Intragenic and intergenic sequences regulating the expression of the 5'-to-5' linked adult αand β-globin genes from large yellow croaker Pseudosciaena crocea

One adult α-globin gene and one β-globin gene have been cloned from the large yellow croaker Pseudosciaena crocea. Linkage analysis indicated that the α- and β-globin genes were oriented head-to-head relative to each other. To identify the regulatory elements present in the intergenic and intragenic regions of the globin complex, the intergenic region alone or together with the β-globin gene first intron was cloned into the luciferase-reporter vector pGL3-Basic respectively, and the chimeric constructs were tran- siently transfected into Vero cells and primary fish erythrocytes. The intergenic region cannot support the high-level expression of luciferase. However, the promoter activity of the intergenic region was strongly stimulated by the positive regulatory elements （PRE） located in the β-globin gene intron 1. Thus, it is proposed that the intergenic promoters and intragenic PRE were necessary for the effective expression of the linked α- and β-globin genes.     

短沟对虾和斑节对虾酚氧化酶原基因的克隆和序列分析

采用RT-PER原理和长片段扩增技术克隆短沟对虾和斑节对虾酚氧化酶原基因。设计合成特异引物、提取血细胞总RNA，反转录后扩增获得的特异片段回收并克隆到pGEM-TEasyVector系统的T载体上，重组子进行碱基序列测定。结果表明，所克隆的短沟对虾酚氧化酶原基因含起始密码子A1B到终止密码子TGA的2055bp，编码684个氨基酸。斑节对虾酚氧化酶原基因含ATG到TGA的2061bp，编码686个氨基酸。短沟对虾酚氧化酶原推算分子量为78153Da，等电点pI为6．25；斑节对虾则为78581Da，pI为5．83，用DNA分析软件分析显示两种对虾酚氧化酶原基因具有高度的同源性，二者的碱基和推测的氨基酸序列同源性分别为93％和92％；对虾与其他节肢动物酚氧化酶原基因的同源性的高低与种类间亲缘关系相关；不同种类间酚氧化酶活性中心重要组成部分的2个铜结合位点、位于铜结合位点内的6个组氨酸以及与铜结合位点相邻的氨基酸序列高度保守。     

龙须菜UDP-葡萄糖焦磷酸化酶基因的克隆及其表达与琼胶产量的关系

对红藻龙须菜(Gracilarialei maneiformis)UDP葡萄糖焦磷酸化酶(UGPase)基因的表达与不同藻株中藻体琼胶产量之间的相关性进行研究。通过PCR扩增的方法得到了龙须菜UGPase的基因序列。该基因cDNA序列的全长为2 200 bp,开放阅读框1 485 bp,编码494个氨基酸;龙须菜UGPase基因的氨基酸序列与其它物种的UGPase氨基酸序列相似性较高;该基因是单拷贝的,缺乏内含子;具有特殊的加尾信号。利用荧光实时定量PCR的方法检测UGPase基因相对表达量。结果表明,该基因的表达与琼胶含量存在显著相关性,即在高琼胶组藻株中的表达量显著高于低琼胶组藻株,对琼胶含量高低具有指示作用,显示了在产业化应用中的潜能。     

