棕囊藻北部湾株的18S rDNA分子鉴定

为研究北部湾棕囊藻(Phaeocystis)藻华的成因,采用PCR克隆了棕囊藻北部湾株核糖体18S r DNA序列。结果表明,棕囊藻北部湾株具有游动单细胞与群体结构两种形态;其18S r DNA序列和NCBI基因库中球形棕囊藻(Phaeocystis globosa)的同源性为99%~100%,在系统进化树上与不同海域来源的球形棕囊藻聚在一大分支上,且与球形棕囊藻间的遗传距离均小于其他种。首次从分子生物学上确定棕囊藻北部湾株为球形棕囊藻。     

赤潮棕囊藻(Phaeocystis globosa)rDNA ITS区序列变异与二级结构分析

测定了南海球形棕囊藻香港株P1、P2和湛江株ZhJ1的rDNAITS区序列(含5.8srDNA),结合Gen Bank的13条同源序列,比对长度为904bp,变异位点271个,简约信息位点221个,平均(A+T)(34.5%)<(G+C)(65.4%).藻株P1、P2和ZhJ1序列存在变异位点20个,序列间相似性为97.9%～98.5%.ITS序列在种间和种内的解析度高于18srDNA和28srDNA基因;构建的NJ树、MP树、贝叶斯推断系统树的结构是一致的,不同种类的棕囊藻单独聚类,不同地理来源的球形棕囊藻混杂分布但相同地理来源的藻株多聚类在一起.RNA二级结构显示,不同藻种间5.8srDNA区结构基本一致,表现出属的特异性;ITS1、2区结构表现较大的种间差异,表明ITS区RNA二级结构可为棕囊藻分类鉴定提供有用的分子结构信息.     

棕囊藻属(Phaeocystis)的分类与生活史(综述)

棕囊藻属Phaeocystis（定鞭藻纲Prymnesiophyceae)的分类问题目前还有争论。其种的分类标准是以初始的群体形态、地理分布、细胞特征等以及分子生物学特征,如染色体倍性,基因组大小等为依据。基于以上各种分类特征,目前比较确定的棕囊藻属藻类有四种：一种是只观察到单细胞形态的凹孔棕囊藻（P.scrobiculata),另外三种是能够形成群体的波切棕囊藻（P.pouchetii)、球形棕囊藻（P.globosa)和南极棕囊藻（P.antarctica)。棕囊藻具有一个复杂的异形生活史,介于几种游离的单细胞（不动的细胞,具有鞭毛的动细胞,小游动孢子以及可能存在的大游动孢子）和群体之间的形态交替。但其生活史中仍有许多不确定的问题。     

棕囊藻属的分类现状

棕囊藻属藻类生活史复杂、地理差异显著、游动单细胞个体微小,至今尚无明确的分类标准。在目前已报道的9个种中,Sournia(1988)认为只有两个种比较可靠,即形成胶群体的P.pouchetii(sensu lato)(包括P.globosa)和不形成胶群体的P.scrobiculata;一些学者将P.puchetii(sensu lato)进一步细分成P.pouchetii(sensu stricto)、P.globosa,P.antarctica等3种,为方便起见,多数学者将形成胶群体的棕囊藻定名为P.pouchetii,或写成未定种的形式Phaeocystis sp.棕囊藻属分类的混乱制约了相关研究的深入,解决棕囊藻分类混乱问题有赖于新的技术和方法的使用。     

实验室培养球形棕囊藻溶血毒素的提取、分离及其生成特征

研究从实验室培养的球形棕囊藻(Phaeocystis globosa)提取溶血毒素的条件,探讨不同生长期球形棕囊藻溶血毒素的生成特征,用薄层色谱法对溶血成份进行了初步分析。结果表明,球形棕囊藻细胞壁的超声波破碎最适条件为：功率600W,4℃下处理30min。对数生长期、平稳期和衰亡期藻细胞适宜处理量分别为每次3000、2000、1000ml;在球形棕囊藻生长的平稳期和衰亡期具有明显的溶血活性,对数生长期溶血活性很低,甚至检测不到。球形棕囊藻溶血毒素至少含有4种糖脂类化合物。     

红树植物秋茄叶片水提物的抑藻效应

通过藻细胞密度的测定,探讨了不同浓度(0.5、1.0和2.0g·L-1)红树植物秋茄(Kandelia candel)新鲜叶片水提物对球形棕囊藻(Phaeocystis globosa)和赤潮异弯藻(Heterosigma akashiwo)的化感抑制效应,研究了高温处理对秋茄提取物化感作用的影响.结果表明:秋茄叶片提取物对两种赤潮藻均具有显著的化感抑制作用,不同浓度提取物化感作用强度不同;5 d内,浓度为2.0g·L-1秋茄叶片提取物对球形棕囊藻和赤潮异弯藻的最大抑制率分别为91.6％和77.0％;球形棕囊藻和赤潮异弯藻对红树植物秋茄提取物的敏感性不同,提取物对球形棕囊藻的抑制效果要优于赤潮异弯藻;经高温处理后,秋茄提取物抑藻效果显著降低(P＜0.05);秋茄叶片水提物影响藻细胞膜结构,使藻细胞体积增大、细胞破裂.     

