牡丹开花相关基因PsAP1的克隆与表达

APETALA1基因对花器官的形成具有重要作用,并且能够调节花期.以牡丹品种赵粉(Paeoniasuffru-ticosaL.cv.Zhaofen)为试材,采用RT-PCR和RACE方法从花瓣中获得了1个牡丹APETALA1基因cDNA全长,命名为PsAP1,GenBank登录号为HM143943.其cDNA全长1103 bp,包含130 bp的5′非编码区、244 bp的3′非编码区和1个长度为729 bp编码242个氨基酸的开放阅读框.序列比对和系统进化分析表明,PsAP1与葡萄的亲缘关系最近,相似性达80%以上,属于MADS家族AP1/SQUA亚家族.相对荧光定量PCR分析表明,PsAP1在花瓣中的表达量最高,在雄蕊中表达量最低.     

斑马鱼胚胎发育过程中TGF-1基因的表达特征分析

为研究转化生长因子 (Transforming growth factor , TGF)1对斑马鱼胚胎发育的调控作用, 通过NCBI获得TGF-1基因序列, TGF-1 cDNA全长1571 bp, 编码377个氨基酸。系统进化树分析发现, TGF-蛋白按照不同的类型严格聚类, 斑马鱼TGF-1与其他鱼类的TGF-1聚集到一个分支, 在进化中非常保守。对斑马鱼胚胎进行RT-PCR和Real-Time PCR检测显示, TGF-1基因为母源表达基因, 在分节期之前的表达水平比较低, 而从咽囊期开始持续高水平的表达。胚胎整体原位杂交发现, TGF-1基因在斑马鱼24 hpf 胚胎中开始有特异信号出现, TGF-1基因的表达主要分布在腮弓、侧线原基、耳囊、嗅觉基板、心脏和前肾等处, 表明TGF-1基因可能参与斑马鱼胚胎免疫调节、循环系统发育和侧线形成。用低氧处理斑马鱼胚胎, 发现低氧处理24h后斑马鱼胚胎发育延迟。利用Real-Time PCR和胚胎整体原位杂交检测发现, 低氧处理后发育延迟的斑马鱼胚胎中TGF-1 mRNA表达量较常氧组显著降低。以上结果表明, TGF-1基因参与斑马鱼胚胎发育调控, 并且可能与低氧处理后斑马鱼胚胎发育延迟有关。研究结果将为深入研究斑马鱼TGF-1基因的功能奠定基础。       

草鱼α1-抗胰蛋白酶基因cDNA全长克隆与表达分析

采用RACE技术获得α1-抗胰蛋白酶基因cDNA全长序列为1 469 bp,开放阅读框为1 329 bp,可编码442个氨基酸。5′非编码区长19 bp,3′非编码区长121 bp。核苷酸序列分析表明,在N端可能存在一个由1～21位氨基酸残基组成的信号肽;与斑马鱼的同源性最好,其次是虹鳟;在系统进化上,与在斑马鱼、虹鳟共聚为一个大支。用半定量RT-PCR分析正常及细菌诱导下草鱼α1-抗胰蛋白酶基因在不同组织中的表达分布。结果显示:正常情况下,草鱼α1-抗胰蛋白酶在肝脏表达最丰富,在脾脏、前肾、前肠、中肠、后肠和也有少量表达;细菌诱导下,肝脏中表达最强,前肾、脾脏、肠道中表达均明显提高,心脏和后肾中也出现较高表达。提示α1-抗胰蛋白酶可能参与了机体对嗜水气单胞菌感染的免疫应答。     

一个含WD结构域蛋白的基因在鱼类卵子发生过程中的差异表达及其特征分析

用抑制性差减杂交结合SMART cDNA合成和RACE-PCR技术的综合方法,从鱼类卵母细胞卵黄形成后期差减文库中克隆到一个新的含WD结构域蛋白的基因.该基因cDNA全长为1870bp,可读框长990bp,编码的蛋白质由329个氨基酸组成,含有6个WD结构域;5′非编码区长210bp,3′非编码区长670bp,有脊椎动物典型的ANNATG起始序列和polyA加尾信号AATAAA.由于其编码的蛋白质与STRAP蛋白(serine-threonine kinase receptor- associated protein)有92%的同源性,所以把它称为FSTRAP (fish STRAP).虚拟Northern杂交表明,FSTRAP在卵母细胞的卵黄形成后期表达,而在卵黄形成前期(Ⅰ时相卵母细胞)不表达.各种组织的RT-PCR显示,FSTRAP在脑、心、肾、肌肉、卵巢、脾和精巢有转录,而在肝脏中未见扩增带.不同发育时相卵母细胞的RT-PCR表明, FSTRAP在第Ⅱ~Ⅴ时相都有转录.Western杂交也显示FSTRAP在除肝组织外的多种组织中都表达,与RT-PCR结果相同,FSTRAP在卵黄形成前期几乎不表达,从第Ⅱ时相开始表达,且与其转录水平一致.     