银鲳(Pampus argenteus)胰岛素样生长因子-I基因的克隆和表达分析

胰岛素样生长因子-I(Insulin-like growth factor-I,IGF-I)是影响脊椎动物生长、发育及代谢的重要调控因子。本研究采用RT-PCR和RACE技术,克隆了银鲳(Pampus argenteus)肝脏IGF-IcDNA序列,应用半定量RT-PCR、Real-time q PCR和原位杂交的方法检测了IGF-I的组织表达特性、在肝脏中的生长表达特性和IGF-I基因在肝脏中的定位。序列分析表明,IGF-I cDNA序列全长836bp,其5′非编码区128bp、3′非编码区92bp,开放阅读框(open reading frame,ORF)605bp,由此推导IGF-I前体蛋白由201个氨基酸组成;前体肽由信号肽、成熟肽、E肽三部分组成,其中信号肽59个氨基酸,成熟肽68个氨基酸,E肽74个氨基酸;成熟肽由B、C、A、D四个区域组成,E肽分析表明,银鲳IGF-I属Ea-4型。同源性比较结果表明,银鲳与同目鲈形目鱼类的IGF-I编码序列同源性较高,为83.52%—91.40%;与哺乳类、鸟类和爬行类的同源性较低。半定量RT-PCR和Real-time q PCR组织特异性表达结果显示,IGF-I m RNA在肝脏组织中的表达量最高,显著高于其它组织,肾脏、心脏、肌肉、脑、鳃、小肠、卵巢次之,嗅球、脾脏和胃中表达较低;半定量RT-PCR和Real-time q PCR不同生长阶段表达结果显示,IGF-I m RNA在30—50g肝脏组织中表达量最高(P0.05);IGF-I m RNA在肝脏中的原位杂交定位结果显示,在肝脏细胞中均有表达,阳性信号主要位于细胞质中,靠近细胞边缘处信号较强。     

缢蛏（Sinonovacula constricta）GST和HSP90基因克隆及其在氨氮胁迫下的表达特征分析

克隆获得缢蛏(Sinonovacula constricta)谷胱甘肽S-转移酶(Sc-GSTσ)和热休克蛋白90(Sc-HSP90)基因的cDNA全长,分析了它们的组织表达差异及其在氨氮胁迫下的表达特征。结果表明,Sc-GSTσ的全长cDNA为1 414 bp,含有639 bp的开放阅读框(Open Reading Frame,ORF),编码212个氨基酸,Sc-GSTσ氨基酸序列与其他物种的GST氨基酸序列同源性为31.88%～43.40%;而Sc-HSP90的全长cDNA为2 752 bp,ORF为2 181 bp,编码726个氨基酸,其氨基酸序列与其他物种HSP90的氨基酸序列同源性为76.77%～87.05%。荧光定量PCR分析发现,Sc-GSTσ和Sc-HSP90在缢蛏各组织中均有表达,两者均在肝胰腺中表达量最高。氨氮胁迫后,Sc-GSTσ和Sc-HSP90 mRNA在肝胰腺中表达均显著上调(p<0.05),表明氨氮胁迫引起机体的应激反应,2个基因可能参与机体解毒或防御过程。但胁迫后期表达量下降推测是机体的防御能力有限,不足以完全保护宿主免受应激诱导的细胞损伤。     

泥蚶锰超氧化物歧化酶基因的研究

Special primers were designed based on the sequence information of superoxide dismutase genes reported in the Iiterature and were used for RT-PCR and rapid amplification of cDNA ends(RACE) to clone Tegillarca granosaMn-superoxide dismutase gene successfully.The length cDNA of Tegillarca granosaMn-superoxide dismutase gene was of 635bp by including a 3^'untranslated region(UTR)of 230bp and an open reading frame(ORF) of 4.5bp encoding a polypeptide of 135 amino acids. TheTegillarca granosaMn-superoxide dismutase gene shares 66%~73% homology with Mizuhopecteb yessoensis^'s,Danio rerio^'s,Xenopus Laevis^'s,Human^'s,Callinectes sapidus^'sSOD CDS, while the corresponding amino acids shares 68%-78% homology and 80%~89% comparability. These indicate the SOD gene is high evolutional conservation in animals.     

Expressed sequence tag analysis and cloning of trehalose-6-phosphate synthase gene from marine alga Laminaria japonica (Phaeophyta)