球形棕囊藻的生长、囊体形态以及囊体细胞的分布

球形棕囊藻(Phaeocystis globosa)是中国近海海区常见有害藻华原因种, 其异型生活史中包含单细胞和球形囊体两种形态。游离单细胞直径一般为几微米, 囊体最大直径可达3 cm, 巨大的体积可能导致囊体具有特殊的结构和细胞分布。以球形棕囊藻汕头株为研究对象, 测定了囊体直径、囊体细胞丰度和游离单细胞丰度, 并探讨球形棕囊藻囊体形态与细胞分布的关系。研究结果表明, 囊体形态在其异型生活史中占优势, 囊体对生物量的贡献介于38%–95%之间, 在对数生长期的后期和稳定期, 囊体细胞与单细胞相比占绝对优势。囊体细胞数量与囊体直径的对数呈线性相关, 回归线斜率为1.34, 该值显著低于世界海区其它球形棕囊藻株系的研究结果, 表明汕头株单位囊体表面上分布的细胞数更少。中国海区的球形棕囊藻囊体结构和细胞分布与其它株系不同, 在爆发球形棕囊藻的海区, 巨大的囊体能够有效地抵御摄食, 可能对区域海洋食物链结构和功能有重要影响。     

高梁叶绿体psaA基因的克隆和核苷酸序列分析

我们克隆了含高梁叶绿体psaA基因的6．7kb Ec0RI酶切片段,进行了限制酶图谱分析和核苷酸序列结构测定,所测核苷酸序列总长为3080bp,其中高梁叶绿体psaA基因序列为2253bp,编码一个含750个氨基酸残基的蛋白质,其分子量为83000Da。高梁psaA基因的核苷酸序列与玉米、水稻,菠菜的同源性分别为99．4％、96．3％和89．4％;推导出的氨基酸序列的同源性分别为99．3“、98．O％和95．7％。C₄植物与C₃植物比较,由于P 700脱辅基蛋白的第493位氮基酸性质的不同(Gly→Arg,ser),而导致了它们的乡肽在疏水性图谱上的差异。     

18SrDNA序列分析鉴定棕囊藻香港株P2为球形棕囊藻

有毒赤潮原因种棕囊藻(Phaeocystis sp.)生活史复杂,且形体微小,缺乏明确可靠的分类标准。1997、1999年中国东南沿海发生两次棕囊藻赤潮,但目前种的鉴定仍存在混乱。本文测定了香港海域一株棕囊藻赤潮原因种P₂的18SrDNA部分核苷酸序列,序列长度为650bp,并对其进行了分子鉴定,结果表明其为球形棕囊藻(P.globosa)。     

球形棕囊藻游离单细胞的密度与囊体形成的关系研究

球形棕囊藻(Phaeocystis globosa Scherffel)主要以囊体形态形成赤潮,由单细胞向囊体形态的转变是赤潮爆发的关键.本研究推测囊体形成的前提是游离单细胞达到一定密度阈值,当密度低于该阈值时,囊体无法形成.基于此,本文探究了不同条件(温度、营养充气搅动、摄食压力、初始密度)下囊体形成时游离单细胞的密...     

Cloning and nucleotide sequence of the Salmonella typhimurium LT2 gnd gene and its homology with the corresponding sequence of Escherichia coli K12

Summary The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative.     

新疆黄精凝集素基因的克隆、序列分析和表达

用百合科黄精属植物凝集素基因的保守序列为引物,从新疆黄精的叶中克隆出黄精凝集素的全长cDNA。序列分析表明, 克隆获得的新疆黄精凝集素(Polygonatum roseum agglutinin, PRA)基因完整的ORF片段大小为550 bp, 编码1 条长159个氨基酸肽链, 没有内含子, 其中N 端的28个氨基酸是信号肽。对新疆黄精凝集素cDNA 序列同源性的分析比较发现, 黄精属植物凝集素基因之间有很高的同源性(92%)。氨基酸序列比对和SWISS-MODEL同源模建分析表明, PRA由12个b-折叠片组成的b-桶结构, 具有与单子叶植物甘露糖结合凝集素相似的空间结构。重组质粒pGEX-4T-1-PRA 和pMAL-p2x-PRA, 分别转化E. coli BL21进行原核表达, 新疆黄精凝集素能够以可溶性融合蛋白形式表达, 分子量约为14 kD。构建真核表达载体pcDNA3-PRA, 免疫小鼠后获得了抗血清。免疫印迹结果显示为单一的条带, 证明该抗血清具有针对PRA抗原的专一性。新疆黄精凝集素基因的克隆、原核和真核的表达以及抗血清的制备, 为进一步研究凝集素蛋白的性质和功能, 并为植物抗病虫基因工程研究提供有用的实验材料。     

