6—DMAP和胚胎干细胞代数对异种重构卵体外发育的影响（简报）

The development of reconstructed oocytes and the survival rate of cloned animal were affected by many factors during nuclear transfer. The genetic constitution and the genetic state of donor nucleus were proposed to be primary factors, which affected the survival rate of cloned animal. In addition, the survival rate of cloned animal might be influenced by nuclear transfer technique itself and passages of donor cells as well as the activation methods of oocytes. We reconstructed oocytes with outbreeding Kunming albino mouse ES cells and enucleated rabbit oocytes, and analyzed the effects of the passages of ES cells and 6-DMAP on the development of interspecific reconstructed oocytes. The interspecific reconstructed ES-rabbit oocytes were activated either by combined two set electric pulses and 6-DMAP or by two set electric pulses alone. The rate of cleavage was significantly higher for the group (86.2%) treated with 6-DMAP than the group (64.2%, P < 0.05) treated with electric pulses only, and the rate of blastocysts was 17.0% and 13.4% respectively, which were not significantly different between two groups. When ES cells that had been passed for 24 and 14 generations were used as donors, the cleavage rates of the reconstructed oocytes were 88.5% and 82.1%, respectively (P > 0.05), and the rates of blastolation were 16.7% and 15.4%, respectively (P > 0.05). The results show that 6-DMAP increases the cleavage rate of reconstructed oocytes derived from ES cells, and affects slightly the developmental rate of blastocysts. There are no differences when high passage and low passage ES cells are used as nuclear donors.     

芒果炭疽病菌β-微管蛋白基因的克隆及其与多菌灵抗药性发生的关系

参照豆科合萌属 (Aeschynomene)作物炭疽病菌的tub1和tub2基因序列设计了 2对引物 ,分别从芒果 (Man gifera)炭疽病菌对多菌灵 (MBC)田间抗药性 (MBCR)和敏感 (MBCS)的菌株中扩增 β_微管蛋白基因。结果只有以tub2为参照设计的引物扩增到了特异片段。进一步对全基因进行了克隆和测序。该基因序列全长 1344bp ,编码4 4 7aa ,其核苷酸和氨基酸序列与豆科合萌属炭疽病菌的tub2基因高度同源。对芒果炭疽病菌抗、感菌株 β_微管蛋白氨基酸序列进行比较分析 ,发现第 181、2 37和 36 3位氨基酸发生了突变 ,而其它位置 (如第 198位或 2 0 0位 )均不变     

硫丹对黄瓜光合色素及几种有关酶活性的影响

用0.1％-0.6％的硫丹喷施黄瓜幼苗4d后,叶绿素总量、叶绿素a、叶绿素b、原叶绿素、类胡萝卜素含量分别下降21.00％-38．57％、21.27％-38.69％、20.22％-38.24％、6．60％-49.24％和13.54％-68.85％;叶绿素酶活性上升16.51％-72．80％;δ-氨基乙酰丙酸（ALA）合酶和脱水酶活性分别下降14.14％-44.61％和4．38％-40．11％。讨论了硫丹影响叶绿素含量的可能机理。     

S-PI对香菇酸性蛋白酶的抑制机理及其促进出菇的生化解释

S-PI对纯化的香菇酸性蛋白酶具有强烈的抑制作用,10μmol／L的S-PI可使酶活降低77％,这种抑制属于可逆竞争抛物线型抑制。经25μmol／L酸性蛋白酶水解6小时后,香菇胞外酸性磷酸酶的活性从24．6单位降低到20．8单位,同时C1酶和Cx酶的活性分别比对照降低了76％和58％,这表明香菇酸性蛋白酶不仅直接左右着香菇发育的氮代谢,同时也间接地影响着碳代谢及能量转换。     

Cloning,expression, and characterization of thermostable Manganese superoxide dismutase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN₃, but not by KCN or H₂O₂. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60°C and the half-life at 80°C was approximately 40 min.     

Comparison of the effects of symmetric and asymmetric temperature elevation and CO2 enrichment on yield and evapotranspiration of winter wheat (Triticum aestivum L.)