A high quality cDNA library was constructed from the brown alga Laminaria japonica,with the titer of 1.2×10 5 pfu/ml.The average insert size of the cDNA library is about 1.6 kb.From the cDNA library,591 cDNA clones were randomly selected and sequenced.As a result,574 EST(expressed sequence tag) sequences were generated.All of the 574 ESTs were submitted to the dbEST database section of GenBank with the accession numbers from CX942625 to CX943198.The cDNA library was screened with a α-32 p labeled 453 bp T P S gene probe,which is a partial sequence yielded from Porphyra yezoensis.Four positive cDNA clones were screened and the sequencing data showed that these four cDNA clones covered majority of L.japonica TPS cDNA sequence.After PCR amplification,sequencing and assembling,the entire ORF(open reading frame) sequence of the T P S gene was obtained,which was named LjTPS.LjTPS encodes a protein containing 908 amino acids with a calculated molecular mass of 101 674 Daltons.The LjTPS gene was successfully expressed in E.coli and rice.The LjTPS gene has potential application both in plant breeding to stress tolerance and in deciphering the T P S gene function and mechanism to stress tolerance.     

Molecular cloning and expression analysis of Crustin-like gene from Chinese shrimp Fenneropenaeus chinensis

A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fennero- penaeus chinensis by 3 ′and 5′ RACE. The full-length cDNA of Crustin-like gene contains a 390 bp open reading frame, encoding 130 amino acids. The deduced peptide contains a putative signal peptide of 17 amino acids and mature peptide of 113 amino acids. The molecular mass of the deduced mature peptide is 12. 3 ku. It is highly cationic with a theoretical isoelectric point of 8.5. The deduced amino acids sequence of this Crustin showed high homology with those of Penaeus （ Litopenaeus ） setferus. Northern blotting showed that the cloned Crustin gene was mainly expressed in haemocytes, gill, intestine, and RNA in situ hybridization indicated that the Crustin gene was constitutively expressed exclusively in haemocytes of these tissues. Capillary elec- trophoresis RT-PCR analysis showed that Crustin was up-regulated dramatically from 12 to 48 h after a brief decrease of mRNA during first 6 h in response to microbe infection. The level of Crustin mRNA began to restore at 72 h post-challenge. This indicated that Crustin gene might play an important role when shrimps are infected by bacterial pathogen.     

红笛鲷(Lutjanus sanguineus)CD4和CD4-2基因克隆与诱导表达

应用RT-PCR和RACE-PCR技术克隆了红笛鲷(Lutjanus sanguineus)CD4和CD4-2基因,并分析了它们在健康鱼不同组织的表达分布及免疫刺激物诱导后的表达变化。CD4基因c DNA序列全长2216bp,包含180bp的5′UTR(untranslated regions)、605bp的3′UTR和1431bp的开放阅读框(open reading frame,ORF),编码476个氨基酸;红笛鲷CD4-2基因c DNA序列全长为1520bp,包含62bp的5′UTR、525bp的3′UTR和933bp的ORF,编码310个氨基酸。CD4分子由信号肽、4个免疫球蛋白样结构域(D1—D4)构成的胞外区、跨膜区和胞浆区组成,CD4-2分子由信号肽、2个免疫球蛋白样结构域(D1—D2)构成的胞外区、跨膜区和胞浆区组成。荧光定量PCR(Real Time Quantitative PCR,q PCR)分析显示红笛鲷CD4和CD4-2基因在健康鱼的胸腺中表达量最高,其次是中肾、鳃、皮肤、脾、头肾和肠。红笛鲷头肾淋巴细胞体外经脂多糖(Lipopolysaccharide,LPS)和刀豆蛋白A(Concanavalin A,Con A)刺激12h后,CD4和CD4-2表达量显著上调(P0.05)。哈维氏弧菌疫苗免疫24h后鳃、头肾、脾脏和肠的表达量显著上升(P0.05)。研究结果为进一步研究鱼类CD4和CD4-2在抗菌免疫反应中的作用提供了基础资料。     

赤点石斑鱼HSP60基因克隆及弧菌应激前后的组织表达特性分析

HSP60(heat shock protein 60,热休克蛋白60)作为高度保守的热休克蛋白家族的一员,不仅可以在应激状态下帮助其他蛋白正确折叠并恢复天然构象,还可作为危险信号作用于天然免疫系统.基于HSP60的EST序列设计特异引物,采用RACE技术成功克隆获得赤点石斑鱼HSP60 cDNA全序列及基因组全序列....     

