奠定了基础。     

半滑舌鳎脑芳香化酶基因cDNA克隆及表达分析

为研究脑型芳香化酶(P450aromB)在半滑舌鳎性别分化中的作用,采用同源克隆策略,从半滑舌鳎脑分离了2184bp长的脑型芳香化酶的全长cDNA,该基因编码498个氨基酸。氨基酸序列和系统发育分析表明,P450aromB属于脑型P450arom,P450aromB的氨基酸序列与其他鱼类脑型P450arom的同源性较高(48.3%-66.1%),与性腺型P450arom的同源性较低(34.2%-49.9%),与自身的性腺型芳香化酶同源性为45.1%。RT-PCR分析表明:P450aromB mRNA的表达具有明显组织特异性,P450aromB只在性腺、脑、鳃和皮肤中表达,且脑中表达量远高于性腺,而在雌雄鱼的其他组织中都不表达。经过甲基睾酮浸浴处理和高温诱导半滑舌鳎由雌性性反转为雄性后,脑中P450aromB的表达量降低,这些结果表明P450aromB参与了半滑舌鳎的性腺分化和性别决定过程。     

两种泥鳅中PdSox8和PdSox9的RFLP分析

以α-32P-dCTP标记的大鳞副泥鳅的PdSox8和PdSox9基因片段为探针,研究了二者在泥鳅和大鳞副泥鳅中的限制性片段长度多态性。结果表明两种探针在6尾大鳞副泥鳅（3♀3 ♂）和6尾泥鳅（3♀3 ♂）的经EcoRI和HindIII完全酶切的基因组DNA中检测到了较丰富的阳性杂交带,并讨论了其产生的可能原因。未发现性特异杂交带。 Abstract：With the α-32 P-dCTP-labeled PdSox8 and PdSox9 fragments of Paramisgurnus dabryanus as probes, the Restricted Fragment Length Polymorphism(RFLP) were analyzed in two species of loaches P. dabryanus and Misgurnus anguillicaudatus. The results showed that the plentiful positive bands were observed in the genomic DNA of 6 individuals(3♀3♂) of P. dabryanus and 6 individuals(3♀3♂) M.anguillicaudatus, which were digested thoroughly by EcoRI and HindIII. The possible reasons were discussed. No sex-specific band was observed.     

人工诱导大鳞副泥鳅雄核发育二倍体克隆鱼的产生

首次获得人工诱导雄核发育二倍体大鳞副泥鳅克隆鱼。方法是:用紫外线210mJ/cm~2辐射浸泡在人工合成卵巢液中的泥鳅卵,使其染色体遗传失活,再与大鳞副泥鳅精子“受精”。在室温26℃条件下“受精”后15min开始,每隔2min一组,将“受精卵”置于39℃温水中热休克处理2min,以阻止第一次有丝分裂的发生,结果表明:二倍诱导率距“受精”后15～19min和27～29min出现两个高峰,最高二倍诱导率为61.1％,经染色体和形态特征鉴定表明,鱼苗为大鳞副泥鳅雄核发育二倍体克隆鱼,其染色体数(2n=48)和外部形态均与父本相同。     

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromBA) and ovary (P450aromAB) and have a different developmental program (BA) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24–48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (, β, and γ). The 5′-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (ab) are opposite to fish pituitary (ba). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30–48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.     

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromBA) and ovary (P450aromAB) and have a different developmental program (BA) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24–48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (α, β, and γ). The 5′-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (ab) are opposite to fish pituitary (ba). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30–48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.     

两种泥鳅中Sox基因的检出(英文)

性别决定基因ＳＲＹ都具有一个高度保守的基因序列──ＨＭＧ－ｂｏｘ,编码Ｓｏｘ蛋白,在胚胎发育过程中起重要作用。本文利用两种引物检测了泥鳅和大鳞副泥鳅的Ｓｏｘ基因。第一对引物特异扩增人类ＳＲＹ基因的保守区。在扩增泥鳅的基因组ＤＮＡ时可见长度分别为２００、５５０、９４０和１０００ｂｐ的四条扩增带。在大鳞副泥鳅中可以扩增出２００、５５０、９００ｂｐ三条带（Ｆｉｇ．１）。Ｓｏｕｔｈｅｒｎ杂交表明２００和５５０ｂｐ两条带可以和ＳＲＹ基因探针杂交（Ｆｉｇ．２）。第二对引物是兼并引物,可以特异扩增Ｓｏｘ基因的ＨＭＧ－ｂｏｘ区域。以基因组ＤＮＡ为模板可以在大鳞副泥鳅中扩增出２２０、５５０、７００和１５００ｂｐ四条带（Ｆｉｇ．３）;而在泥鳅中则为２２０、５３０、５７０和１５００ｂｐ四条带（Ｆｉｇ．４）。ＰＣＲ产物的长度差异被认为是由于部分Ｓｏｘ基因的ＨＭＧ－ｂｏｘ区域中存在着内含子。没有发现性别特异扩增带。以上结果说明哺乳动物与性决定有关的组分在脊椎动物中是广泛存在的。但这些组分在鱼类中是否与性决定有关,或仅是哺乳动物性决定因子的进化前体尚不得而知。无论如何广泛深入研究鱼类的Ｓｏｘ基因对于揭示性决定系统和因子的进化方式是十分     

Simultaneous formation of haploid, diploid and triploid eggs in diploid–triploid mosaic loaches

In the loach Misgurnus anguillicaudatus , very few diploid–triploid mosaic individuals, which are generated by accidental incorporation of the sperm nucleus into diploid eggs produced by clonal diploid loach, occur in nature. Ploidy examination of gynogenetic progeny induced by activation with ultraviolet-irradiated goldfish sperm indicated that diploid–triploid mosaic females laid haploid, diploid and triploid eggs, simultaneously. In addition, triploid eggs exhibited larger egg sizes. Microsatellite genotyping of diploid–triploid mosaics revealed that triploid genotypes of mosaic mothers possessed two alleles specific to the clonal diploid and one allele from normal diploid male. Diploid eggs from a mosaic mother had genotypes absolutely identical to the diploid clone. Most genotypes of triploid eggs were identical to the mosaic mother, and one of the three alleles of the mosaic mother was transmitted to haploid eggs. These results suggested that diploid germ cells, which had a clonal genome, were differentiated into clonal diploid eggs, and triploid and haploid eggs were produced from triploid germ cells in the same ovary of mosaic individuals.     

桔小实蝇CYP4D46的克隆及其组织特异性表达研究

从桔小实蝇Bactrocera dorsalis(Hendel)成虫体内提取总RNA,利用RT-PCR和cDNA末端快速扩增技术获得了一个新的细胞色素P450基因cDNA序列全长.该基因经细胞色素P450基因命名委员会命名为CYP4D46(Gen-Bank登录号:GU292422),其cDNA全长为1717 bp,包含1530 bp的完整开放阅读框(ORF),编码510个氨基酸,理论分子量约为58.40 kD,等电点为8.82.系统发育分析表明该基因与昆虫第4家族P450基因具有较高的同源性.实时定量PCR分析发现,CYP4D46基因在脂肪体中的相对表达量较高,分别是马氏管和中肠内的756倍和60倍,说明CYP4D46可能与脂肪体的重要生理功能相关.