抗冻蛋白应用于水稻悬浮细胞超低温保存的研究

本文将鱼类抗冻蛋白应用于植物细胞的超低温保存。结果表明,在水稻悬浮细胞的两步法保存中,浓度为0.01mg/ml的抗冻蛋白具有特别的负作用,相对高浓度的抗冻蛋白则能减小细胞存活率的波动性。在玻璃化法保存中,浓度为0.2mg/ml的抗冻蛋白能改善保存效果,更高浓度的抗冻蛋白(>5mg/ml)反而会降低保存效果。环境冰晶量、抗冻蛋白浓度、低温保护剂浓度和细胞膜组成等是影响抗冻蛋白使用效果的几大因素。作者在机理分析中认为,一方面,抗冻蛋白能和冰晶作用,抑制重冰晶,防止去玻璃化;另一方面,抗冻蛋白也能和细胞膜作用,诱发膜表面冰晶形成。     

Profiling cellular protein complexes by proximity ligation with dual tag microarray readout

Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.     

中国红豆杉紫杉烯合酶cDNA的分离、表达和鉴定

紫杉烯合酶是一种二萜环化酶 ,催化牛儿基牛儿基焦磷酸形成紫杉醇生物合成过程中的中间体紫杉烯。利用PCR扩增同源探针筛选cDNA文库 ,克隆了一个编码中国红豆杉 (Taxuschinensis (Pilg .)Rehd .)紫杉烯合酶 3′端的 2 15 1bp的cDNA片段 ,也通过PCR扩增得到了该基因 5′端的 6 11bp的cDNA片段 ,将这两个cDNA片段拼接在一起 ,得到长 2 712bp的cDNA片段 ,具有一个 2 5 86个碱基的开放阅读框架 (ORF) ,编码包括质体转移肽在内的共 86 2个氨基酸残基 ;该酶与太平洋红豆杉紫杉烯合酶有 97%的同源性 (identity) ,与其他植物萜类环化酶也有较高的同源性。利用融合表达载体pET_32a在大肠杆菌BL2 1trxB中表达 ,所表达的融合蛋白以包含体形式存在。包含体经过变性、复性和再折叠 ,利用His残基亲和凝胶柱层析得到融合的紫杉烯合酶。用毛细管气相色谱 质谱联用对酶促反应产物进行分析 ,结果表明 ,融合的紫杉烯合酶能催化产生 4(5 ) ,11(12 )_紫杉烯     

基于最小限制水分范围评价不同耕作方式对土壤有机碳的影响

以2009年吉林省德惠市中层黑土上进行了8a的田间定位试验小区土壤为研究对象,对免耕和秋翻两种耕作方式及玉米-大豆轮作和玉米连作两种种植方式下耕层有机碳进行分析,分别采用加权平均和分层两种方法计算最小限制水分范围(LLWR),用其评价不同耕作方式对土壤有机碳的影响.结果表明,免耕在玉米-大豆轮作和玉米连作下0-5 cm土壤有机碳含量分别比秋翻增加了15.2％和11.5％ (P＜0.05).采用加权平均法计算的LLWR值为0.148-0.166 cm3/cm3,不同耕作方式下玉米-大豆轮作的LLWR高于玉米连作且在两种种植方式下均表现出免耕小于秋翻的特点;利用分层法计算得到的LLWR值介于0.130-0.173 cm3/cm3之间,玉米-大豆轮作和玉米连作下免耕0-5 cm LLWR均显著小于秋翻,而5-30 cm LLWR数值免耕大于秋翻(P＞0.05);玉米-大豆轮作下0-30 cm各层LLWR均高于玉米连作.由于LLWR可以评价不同耕作方式对土壤有机碳的影响,因此采用加权平均法计算的LLWR可以客观的反映不同耕作处理尤其是种植方式对土壤有机碳的影响;而采用分层法计算的LLWR则更清晰的刻画了土壤表层与亚表层固碳能力的差异.     

翘嘴鳜肌球蛋白轻链3b基因(MLC3b)的分子克隆和特征分析

翘嘴鳜以其优良肉质性状近期已成为中国极具商品价值和大力推广的人工养殖名贵鱼类之一。为了解其控制优良肉质性状的遗传基础,本文采用同源克隆RT-PCR方法,获得该鱼肌球蛋白轻链MLC3b基因cDNA序列,该基因cDNA序列为453bp,编码150个氨基酸残基,通过PROSITE tools软件预测显示,该轻链具有两个保守的EF-手相结构和一个C-末端保守序列,且该MLC3轻链N端没有高等脊椎动物特有标志序列。MLC3b基因cDNA序列与MLC3a编码区的同源性达97.7%。本研究结果将为名贵鱼类肉质结构基因及功能的研究提供分子生物学基础。     

