球根白丝膜菌γ-actin基因的克隆及表达分析

根据真菌肌动蛋白(actin)基因保守区序列设计引物,用简并PCR法和RACE技术分离得到球根白丝膜菌(Leucocortinarius bulbiger)γ-肌动蛋白基因(Lb-act)的全长cDNA序列。该序列全长为1 357 bp,包含一个1 137 bp的开放阅读框(ORF),编码378个氨基酸,5'端非翻译区(5'UTR)92 bp,3'UTR长度128 bp。Port Param软件在线分析结果表明,该cDNA所编码的蛋白质理论等电点为5.12,相对分子质量为95.022 kD,具有真菌γ-actin基因3个保守特征序列。Blast同源性检索结果表明,Lb-act氨基酸序列与担子菌肌动蛋白序列有较高的相似性,其与双色蜡蘑的肌动蛋白氨基酸序列的亲缘关系最近。Lb-act基因在不同碳源及磷水平培养条件下表达量基本一致,验证了该基因作为分子内标的可靠性。     

兴安落叶松肌动蛋白基因的分离与序列分析

以兴安落叶松(Larix gmelinii)实生苗为材料,提取总RNA和基因组DNA;利用兼并PCR及RACE技术克隆肌动蛋白基因(Lg-act).序列分析的结果显示,该基因cDNA全长1662 bp,编码377个氨基酸;基因组DNA长度约为2564bp,包含4个内含子,5个外显子.所克隆的Lg-act cDNA序列与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性均在77％以上,氨基酸序列的相似性高达93％以上.根据高等植物肌动蛋白相似性构建的系统树显示,兴安落叶松肌动蛋白与挪威云杉肌动蛋白之间的亲缘关系较为密切,在进化中分化时间最为接近.     

悬钩子属植物肌动蛋白基因片段的克隆与表达

目的:克隆悬钩子属植物肌动蛋白基因(actin),为研究该物种中其他基因的表达和调控提供内标基因.方法:利用一对特异性引物从黑莓、悬钩子杂种和树莓品种中克隆actin cDNA片段,对其进行序列分析和半定量RT-PCR表达分析.结果:从3个品种中均获得一条783 bp actin cDNA片段,编码260个氨基酸,3条actin片段与其他植物核苷酸序列的同源性都在82%以上,与其他植物氨基酸序列同源性也均在95%以上;树莓和悬钩子杂种品种的actin核苷酸和氨基酸序列同源性均达99%,系统进化分析也发现两者亲缘关系较近些;表达分析发现actin基因在各品种不同组织中均有一定的表达量.结论:首次克隆了悬钩子属植物肌动蛋白基因actin,将来自黑莓和树莓品种的序列登录在GenBank,登录号分别为HQ439556和HQ439557.     

中华蜜蜂蜂毒镇静肽基因的cDNA克隆和表达

从中华蜜蜂 (Apisceranacerana)工蜂毒腺中快速抽提总RNA ,用RT PCR扩增得到大小约为2 5 0bp的cDNA片段 ,测序得到的片段长度为 2 34bp ,为蜂毒前镇静肽原 (preprosecapin)基因编码区的cDNA .以 3′RACE方法 ,扩增和测定了 3′端非编码区 2 19bp序列 .中蜂前镇静肽原cDNA序列与已报道的欧洲意蜂该基因cDNA序列具有 92 %同源性 ,氨基酸序列具有 87%同源性 .代表成熟肽镇静肽的最后 2 5个氨基酸序列 ,中蜂与意蜂同源性为 88% .3′端非编码区cDNA序列与欧洲意蜂序列有 73 1%同源性 .将中华蜜蜂蜂毒镇静肽成熟肽编码区与 3′非编码区部分克隆 ,构建了镇静肽与谷胱甘肽转移酶融合表达的载体pGEX AcSecapin .将载体转化大肠杆菌BL2 1(DE3)进行融合表达 .表达产物与抗GST抗体在 2 9kD处有很强的交叉反应 .大肠杆菌超声破碎后的上清液用SDS PAGE检测到表达的蛋白多为可溶性融合蛋白 ,通过亲和层析柱纯化和凝血酶的切割得到了镇静肽蛋白     

建兰Actin基因cDNA全长的克隆及其表达分析

根据单子叶植物的肌动蛋白基因(Actin)的保守区序列设计引物,采用RT-PCR和RACE技术从建兰(Cymbidium ensifolium)中分离出Actin基因cDNA全长.序列分析结果表明,建兰Actin基因长度为1 434 bp,编码区长度为1 134 bp,编码377个氨基酸,将其命名为CeActin,GenBank登录号为JN613147.CeActin推导的氨基酸序列与其他植物的同源性都较高,具有高度的保守性.采用半定量RT-PCR技术分析CeActin在建兰各组织及花不同发育时期的表达情况,结果表明,表达量没有明显差异,表明CeActin基因可作为内参基因.     