羊草OEE1基因的克隆及盐胁迫下的表达

从羊草(Leymus chinensis )叶片cDNA文库中克隆得到可能编码33 kD的光系统Ⅱ(PSⅡ)外周蛋白(oxygen-evolving enhancer protein1,OEE1)全长cDNA(GenBank登录号为EF583851),命名为LcOEE1.序列分析结果表明,该cDNA全长1 107 bp,5′非编码区为32 bp,3′非编码区为71 bp,编码区长987 bp,编码328个氨基酸.BALSTp比对发现,该基因氨基酸序列与已报道的小麦和水稻中的OEE1序列具有95%和94%的相似性.聚类分析表明,该基因与小麦和水稻的亲缘关系较近,与拟南芥和菠菜OEE1基因的亲缘关系较远.Northern杂交结果表明,在200 mmol/L的NaCl处理7 d的幼叶中,OEE1 mRNA的表达量明显高于未处理的对照,说明羊草中OEEl基因受盐诱导.     

普通烟草K^＋通道基因NKT4的克隆、序列和表达分析

通过比对拟南芥、胡萝卜、番茄和马铃薯的K+通道氨基酸序列得到了保守序列,设计1对简并引物,利用RT-PCR获得3条490bp的普通烟草K+通道基因中间片段.以其中一条中间片段设计特异性引物,应用RACE方法得到5′末端和3′末端cDNA序列.通过拼接并结合全长克隆及测序验证,获得一个未报道的普通烟草K+通道基因,并将其命名为NKT4(GenBank登录号为FJ233071).NKT4的cDNA全长为2937bp,其中5′非编码区45bp、编码区2679bp、3′非编码区213bp;编码区编码892个AA.构建了一个烟草、拟南芥及相关植物K+通道蛋白的系统进化树.基因表达分析表明,NKT4主要在烟草主根和侧根中表达,在烟草叶中也有少量表达.     

棉花咖啡酰辅酶A-O-甲基转移酶基因的克隆及特征分析

根据棉花纤维特异表达cDNA文库得到的咖啡酰辅酶 A-O-甲基转移酶基因EST序列设计引物,采用RT-PCR技术从棉花中克隆了一个CCoAOMT基因,命名为GhCCoAOMT2.GhCCoAOMT2基因cDNA(GenBank登录号为FJ376606)具有一个747 bp的开放阅读框,5′非编码区为12 bp,3′非编码区为243 bp,编码248个氨基酸,预测分子量约为28.023 kD,等电点为5.39.GhCCoAOMT2基因组序列长度为1 442 bp,包含4个外显子和3个内含子.氨基酸同源分析发现,GhCCoAOMT2与来自毛白杨、烟草和苎麻的CCoAOMT同源性较高.半定量RT-PCR检测表明,GhCCoAOMT2基因在棉花各个组织中都有表达,其中茎部的表达量最高.原核表达分析表明,最佳诱导表达条件为0.2 mmol/L IPTG在37℃下诱导6 h.     

珊瑚藻R-藻红蛋白γ亚基基因的克隆(英文)

根据珊瑚藻(Corallina afficinalis L.)R-藻红蛋白γ亚基N末端部分氨基酸序列(P83592)设计简并引物,结合RACE方法,扩增获得g亚基的全长cDNA序列。结果表明,序列全长为2308 bp(AY209894),5′非编码区长1203bp,3′非编码区长145 bp,编码区长960 bp,编码320个氨基酸组成的前体,包含71个氨基酸构成的信号肽和249个氨基酸组成的成熟蛋白。成熟蛋白序列内部存在重复序列与前人的报道一致。珊瑚藻亚基cDNA序列不同克隆子的测序结果表明,g亚基cDNA序列存在不同的3′末端,说明该基因可能存在多个拷贝或存在转录后加工。此外,扩增获得g亚基DNA序列(AY308999),比较表明编码区内部没有内含子存在。本文是对珊瑚藻R-藻红蛋白g亚基基因序列的首次报道。     

