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以曼氏无针乌贼（Sepiellamaindroni）肌肉组织为研究材料,利用Clontech公司的cDNA文库构建试剂盒构建了曼氏无针乌贼肌肉cDNA文库．对文库质量的分析结果表明：cDNA文库的库容量约为1．2×10。克隆（cfu／dm^3）,重组率达96％,插入片段平均长度大于l000bp．挑取568个阳性克隆进行5’末端测序,其中475条ESTs长度大于100bp,初步拼接后得到200个单基因簇,包括41个重叠群和159个单拷贝EST,冗余度为57．89％．经检索,53条ESTs（占比为26．5％）在BLASTx上无明显的同源性（E值≥1．00×10^-10）,为新基因．147条ESTs（占比为73．5％）与已报道的基因有较高的同源性;其中与能量相关基因的ESTs丰度最高,占总数的23．0％．细胞信号传导、细胞骨架和蛋白质代谢的次之,比例分别为17．5％、12．5％、9．5％,其中包括热激蛋白70、转铁蛋白等．这些EST数据有助于进一步筛选和克隆曼氏无针乌贼和其他头足动物的肌肉特异性表达基因． 相似文献
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通过对半滑舌鳎雌鱼fosmid文库进行有序混合,成功构建了两步-三维PCR筛选体系,为后续的基因筛选奠定了基础。通过对半滑舌鳎W染色体文库序列进行引物设计,并分别在雌、雄鱼基因组DNA中筛选,获得1对雌鱼特异性引物。使用该引物对fosmid文库进行筛选,得到1个雌鱼特异性克隆,并通过荧光原位杂交技术将克隆定位到了W性染色体上,证实了使用该体系进行文库筛选的有效性以及雌鱼引物的特异性,建立了半滑舌鳎遗传性别的分子及细胞遗传学鉴定方法,为后续半滑舌鳎基因组及性染色体的研究奠定了基础。 相似文献
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XUAN Jinsong FENG Yanbin WENG Manli ZHAO Ge SHI Jinfeng YAO Jianting WANG Xiuliang GUO Baotai QIAO Lixian DUAN Delin WANG Bin 《海洋学报(英文版)》2012,31(6):139-148
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. 相似文献
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The aquaculture of sea cucumber Apostichopus japonicus (Echinodermata, Holothuroidea) has grown rapidly during recent years and has become an important sector of the marine industry in Northern China. However, with the rapid growth of the industry and the use of non-standard culture techniques, epidemic diseases of A. japonicus now pose increasing problems to the industry. To screen the genes with stress response to bacterial infection in sea cucumber at a genome wide level, we constructed a cDNA library from A. japonicus Selenka (Aspidochirotida: Stichopodidae) after infecting them with Vibrio sp. for 48 h. Total RNA was extracted from the intestine, mesentery and coelomocyte of infected sea cucumber using Trizol and mRNA was isolated by Oligotex mRNA Kits. The ligated cDNAs were transformed into DH5α, and a library of 3.24×105 clones (3.24×105 cfu mL-1) was obtained with the sizes of inserted fragments ranging from 0.8 to 2.5 kb. Sequencing the cDNA clones resulted in a total of 1106 ESTs that passed the quality control. BlastX and BlastN searches have identified 168 (31.5%) ESTs sharing significant homology with known sequences in NCBI protein or nucleotide databases. Among a panel of 25 putative immunity-related genes, serum lectin isoform, complement component 3, complement component 3-like genes were further studied by real-time PCR and they all increased more than 5 fold in response to Vibrio sp. challenge. Our library provides a valuable molecular tool for future study of invertebrate immunity against bacterial infection and our gene expression data indicates the importance of the immune system in the evolution and development of sea cucumber. 相似文献
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硬脂酰辅酶A去饱和酶(SCD)是脂肪代谢的关键酶。本研究采用RT-PCR和RACE技术克隆了曼氏无针乌贼(Sepiella maindroni)SCDcDNA的全序列,序列全长1513bp,由261bp的5′非翻译区、编码306个氨基酸的921bp开放阅读框和331bp的3′非翻译区组成。在线翻译所得多肽理论分子量为34.92kDa,等电点为8.95,是疏水性蛋白,含有丰富的螺旋结构(45.10%),存在4个跨膜区。其氨基酸序列与真蛸(Octopus vulgaris)和长牡蛎(Crassostrea gigas)相似性达到91%,与其它非软体动物也表现为50%以上的相似性,说明SCD结构相对保守;系统进化树结果表明曼氏无针乌贼和真蛸及牡蛎进化关系最近,与鱼类稍远,与人及大鼠等哺乳动物亲缘关系最远。SCD基因是改善曼氏无针乌贼肉质的重要候选基因,其成功克隆及相关分析对于深入探讨软体动物脂肪酸代谢相关基因在生物体内作用机制及调控机理具有重要意义。 相似文献
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采用生物素磁珠富集法,用生物素标记的(GT)15和(CT)15两种探针与细角螺基因组 MseI 酶切的300~1200bp 片段杂交,杂交复合物与链霉亲和素磁珠结合,捕获含有重复序列的微卫星片段。最后将磁珠捕获到的重复序列与PMD19-T载体连接后克隆到DH5α中构建微卫星基因组文库。通过PCR检测出354个阳性克隆,从中随机选取248个片段大于500bp的阳性克隆进行测序,结果显示,在220个成功测序的阳性克隆中共获得278个微卫星序列,其中完美型171个,占61.5%;非完美型81个,占29.1%;复合型26个,占9.4%。除探针使用的GT和CT的重复序列外,还筛选到三碱基GTT、TGG、GAA、CAA;四碱基TCTA、ACAG、TAGA、GTGA、GTCT、GATA及五碱基TTTTG的重复序列。在278条序列中共有82条可以设计引物。 相似文献
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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… 相似文献