血红密孔菌(Pycnoporussanguineus)漆酶基因的克隆与序列分析

为克隆血红密孔菌 (Pycnoporussanguineus)漆酶基因 ,根据真菌漆酶氨基酸序列保守区设计了 1对简并引物 .以血红密孔菌基因组DNA为模板 ,PCR扩增出长 12 2 7bp的漆酶基因片段 .以此序列为基础 ,通过 5′及 3′RACE技术克隆出漆酶全长cDNA序列 ,序列长为 190 2bp ,其 5′端和 3′端非编码区长分别为 5 1bp和 2 97bp ,开放阅读框长 15 5 4bp ,编码 5 18个氨基酸的蛋白 .该蛋白具有 4个铜离子结合区域 ,预测其相对分子量为 5 6 313 2 ,等电点为 5 5 9,其氨基酸序列与Pycnoporuscinnabarinus漆酶 (lcc3 2 )的同源性最高 ,为 96 % .以该cDNA编码区的两端序列为引物 ,PCR扩增得到漆酶的长度为 2 15 4bp的全长DNA序列 ,序列中包括 10个内含子序列 ,长为 5 2～ 70bp     

大口黑鲈脂代谢相关基因NPY、UCP2、LPL、HL克隆与分子进化分析

采用RT-PCR和RACE方法克隆了大口黑鲈NPY基因cDNA全序列及UCP2、LPL、HL基因cDNA核心片段。序列分析结果表明,大口黑鲈NPY基因cDNA全序列长664 bp,其中5′端非翻译区(5′-UTR)长53 bp,3′端非翻译区(3′-UTR)长311 bp,开放阅读框(ORF)长300 bp,编码99个氨基酸,即前体NPY。大口黑鲈前体NPY包括三个部分,28个氨基酸组成的信号肽、36氨基酸组成的成熟NPY以及32个氨基酸组成的由Gly-Lys-Arg指示的NPY C端肽（CPON）。大口黑鲈UCP2、LPL、HL基因cDNA核心片段长度分别为737 bp、509 bp和666 bp,各自编码245个氨基酸、169个氨基酸和222个氨基酸。将4个基因的氨基酸序列分别与其他物种的氨基酸序列进行同源性比较,并通过MEGA3构建系统树,对这4个脂代谢相关基因的分子进化特征进行了探讨。     

一个新的油菜NHX基因电子克隆及序列分析

基于电子克隆的方法,从甘蓝型油菜中获得一个新的反向转运蛋白基因cDNA序列,暂被命名为BnNHX6。BnNHX6包含一个完整的长为1593bp的开放阅读框架,编码530个氨基酸。BnNHX6蛋白属于跨膜蛋白,有9个跨膜区,含有信号肽,预测在质膜上。通过同源比对和进化分析发现,BnNHX6的氨基酸序列与拟南芥AtNHX5和AtNHX6、西红柿LeNHX2、毛白杨PtNHX2基因所编码的氨基酸序列高度同源,同源性分别为78.4%、92.6%、77.1%、76.9%,亲缘关系较近;但与已报道的油菜BnNHX1同源性仅为24.9%,亲缘关系很远,表明BnNHX6是一个新的油菜反向转运蛋白基因。     

虾夷扇贝β-actin基因的克隆和序列分析

为进一步研究虾夷扇贝功能基因的表达调控。利用SMART cDNA文库构建试剂盒成功构建了健康虾夷扇贝外套膜和肾脏两种组织的cDNA文库。对随机选取的4009个克隆进行5′端测序,比对,筛选出1条β肌动蛋白同源序列,对此EST序列两端进行扩增、测序,得到肌动蛋白基因cDNA全长序列。肌动蛋白基因cDNA全长1536bp(不包括polyA),5′端非编码区84bp,3′端非翻译区321bp,阅读框1131bp,编码377个氨基酸。在基因组DNA中,该基因被一个内含子分为两段,内含子位于第41和第42个氨基酸之间,长度为1498bp。系统发育分析显示该肌动蛋白属于β类型。本研究得到的虾夷扇贝β-肌动蛋白基因可以被用于作为定量某种虾夷扇贝mRNA的标准,这为继续研究虾夷扇贝其它功能基因,及其分子生物的进一步研究、促进其他相关分子发育和系统进化研究奠定了基础。     