拟南芥SDIR1基因转录的选择性剪接

采用PCR及RT-PCR法分别克隆了拟南芥SDIR1基因的DNA和cDNA序列。根据序列比对分析结果,发现了3种不同的转录本,提示SDIR1基因的转录中存在选择性剪接。3种转录本的长度分别为822bp、691bp和666bp,依次命名为：SDIR1-822、SDIR1-691、SDIR1-666。与SDIR1基因的DNA序列及已报道的SDIR1cDNA序列比较,除转录本SDIR1-822包含了完整的编码序列外,其余2种转录本的编码序列都存在不同长度的缺失。其中,SDIR1-691缺失了131bp的片段：第2外显子3′端缺失33bp,第3外显子53bp全部缺失,第4外显子5′端缺失45bp;转录本SDIR1-666缺失了156bp的片段：第3外显子3′端缺失18bp,第4外显子5′端缺失138bp。进而随机挑取101个克隆子对三种转录本的表达比例进行初步分析,结果表明3种分子的比值为SDIR1-822：SDIR1-691：SDIR1-666=26.00：1.33：1.00,反映出SDIR1基因不同转录本在拟南芥中的相对表达量。     

棉花Gh4CL1基因克隆及表达

根据棉花纤维特异表达cDNA文库分析得到的4-香豆酸辅酶A连接酶基因EST序列设计引物,采用RT-PCR技术从棉花中克隆了1个4CL基因,命名为Gh4CL1(GenBank登录号为FJ479707).结果表明:Gh4CL1基因cDNA全长2 331 bp,具有1个1 722 bp的开放阅读框,5′非编码区为64 bp,3′非编码区为445 bp,编码573个氨基酸,预测分子量约为61.951 kD,等电点为5.70.氨基酸同源性分析发现,Gh4CL1与来自白杨、大豆和紫草的4CL同源性较高.半定量RT-PCR检测表明,Gh4CL1基因在不同发育阶段的棉纤维中均有表达,在开花后20 d的棉纤维中表达量最大,说明该基因可能参与调控棉纤维细胞的伸长和次生壁的增厚.Gh4CL1基因在棉花花瓣中表达量最高,在其他组织中低水平表达或不表达.     

Zar1 represses translation in Xenopus oocytes and binds to the TCS in maternal mRNAs with different characteristics than Zar2

Maternal mRNAs are translationally regulated during early development. Zar1 and its closely related homolog, Zar2, are both crucial in early development. Xenopus laevis Zygote arrest 2 (Zar2) binds to the Translational Control Sequence (TCS) in maternal mRNAs and regulates translation. The molecular mechanism of Zar1 has not been described. Here we report similarities and differences between Xenopus Zar1 and Zar2. Analysis of Zar sequences in vertebrates revealed two Zar family members with conserved, characteristic amino acid differences in the C-terminal domain. The presence of only two vertebrate Zar proteins was supported by analyzing Zar1 synteny. We propose that the criteria for naming Zar sequences are based on the characteristic amino acids and the chromosomal context. We also propose reclassification of some Zar sequences. We found that Zar1 is expressed throughout oogenesis and is stable during oocyte maturation. The N-terminal domain of Zar1 repressed translation of a reporter construct in immature oocytes. Both Zar1 and Zar2 bound to the TCS in the Wee1 and Mos 3′ UTRs using a zinc finger in the C-terminal domain. However, Zar1 had much higher affinity for RNA than Zar2. To show the functional significance of the conserved amino acid substitutions, these residues in Zar2 were mutated to those found in Zar1. We show that these residues contributed to the different RNA binding characteristics of Zar1 compared to Zar2. Our study shows that Zar proteins have generally similar molecular functions in the translational regulation of maternal mRNAs, but they may have different roles in early development.     

Xenopus laevis zygote arrest 2 (zar2) encodes a zinc finger RNA-binding protein that binds to the translational control sequence in the maternal Wee1 mRNA and regulates translation

Zygote arrest (Zar) proteins are crucial for early embryonic development, but their molecular mechanism of action is unknown. The Translational Control Sequence (TCS) in the 3' untranslated region (UTR) of the maternal mRNA, Wee1, mediates translational repression in immature Xenopus oocytes and translational activation in mature oocytes, but the protein that binds to the TCS and mediates translational control is not known. Here we show that Xenopus laevis Zar2 (encoded by zar2) binds to the TCS in maternal Wee1 mRNA and represses translation in immature oocytes. Using yeast 3 hybrid assays and electrophoretic mobility shift assays, Zar2 was shown to bind specifically to the TCS in the Wee1 3'UTR. RNA binding required the presence of Zn(2+) and conserved cysteines in the C-terminal domain, suggesting that Zar2 contains a zinc finger. Consistent with regulating maternal mRNAs, Zar2 was present throughout oogenesis, and endogenous Zar2 co-immunoprecipitated endogenous Wee1 mRNA from immature oocytes, demonstrating the physiological significance of the protein-RNA interaction. Interestingly, Zar2 levels decreased during oocyte maturation. Dual luciferase reporter tethered assays showed that Zar2 repressed translation in immature oocytes. Translational repression was relieved during oocyte maturation and this coincided with degradation of Zar2 during maturation. This is the first report of a molecular function of zygote arrest proteins. These data show that Zar2 contains a zinc finger and is a trans-acting factor for the TCS in maternal mRNAs in immature Xenopus oocytes.     