cDNA cloning and mRNA expression of the translationally controlled tumor protein (TCTP) gene from Japanese sea perch (Lateolabrax japonicus)

1 Introduction T he translationally controlled tum or protein(TC TP) w as firstdescribed as a grow th-related proteinin m ouse E hrilisch ascites tum or cells and ery-throleukem ia cells (Y enofsky etal., 1983). Subse-quently, TC TP w as founded to be present in m anycells (Sanchez etal., 1997; G ross etal., 1989; C hunget al., 2000; V ercoutter-Edouart et al., 2001) exceptthe hum an kidney cell(Sanchez etal.,1997;G achetetal., 1999). H om ologues of TC TP have been reportedfrom sever…     

桐花树多酚氧化酶同源基因片段的克隆及序列分析

以桐花树(Aegiceras corniculatum (L.) Blanco)叶为材料, 采用同源PCR克隆策略, 扩增桐花树多酚氧化酶(Polyphenol Oxidase, PPO)铜结合区之间的cDNA序列.该序列长度597 bp (Genbank accession No. GQ404509), 编码了一段由199 个氨基酸残基组成的多酚氧化酶保守区片段.桐花树PPO保守区片段具有PPO铜结合区A(CuA)的特征信号序列Hnswl.FFpFHRyyLyffE.同源比对表明, 该序列与GenBank上其他已有的植物多酚氧化酶氨基酸序列的同源性都在65%以上.     

太平洋牡蛎内切葡聚糖酶基因的克隆、序列特性和表达分析

内切葡聚糖酶是一种重要的纤维素酶,在纤维素水解过程中起到重要作用。本实验利用RT-PCR和cDNA末端快速扩增技术(RACE)克隆获得了太平洋牡蛎(Crassostrea gigas)内切葡聚糖酶基因,并对其基因结构和系统进化进行了分析。内切葡聚糖酶cDNA全长802bp,开放阅读框架630bp,编码209个氨基酸。基因组DNA全长505 6bp,含有两段内含子长度分别为205 3和231 3bp。cDNA具有与已发现的内切葡聚糖酶基因相同的保守区结构和功能域,并且与其他已知贝类的内切葡聚糖酶有较高的同源性。利用RT-PCR方法研究了该基因在太平洋牡蛎消化腺、外套膜、鳃、肌肉、性腺和唇瓣组织中的表达,发现内切葡聚糖酶基因仅在消化腺中表达,而在其他组织中没有表达。     

凡纳滨对虾P23蛋白基因的筛选、表达和生物信息学分析

克隆凡纳滨对虾极端体重个体的组织差异表达基因P23基因并进行生物信息学分析, 为进一步研究该基因的功能以及凡纳滨对虾的分子选育等研究提供信息。以凡纳滨对虾雌虾极端体重个体腹部肌肉为实验材料, 利用抑制消减杂交(SSH)技术构建极大体重雌性个体和极小体重雌性个体腹部肌肉组织正反向消减cDNA文库:正库(以极大体重个体为试验组, 以极小体重个体为驱动组, L-S)和反库(以极小体重个体为试验组, 以极大体重个体为驱动组, S-L)消减cDNA文库, 并采用实时定量技术分析P23基因的组织表达规律, 利用生物信息学对其功能和结构进行预测。以β-actin为看家基因检测两个文库的消减效率分别为210和25, 实时定量技术分析结果表明P23基因在体重的调节中起上调作用。P23基因编码区序列全长495bp, 编码165个氨基酸, GenBank登录号:JF806619。生物信息学分析发现P23蛋白部分序列含有强疏水性, 无跨膜螺旋结构, 两种信号肽预测都显示P23含有信号肽。     