白桦肌动蛋白(Actin)基因全长cDNA克隆与序列分析

以白桦(Betula platyphylla Suk.)次生木质部为材料,用改良CTAB方法提取总RNA。根据植物肌动蛋白(Actin)基因编码区的保守序列设计引物后进行RT-PCR,并采用RACE技术扩增出Actin基因全长序列。该基因cDNA全长1 785 bp,序列分析表明,该基因编码区1 134 bp,编码377个氨基酸,5′非编码区157 bp,3′非编码区495 bp。所得序列与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性均在80%以上,氨基酸序列的相似性高达96%以上。此基因已在GenBank注册（EU588981）。根据高等植物肌动蛋白相似性构建了进化树,表明白桦肌动蛋白与蓖麻肌动蛋白之间的亲缘关系最为密切,在进化中分化时间最为接近。     

Molecular identification and characterization of the Arabidopsis AtADF1, AtADF5 and AtADF6 genes

Actin depolymerizing factor (ADF) is a key regulator of the organization of the actin cytoskeleton during various cellular activities. We found that ADF genes in Arabidopsis form a large family consisting of at least nine members, four of which were cloned and sequenced in this study. Comparison of genomic and cDNA sequences showed that the AtADF1, AtADF5, and AtADF6 genes all contain two introns at conserved positions. Analysis of transgenic Arabidopsis plants carrying promoter-GUS fusion constructs revealed that AtADF1 and AtADF6 are expressed in the vascular tissues of all organs, whereas expression of AtADF5 is restricted to the root tip meristem. GFP-AtADF1, GFP-AtADF5, and GFP-AtADF6 fusion proteins were found to bind to actin filaments in vivo, and to reorganize the actin cytoskeleton when transiently expressed in plant cells.     

Nucleotide sequence and expression of a cDNA encoding chick brain actin depolymerizing factor

Chick brain actin depolymerizing factor (ADF) is a 19-kDa protein that severs actin filaments and binds actin monomers. We have obtained a cDNA encoding ADF by screening a chick embryo lambda gt11 cDNA library with both a rabbit anti-ADF antiserum and two oligonucleotide probes. Several non-full-length clones of 636 bases and one full-length clone of 1886 bases were isolated and sequenced. The full-length cDNA encodes a protein of 165 amino acids with a calculated molecular weight of 18,520. The deduced amino acid sequence shows 73% identity with the porcine brain actin binding protein cofilin. The coding region of the ADF cDNA has been placed in an expression vector, and the resulting protein shows immunoreactivity with an anti-ADF antiserum but not with an anti-cofilin antibody. The expressed ADF has been purified and has an actin depolymerizing activity identical with that of brain ADF. Like cofilin, ADF contains a sequence similar to the nuclear transport signal sequence of the SV40 large T antigen and a calcium/calmodulin-dependent protein kinase II phosphorylation consensus sequence. Northern blots of both embryonic chick brain and muscle RNA revealed two ADF mRNAs of length 2.1 and 0.9 kilobases. Southern blots suggest that the ADF gene is present in a single copy within the chicken genome. ADF contains regions of homology with other actin binding proteins including tropomyosin, gelsolin, and depactin.     

Sequence of cDNAs encoding actin depolymerizing factor and cofilin of embryonic chicken skeletal muscle: two functionally distinct actin-regulatory proteins exhibit high structural homology.