新疆黄精凝集素基因的克隆、序列分析和表达

用百合科黄精属植物凝集素基因的保守序列为引物,从新疆黄精的叶中克隆出黄精凝集素的全长cDNA。序列分析表明, 克隆获得的新疆黄精凝集素(Polygonatum roseum agglutinin, PRA)基因完整的ORF片段大小为550 bp, 编码1 条长159个氨基酸肽链, 没有内含子, 其中N 端的28个氨基酸是信号肽。对新疆黄精凝集素cDNA 序列同源性的分析比较发现, 黄精属植物凝集素基因之间有很高的同源性(92%)。氨基酸序列比对和SWISS-MODEL同源模建分析表明, PRA由12个b-折叠片组成的b-桶结构, 具有与单子叶植物甘露糖结合凝集素相似的空间结构。重组质粒pGEX-4T-1-PRA 和pMAL-p2x-PRA, 分别转化E. coli BL21进行原核表达, 新疆黄精凝集素能够以可溶性融合蛋白形式表达, 分子量约为14 kD。构建真核表达载体pcDNA3-PRA, 免疫小鼠后获得了抗血清。免疫印迹结果显示为单一的条带, 证明该抗血清具有针对PRA抗原的专一性。新疆黄精凝集素基因的克隆、原核和真核的表达以及抗血清的制备, 为进一步研究凝集素蛋白的性质和功能, 并为植物抗病虫基因工程研究提供有用的实验材料。     

Two adjacent nuclear genes are required for functional complementation of a chloroplast trans-splicing mutant from Chlamydomonas reinhardtii

The chloroplast tscA gene from Chlamydomonas reinhardtii encodes a co-factor RNA that is involved in trans-splicing of exons 1 and 2 of the psaA mRNA encoding a core polypeptide of photosystem I. Here we provide molecular and genetic characterization of the trans-splicing mutant TR72, which is defective in the 3'-end processing of the tscA RNA and consequently defective in splicing exons 1 and 2 of the psaA mRNA. Using genomic complementation, two adjacent nuclear genes were identified, Rat1 and Rat2, that are able to restore the photosynthetic growth of mutant TR72. Restoration of the photosynthesis phenotype, however, was successful only with a DNA fragment containing both genes, while separate use of the two genes did not rescue the wild-type phenotype. This was further confirmed by using a set of 10 gene derivatives in complementation tests. The deduced amino acid sequence of Rat1 shows significant sequence homology to the conserved NAD+-binding domain of poly(ADP-ribose) polymerases of eukaryotic organisms. However, mutagenesis of conserved residues in this putative NAD+-binding domain did not reveal any effect on restoration efficiency. Immunodetection analyses with enriched fractions of chloroplast proteins indicated that Rat1 is associated with chloroplast membranes. Using the yeast three-hybrid system, we were able to demonstrate the specific binding of tscA RNA by the Rat1 polypeptide. We propose that the two nuclear factors Rat1 and Rat2 are involved in processing of chloroplast tscA RNA and in subsequent splicing of psaA exons 1 and 2.     

Cloning and sequencing analysis of three amylase cDNAs in the shrimpPenaeus vannamei (Crustacea decapoda): evolutionary aspects

InPenaeus vannamei, α-amylase is the most important glucosidase and is present as at least two major isoenzymes which have been purified. In order to obtain information on their structure, a hepatopancreas cDNA library constructed in phage lambda-Zap II (Strategene) was screened using a synthetic oligonucleotide based on the amino acid sequence of a V8 staphylococcal protease peptide ofP. vannamei α-amylase. Three clones were selected: AMY SK 37 (EMBL sequence accession number: X 77318) is the most complete of the analyzed clones and was completely sequenced. It contains the complete cDNA sequence coding for one of the major isoenzymes of shrimp amylase. The deduced amino acid sequence shows the existence of a 511-residue-long pre-enzyme containing a highly hydrophobic signal peptide of 16 amino acids. Northern hybridization of total RNA with the amylase cDNA confirms the size of the messenger at around 1,600 bases. AMY SK 28, which contains the complete mature sequence of amylase, belonged to the same family characterized by a common 3′ terminus and presented four amino acid changes. Some other variants of this family were also partially sequenced. AMY SK 20 was found to encode a minor variant of the protein with a different 3′ terminus and 57 amino acid changes. Phylogenetic analysis established with the conserved amino acid regions of the (β/α) eight-barrel domain and with the total sequence ofP. vannamei showed close evolutionary relationships with mammals (59–63% identity) and with insect α-amylase (52–62% identity). The use of conserved sequences increased the level of similarity but it did not alter the ordering of the groupings. Location of the secondary structure elements confirmed the high level of sequence similarity of shrimp α-amylase with pig α-amylase. Correspondence to: A. Van Wormhoudt     