口虾蛄高血糖激素基因全长cDNA的克隆及表达分析

本文首次采用RACE技术获得口虾蛄(Oratosquilla oratoria)眼柄部高血糖激素(crustacean hyperglycemic hormone,CHH)基因cDNA全长序列,共2 421 bp,5'UTR长度为180 bp,3'UTR 为1 821 bp,开放阅读框(ORF)长420 bp,编码139个氨基酸。预测蛋白二级结构α-螺旋23.74%,延伸链占23.02%,无规则卷曲占53.24%,预测分子量为15.47 kD,等电点(pI)为7.049。CHH成熟肽包含6个CHH家族中位置保守的Cys残基,C端为GK,推断为ES型CHH基因。聚类分析表明:口虾蛄CHH和十足目CHH聚为同一个分支的两亚群,具有较近的亲缘关系。荧光定量PCR分析结果表明,CHH基因在肠中表达量最高,在眼柄、触角中有较高的表达量,在肌肉中表达量相对较低,在卵巢中表达最低,而在精巢中不表达。     

三斑石斑鱼(Epinephelus trimaculatus)两种芳香化酶cDNA的克隆及其表达的组织特异性

通过设计引物,从三斑石斑鱼的卵巢中克隆鉴定出性腺芳香化酶(EtP450arom a)和脑芳香化酶(EtP450arom b)的cDNA序列,EtP450arom a开放阅读框为1557bp,编码519个氨基酸残基;EtP450arom b开放阅读框为1530bp,共编码510个氨基酸残基;性腺芳香化酶(EtP450arom a)和脑芳香化酶(EtP450arom b)的氨基酸序列的同源性为63.3%,并且包含了芳香化酶经典的功能保守结构。构建了芳香化酶的系统进化树。采用半定量RT-PCR方法研究了性腺芳香化酶(EtP450arom a)和脑芳香化酶(EtP450arom b)在雌性成鱼各组织中的mRNA的表达情况。组织表达结果显示,性腺芳香化酶EtP450arom a只在性腺中特异性表达,而脑芳香化酶EtP450arom b主要在脑区和垂体中表达,表明三斑石斑鱼两种芳香化酶基因的组织特异性及功能的差异。     

蛋白核小球藻核酮糖-1,5-二磷酸羧化/加氧酶小亚基部分基因序列的克隆与分析

以单细胞绿藻蛋白核小球藻(Chlorella pyrenoidosa)(藻株编号为NMBluh015-1)为实验材料,利用几种绿藻中的核酮糖-1,5-二磷酸羧化/加氧酶(Rubisco)的小亚基(rbcS)的保守序列,设计简并引物,克隆到rbcS的一条cDNA(245 bp)和3条DNA序列(其编号为CP1、CP2和CP3,长度依次为1425 bp、810 bp、705 bp)。序列比较结果表明,该蛋白核小球藻rbcS cDNA核苷酸序列与普通小球藻(C.vulgaris)、杜氏藻(Dunaliella tertiolecta)(rbcS1和rbcS2)及蛋白核小球藻"太阳小球藻"株的相似性依次为93%、92%(91%)和90%,而其编码的氨基酸序列与杜氏藻相似性最高(80%);3条rbcS DNA序列与绿藻及高等植物rbcS部分序列表现了较高的同源性,其中2条DNA(CP2和CP3)部分区段同源性很高。实验结果表明扩增得到的cDNA和DNA序列为蛋白核小球藻rbcS基因。     

蓝点马鲛(Scomberomorus niphonius)两种Wap65基因克隆及蛋白功能的研究

采用同源克隆及Race技术获取了蓝点马鲛Wap65-1和Wap65-2基因的c DNA全长序列,长度分别为1659 bp和1725 bp,各自编码426和436个氨基酸。通过Clust W同源性及进化树分析表明:蓝点马鲛Wap65-1与Wap65-2基因的同源性为60%,处于不同的分支上。进一步对该鱼的幼体进行热诱导,通过q RT-PCR分析表明:两种基因在高温诱导下均有上调,而Wap65-2基因上调呈现显著性差异(P0.05)。在此基础上,构建两种基因冷休克表达系统,成功实现了两种蛋白的可溶性表达,而且发现Wap65-2对高温胁迫下E.coli BL21(DE3)的存活具有较强的保护作用。本研究为蓝点马鲛耐热品种的培育奠定了理论基础。