Two actin-regulatory proteins of 19 and 20 kDa are involved in the regulation of actin assembly in developing chicken skeletal muscle. They are homologous with actin depolymerizing factor (ADF) and cofilin, a pH-dependent actin-modulating protein, which were originally discovered in chicken and mammalian brain, respectively. In this study, full-length cDNA clones were isolated by screening a lambda gt11 cDNA library constructed from poly(A+) RNA of embryonic chicken skeletal muscle with the antibodies specific for each protein, and their complete sequences were determined. The chicken cofilin cDNA encoded a protein of 166 amino acids, the sequence of which had over 80% identity with that of porcine brain cofilin. The amino acid sequence of the ADF was 165 amino acids and showed about 70% identity with either chicken or mammalian cofilin, in spite of the fact that ADF and cofilin are functionally distinct. Like chicken and mammalian cofilin, ADF contained a sequence similar to the nuclear transport signal sequence of SV40 large T antigen. ADF and cofilin shared a hexapeptide identical with the amino-terminal sequence of tropomyosin as well as the regions homologous to other actin-regulatory proteins, including depactin, gelsolin, and profilin. The overall nucleotide sequences and Southern blot analysis of genomic DNA, however, indicated that the two proteins were derived from different genes.     

棉花PTS2受体基因(GhPex7)的克隆及表达分析

利用cDNA—AFLP差异片段F010,通过RACE延伸、EST、检索等方法获得了一个棉花过氧化物酶体定位信号2受体蛋白基因(peroxisomal targetingsignal type 2 receptor,GhPex7p)的编码序列。该cDNA包含一个954bp的开放阅读框,编码317个氨基酸,推测其等电点为5．603。同源性分析表明：推测GhPex7与拟南芥、酵母、果蝇、小鼠和人的Pex7p基因存在序列相似性,其中与拟南芥的同源性最高,为83％,并具有3段WD-40蛋白家族的保守域,与拟南芥AtPex7的编码蛋白同类。Southern杂交结果表明该基因在陆地棉基因组中存在两个拷贝。Northern blotting和RT-PCR分析表明该基因在棉花根、茎、叶、花、胚珠和纤维中均表达,但茎、叶组织中的表达水平明显高于胚珠和纤维。     

GhADF6-mediated actin reorganization is associated with defence against Verticillium dahliae infection in cotton

Several studies have revealed that actin depolymerizing factors (ADFs) participate in plant defence responses; however, the functional mechanisms appear intricate and need further exploration. In this study, we identified an ADF6 gene in upland cotton (designated as GhADF6) that is evidently involved in cotton's response to the fungal pathogen Verticillium dahliae. GhADF6 binds to actin filaments and possesses actin severing and depolymerizing activities in vitro and in vivo. When cotton root (the site of the fungus invasion) was inoculated with the pathogen, the expression of GhADF6 was markedly down-regulated in the epidermal cells. By virus-induced gene silencing analysis, the down-regulation of GhADF6 expression rendered the cotton plants tolerant to V. dahliae infection. Accordingly, the abundance of actin filaments and bundles in the root cells was significantly higher than that in the control plant, which phenocopied that of the V. dahliae-challenged wild-type cotton plant. Altogether, our results provide evidence that an increase in filament actin (F-actin) abundance as well as dynamic actin remodelling are required for plant defence against the invading pathogen, which are likely to be fulfilled by the coordinated expressional regulation of the actin-binding proteins, including ADF.     

Two different genes encoding acetylcholinesterase existing in cotton aphid (Aphis gossypii).

Two acetylcholinesterase (AChE) genes, Ace1 and Ace2, have been cloned from cotton aphid, Aphis gossypii Glover, using the rapid amplification of cDNA ends (RACE) technique. To the best of our knowledge, this should be the first direct molecular evidence that multiple AChE genes exist in insects. The Ace1 gene was successfully amplified along its full length of 2371 bp. The open reading frame is 2031 bp long and encodes 676 amino acids (GenBank accession No. AF502082). The Ace2 gene was amplified as a mega-fragment of 2130 bp lacking part of 5'-end untranslated region (UTR). The open reading frame is 1992 bp long and ecodes a protein of 664 amino acids (GenBank accession No. AF502081). Both genes have the conserved amino acids and features shared by the AChE family, but share only 35% identity in amino acid sequence. The Ace1 gene is highly homologous to the AChE gene of Schizaphis graminum (AF321574) with 95% identity, and Ace2 to that of Myzus persicae (AF287291) with 92% identity. Phylogenetic analysis showed that the two cloned AChEs of A. gossypii are different in evolution. The phylogenetic tree generated by the PHYLIP program package inferred that AChE2 of A. gossypii is a more ancestral form of AChE. Homology modeling of structures using Torpedo californica (2ACE_) and Drosophila melanogaster (1Q09:A) native acetylcholinesterase structure as main template indicated that the two AChEs of Aphis gossypii might have different three-dimensional structures. Alternative splicing of Ace1 near the 5'-end resulting in two proteins differing by the presence or absence of a fragment of four amino acids is also reported.     