赖型钩端螺旋体外膜蛋白基因结构比较性研究

用ＰＣＲ方法扩增不同毒力赖型钩体ＯｍｐＬ１基因片段,进行序列测定,用相关软件比较分析核苷酸序列、蛋白质二级结构以及限制性内切酶谱,不同毒力赖型钩体能扩增出９６０ｂｐ的片段,非致病Ｐａｔｏｃ株未能扩出相应片段,中国赖型参考株ＯｍｐＬ１序列（ＧｅｎｅＢａｎｋ Ｎｏ．ＡＦ２５０３１８）与流感伤寒型相应序列比较有９８个核苷酸差异,同源性为８９．８％,二级结构预测和氨基酸疏水图显示变异主要发生在跨膜蛋白的膜     

鸡补体分子C3d的基因克隆及结构分析

目的:克隆鸡补体分子C3d基因并解析其结构特点。方法:将已发表的人、小鼠、地鼠、奶牛、野兔、猪、猩猩、绵羊的C3d基因同鸡的C3α链进行序列比较分析,发现在鸡的C3α链上有一段约897bp的序列同上述动物有较高的同源性,在上下端保守区域设计一对引物788bp,应用RT-PCR扩增鸡C3d部分基因,并克隆到pMD18-T载体中,测序正确后再在上下端分别设计一对引物,理论长度分别为378和336bp,最后用3种PCR产物延伸扩出C3d全长序列。结果:获得了鸡C3d基因重组质粒pMD18-C3d,序列分析表明所获的鸡C3dcDNA全长为993bp,编码331个氨基酸残基。鸡与上述人或动物C3d核苷酸的同源性分别为66.6%、66.2%、67.7%、66.2%、67.1%、67.0%、59.6%、67.1%,与其编码的氨基酸的同源性分别为61.5%、61.9%、61.2%、61.9%、56.5%、61.9%、54.5%、61.5%;而哺乳动物间C3d的核苷酸和氨基酸的同源性则分别为74.2%～100%和72.2%～100%。进化树反映出C3d基因具有种的多样性,亲缘关系越近,进化关系也越近。结论:鸡C3d与其他动物的C3d在抗原结合位点上没有氨基酸的变化,而与CR2结合的28肽区氨基酸差异明显,说明鸡C3d结合抗原没有专一性,而结合免疫细胞则有种的特异性,由此可以推测鸡C3d只能增强鸡的特异性免疫反应。     

Studies on the bacteriophage MS2: XXXIII. Comparison of the nucleotide sequences in related bacteriophage RNAs

We have recently determined the complete nucleotide sequence of the MS2 bacteriophage RNA molecule (Min Jou et al., 1972; Fiers et al., 1975,1976). Extensive segments of the sequence of the closely related phages R17 (23.9%) and f2 (11.5%) have been studied in other laboratories (Table 1). A comparison between the sequences of the phages MS2, R17 and f2 can now be given a functional significance by referring to the complete MS2 RNA sequence (Fig. 1).The estimation of the over-all degree of variation amounts to 3.9% (MS2-R17), 3.4% (MS2-f2) and 3.7% (R17-f2). All the differences observed can be accounted for by single base substitutions: transitions are highly predominant (86%). No change is found in the untranslated terminal regions and only two mutations occur in the intercistronic regions. From a total of 34 observed variable sites located in translated regions, 25 are neutral point mutations and only 9 lead to an (mostly rather conservative) amino acid change. The amount of variation in double-stranded regions (16 out of 36 cases) is much lower than the relative degree of secondary structure of the RNA (roughly two-thirds) would predict. Hence, there is clearly a selective pressure to preserve at least certain aspects of the three-dimensional conformation.     

The porcine hormone-sensitive lipase gene: sequence, structure, polymorphisms and linkage mapping

The porcine hormone-sensitive lipase gene and its cDNA have been isolated and sequenced. Several putative regulatory sequences have been detected in the promotor region. The deduced amino acid sequence is 85% identical to the corresponding human, mouse and rat sequence. A search for polymorphisms revealed one intronic and one exonic polymorphism, the latter resulting in a conservative amino acid substitution. Linkage mapping located the LIPE gene close to the calcium release channel (CRC) locus on chromosome 6.