海洋真菌Aspergillus sp.SCSGAF 0076与细菌Bacillus sp.MNMCCE001共培养次生代谢产物

【目的】阐明海洋真菌Aspergillus sp.SCSGAF 0076与细菌Bacillus sp.MNMCCE 001在平板上共培养时产生的主要抗菌物质和红色素的化学结构。【方法】在固体淀粉培养基上分别进行Aspergillus sp.SCSGAF 0076纯培养、以及SCSGAF 0076和Bacillus sp.MNMCCE 001共培养,培养3 d后分别对纯培养和共培养的培养基进行萃取浓缩得到浸膏,运用高效液相色谱分析比较纯培养和共培养所得浸膏的化学分成的差异,并采用抗菌活性追踪法,结合硅胶柱层析、凝胶柱层析和高效液相色谱等分离方法从共培养浸膏中分离纯化抗菌物质和红色素,运用波谱解析法鉴定化合物的结构。【结果】通过高效液相色谱分析发现,共培养与纯培养的主要次生代谢产物没明显差异,但抗菌物质和红色素的含量差别明显。从这两株菌共培养的发酵产物中分离鉴定了4个化合物,包括抗菌物质青霉酸(1)、青霉酸类似物5(6)-dihydropenicillic acid(2)和9-chloro-8-hydroxy-8,9-deoxyasperlactone(3)、以及红色素viopurpurin(4)。【结论】初步阐明了Aspergillus sp.SCSGAF 0076与Bacillus sp.MNMCCE 001共培养时由Aspergillus sp.SCSGAF 0076产生的主要抗菌活性化合物是青霉酸,伴随明显增多的主要红色素是viopurpurin,二者产率均比Aspergillus sp.SCSGAF 0076纯培养时高。     

四数獐牙菜的胚胎学及其系统学意义

四数獐牙菜花药四室,药壁发育为双子叶型;绒毡层腺质型。小孢子母细胞减数分裂为同时型, 四分体的排列方式为四面体形;成熟花粉为3-细胞。子房2心皮,2心皮连接处强烈内凸,4列胚珠。薄 珠心,单珠被,直生胚珠。胚囊发育为蓼型。胚乳发育为核型。胚胎发育为茄型酸浆I变型。反足细胞 在胚囊成熟时期宿存。果实成熟时,种子只发育至球型胚阶段。承珠盘存在。与已研究的獐牙菜种类 的胚胎特征相比较,该种存在三个显著的特点：直生胚珠,心皮连接处强烈内凸,承珠盘存在。这三个特 征目前仅在龙胆族中的花锚属中发现。经过性状分析,它们均是演征,此三个共有演征表明四数獐牙菜 与花锚属是一单系群,它们的亲缘关系最近。这与Yun &; Kupfer(1995)的分子证据部分吻合,因为分 子系统树上的花锚单系支包括花锚、四数獐牙菜和北美的密花属(广义獐牙菜属中的密花组),而后者的 胚胎学特征未有任何报道。胚胎学研究暗示四数獐牙菜的分类学等级有待重新评价,广义的獐牙菜属 有可能不是一单系发生群。     

黄土区土壤放射性的生物循环

研究了不同类型土壤中放射性物质的生物学循环和平衡问题。结果表明,土壤中放射性核素剖面分布的总趋势是沿发生层往下逐步减少。其生物积聚系数因不同核素而变动在1.06—1.44间。放射性物质在各类植物体内的含量顺序是:牧草>农业作物≥灌木≥果树>乔木。植物对α放射性的富集系数一般均小于0.10,而β放射性的富集系数大都处于0.5—1.0间.不同土地利用情况下土壤放射性可分为积累、消耗和中间3种平衡类型。     

A phylogenetic approach to the identification of phosphoglucomutase genes

The expanding molecular database provides unparalleled opportunities for characterizing genes and for studying groups of related genes. We use sequences drawn from the database to construct an evolutionary framework for examining the important glycolytic enzyme phosphoglucomutase (PGM). Phosphoglucomutase plays a pivotal role in the synthesis and utilization of glycogen and is present in all organisms. In humans, there are three well-described isozymes, PGMI, PGM2, and PGM3. PGM1 was cloned 5 years ago; however, repeated attempts using both immunological approaches and molecular probes designed from PGM1 have failed to isolate either PGM2 or PGM3. Using a phylogenetic strategy, we first identified 47 highly divergent prokaryotic and eukaryotic PGM-like sequences from the database. Although overall amino acid identity often fell below 20%, the relative order, position, and sequence of three structural motifs, the active site and the magnesium-- and sugar-binding sites, were conserved in all 47 sequences. The phylogenetic history of these sequences was complex and marked by duplications and translocations; two instances of transkingdom horizontal gene transfer were identified. Nonetheless, the sequences fell within six well-defined evolutionary lineages, three of which contained only prokaryotes. Of the two prokaryotic/eukaryotic lineages, one contained bacterial, yeast, slimemold, invertebrate, and vertebrate homologs to human PGM1 and the second contained likely homologs to human PGM2. Indeed, an amino acid sequence, derived from a partial human cDNA, that fell within the second cross-kingdom lineage bears several characteristics expected for PGM2. A third lineage may contain homologs to human PGM3. On a general level, our phylogenetic-based approach shows promise for the further utilization of the extensive molecular database.