Cloning and expression analysis of zygote arrest 1 (<Emphasis Type="Italic">Zar1</Emphasis>) in New Zealand white rabbits

Zygote arrest 1 (Zar1) is an oocyte-specific maternal-effect gene. Previous studies indicate that Zar1 plays important role in early embryo development, but little is known about its function in rabbit. The objectives of this study were to clone the New Zealand white rabbit Zar1 gene and to investigate its expression in various organs in groups of animals with different reproductive traits. We obtained a 709-bp Zar1 cDNA fragment consisting of an 8-bp exon 1, 161-bp exon 2, 75-bp exon 3, 271-bp exon 4 and 194-bp 3 ^' sequences. The rabbit Zar1 nucleotide sequence showed per cent identities of 91, 88, 88, 87, 86, 87, 76 and 82% with Zar1 orthologues in human, cattle, sheep, pig, mouse, rat, zebrafish and Xenopus laevis, respectively, indicating a high homology with other species and evolutionary conservation. Quantitative real-time polymerase chain reaction analyses revealed nonoocyte-specific Zar1 expression, with expression in spleen, lung, ovary, uterus, heart, liver and kidney. The expression level was highest in the lung. This study may lay the theoretical foundation for the study of ZAR1’s biological function.     

NP c 1L l 在高脂血症和动脉粥样硬化中的表达分析

目的：研究NPC1L1（Niemann-Pick C1 Like 1）mRNA在单纯高脂血症大鼠和动脉粥样硬化大鼠小肠组织中的表达与差异,探讨其与脂质代谢和动脉粥样硬化之间的关系。方法：通过半定量RT-PCR方法分别检测正常普食组、单纯高脂饲养组和动脉粥样大鼠组小肠组织中NPC1L1 mRNA的表达差异。结果：三个组别大鼠小肠组织中均存在NPC1L2 mRNA,单纯高脂饮食和动脉粥样大鼠小肠组织中NPC1L1 mRNA表达明显高于正常对照大鼠（P〈0．01）;单纯高脂饮食和动脉粥样大鼠小肠组织中NPC1L1 mRNA表达之间无明显差异（P〉0．05）。结论：血脂代谢紊乱与小肠组织中NPC1L1的高表达有关,NPC1L1可能参与了血脂紊乱的病理生理过程;NPC1L1与促成动脉粥样硬化的发生无明显相关性。     

重组原核表达载体pQE30-HPV58L1的构建及鉴定

目的：构建重组原核表达载体以获得HPV58L1活性蛋白,为进一步研制HPV58基因工程疫苗打下基础。方法：用聚合酶链反应（PCR）扩增HPV58L1完整编码区基因,将PCR扩增产物克隆至pUC19质粒中并测序。利用pQE30质粒载体构建重组原核表达载体pQE30-HPV58L1,并通过酶切电泳验证重组结果的正确性。结果：PCR扩增出1.6Kb特异性片段,经克隆至pUC19后测序表明序列同源性与Gen-Bank报道一致。重组质粒pQE30-HPV58L1酶切后显示其大小约5.1Kb,酶切图谱与预期相同。结论：成功构建了重组原核表达载体pQE30-HPV58L1。     

L1CAM regulates DNA damage checkpoint response of glioblastoma stem cells through NBS1

Glioblastomas (GBMs) are highly lethal brain tumours with current therapies limited to palliation due to therapeutic resistance. We previously demonstrated that GBM stem cells (GSCs) display a preferential activation of DNA damage checkpoint and are relatively resistant to radiation. However, the molecular mechanisms underlying the preferential checkpoint response in GSCs remain undefined. Here, we show that L1CAM (CD171) regulates DNA damage checkpoint responses and radiosensitivity of GSCs through nuclear translocation of L1CAM intracellular domain (L1-ICD). Targeting L1CAM by RNA interference attenuated DNA damage checkpoint activation and repair, and sensitized GSCs to radiation. L1CAM regulates expression of NBS1, a critical component of the MRE11-RAD50-NBS1 (MRN) complex that activates ataxia telangiectasia mutated (ATM) kinase and early checkpoint response. Ectopic expression of NBS1 in GSCs rescued the decreased checkpoint activation and radioresistance caused by L1CAM knockdown, demonstrating that L1CAM signals through NBS1 to regulate DNA damage checkpoint responses. Mechanistically, nuclear translocation of L1-ICD mediates NBS1 upregulation via c-Myc. These data demonstrate that L1CAM augments DNA damage checkpoint activation and radioresistance of GSCs through L1-ICD-mediated NBS1 upregulation and the enhanced MRN-ATM-Chk2 signalling.