Down-regulation of GhADF1 gene expression affects cotton fibre properties

Cotton fibre is the most important natural fibres for textile industry. To date, the mechanism that governs the development of fibre traits is largely unknown. In this study, we have characterized the function of a member of the actin depolymerizing factor (ADF) family in Gossypium hirsutum by down-regulation of the gene (designated as GhADF1 ) expression in the transgenic cotton plants. We observed that both the fibre length and strength of the GhADF1 -underexpressing plants increased as compared to the wild-type fibre, and transgenic fibres contained more abundant F-actin filaments in the cortical region of the cells. Moreover, the secondary cell wall of the transgenic fibre appeared thicker and the cellulose content was higher than that of the control fibre. Our results suggest that organization of actin cytoskeleton regulated by actin-associated proteins such as GhADF1 plays a critical role in the processes of elongation and secondary cell wall formation during fibre development. Additionally, our study provided a candidate intrinsic gene for the improvement of fibre traits via genetic engineering.     

白毛黑眼兔眼部虹膜Trp1、Trp2基因克隆与分析

目的 克隆日本大耳白兔白毛黑眼系(白毛黑眼兔)眼部虹膜Trp1、Trp2基因,获取其完整的外显子序列.进一步推测这两个基因编码的蛋白,并分析其特征.方法 从白毛黑眼兔的黑色虹膜组织中提取RNA,并反转录成cDNA.利用来自小鼠、大鼠和人的同源引物,扩增获得白毛黑眼兔Trp1、Trp2基因外显子片段.然后对已知片段进行3' RACE和5'RACE,从而获得白毛黑眼兔Trp1、Trp2基因外显子完整序列.利用相关软件对获得序列进行翻译和分析.结果 首次获得了白毛黑眼兔Trp1、Trp2基因外显子全序列.该实验兔Trp1基因的编码序列全长1604个碱基,其核苷酸序列与人的相似度为87.9％,与小鼠的相似度为82.7％.TRP1成熟蛋白包含513氨基酸,氨基酸序列与人的相似度为89.8％,与小鼠的相似度为86.6％.该实验兔Trp2基因序列全长1554个碱基,其核苷酸序列与人的相似度为83.2％,与小鼠的相似度为81.9％.TRP2成熟蛋白包含494个氨基酸,其序列与人的一致度为84.2％,与小鼠的一致度为84.4％.结论 本研究获得的TRP1、TRP2的序列与已知的家兔酪氨酸相关蛋白家族成员TYR的序列进行比对,结果显示这三种蛋白之间有较高的相似度,并且TRP1和TRP2蛋白序列表现出了酪氨酸酶家族结构上的保守性和特有的结构特征.     

cDNA文库与RACE方法结合克隆一个马铃薯病程相关蛋白基因cDNA

为克隆马铃薯晚疫病抗性相关基因,深入研究马铃薯晚疫病抗性机制,应用SMART LD—PCR技术,以晚疫病菌混合小种诱导48h的水平抗性马铃薯(Solanum tuberosum L．)(R—gene—free)叶片为材料,构建了一个富集晚疫病抗性相关基因的cDNA文库。为提高克隆全长cDNA的效率,将cDNA文库与RACE技术结合,依据本实验室得到的病原诱导表达片段测序结果,在其内部设计两个特异引物,与文库载体臂上的通用引物配对,以文库噬菌体DNA为模板,用高保真PCR分别扩增出cDNA5′端与3′端,从而简便、快捷地得到全长cDNA序列。采用此方法,在马铃薯中克隆了一个受晚疫病菌诱导表达的cDNA,该cDNA长904bp,5′端有29bp的非翻译区,3′端具有完整的polyA尾,包含一个678bp的完整开放阅读框架,编码226个氨基酸(GenBank登录号：AY 185207)。BLAST检索发现其氨基酸序列与烟草一个新的病程相关蛋白基因NtPRp27具有90％的同源性,在马铃薯中尚未发现与之同源的已知基因。Northern杂交结果表明,水杨酸(SA)、茉莉酸(JA)、茉莉酸甲酯(MeJA)、机械伤害和渗透胁迫都能诱导该基因表达。该基因可能是马铃薯一个新的病程相关蛋白基因。