Under the changing climate, asymmetric warming pattern would be more likely during day and night time, instead of symmetric one. Concurrently, the growth responses and water use of plants may be different compared with those estimated based on symmetric warming. In this work, it was compared with the effects of symmetric (ETs) and asymmetric (ETa) elevation of temperature alone, and in interaction with elevated carbon dioxide concentration (EC), on the grain yield (GY) and evapotranspiration in winter wheat (Triticum aestivum L.) based on pot experiment in the North China Plain (NCP). The experiment was carried out in six enclosed‐top chambers with following climate treatments: (1) ambient temperature and ambient CO₂ (CON), (2) ambient temperature and elevated CO₂ (EC), (3) elevated temperature and ambient CO₂ (ETs; ETa), and (4) elevated temperature and elevated CO₂ (ECETs, ECETa). In symmetric warming, temperature was increased by 3°C and in asymmetric one by 3.5°C during night and 2.5°C during daytime, respectively. As a result, GY was in ETa and ETs 15.6 (P < 0.05) and 10.3% (P < 0.05) lower than that in CON. In ECETs and ECETa treatments, GY was 14.9 (P < 0.05) and 9.1% (P < 0.05) higher than that in CON. Opposite to GY, evapotranspiration was 7.8 (P < 0.05) and 17.9% (P < 0.05) higher in ETa and ETs treatments and 7.2 (P < 0.05) and 2.1% (P > 0.05) lower in ECETs and ECETa treatments compared with CON. Thus, GY of wheat could be expected to increase under the changing climate with concurrent elevation of CO₂ and temperature as a result of increased WUE under the elevated CO₂. However, the gain would be lower under ETa than that estimated based on ETs due to higher evapotranspiration.     

微生物来源的γ内酰胺酶研究进展

γ-内酰胺酶属于酰胺酶,其中的(+)γ-内酰胺酶能够高效率的动力学拆分外消旋体γ-内酰胺,获得光学纯的(-)γ-内酰胺。光学纯的(-)γ-内酰胺是制备抗病毒药物碳环核苷化合物的重要手性中间体。目前报道共有7个来源于微生物的γ-内酰胺酶,其中来源于Aureoacterium sp.的(-)γ-内酰胺酶的晶体结构获得了解析。根据晶体结构推测的(-)γ-内酰胺酶的催化机理与α/β水解酶超家族的催化机理是类似的。但是,目前还没有(+)γ-内酰胺酶的晶体结构模型的数据及机理的描述。γ-内酰胺酶的研究方向主要包括γ-内酰胺酶的蛋白质工程改造,对不同对映体选择性的γ-内酰胺酶的催化机理的阐述,以及γ-内酰胺酶在生物体内的功能研究。     

人胎盘滋养层细胞培养与体外hCG释放的研究

本研究的目的是了解细胞滋养层细胞和合胞体滋养层细胞体外分化和生物学特性。方法:采用酶消化和Percoll密度梯度离心法,对人足月胎盘细胞滋养层细胞进行分离、纯化和体外培养。采用放射免疫法(RIA)检测细胞培养上清液hCG含量的变化。结果:经分离和纯化的细胞滋养层细胞在体外培养中生长良好,通过细胞分裂和融合形成合胞体滋养层细胞,随着合胞体滋养层细胞的生长,细胞培养上清液中hCG含量显著升高。我们认为从胎盘中分离和纯化的细胞滋养层细胞在体外培养中可分化和融合形成合胞体滋养层细胞,体外hCG含量的增加与合胞体滋养层细胞生长有关。     

Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells

DNA methylation at the 5 position of cytosine (5mC) in the mammalian genome is a key epigenetic event critical for various cellular processes. The ten-eleven translocation (Tet) family of 5mC-hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), offers a way for dynamic regulation of DNA methylation. Here we report that Tet1 binds to unmodified C or 5mC- or 5hmC-modified CpG-rich DNA through its CXXC domain. Genome-wide mapping of Tet1 and 5hmC reveals mechanisms by which Tet1 controls 5hmC and 5mC levels in mouse embryonic stem cells (mESCs). We also uncover a comprehensive gene network influenced by Tet1. Collectively, our data suggest that Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting 5mC to 5hmC through hydroxylase activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 targets, ultimately contributing to mESC differentiation and the onset of embryonic development.     

A new genotyping method for detecting low abundance single nucleotide mutations based on gap ligase chain reaction and quantitative PCR assay

We tested applicability of a new genotyping technique to detect a low abundance CD17 (A → T) mutation of β-globin gene. The technique utilized a combined gap ligase chain reaction (Gap-LCR) and quantitative PCR (qPCR) methods. One pair of Gap-LCR primers was modified by adding specific sequences to the 5′ end of the upstream and the 3′ end of the downstream primer which served as a combining sequence for qPCR. First, specific mutation is detected using Gap-LCR; then, ligation products are detected by qPCR. Our results show that the amount of LCR products is directly proportional to the amount of template DNA. We further demonstrate that this technique detects a low abundance mutant DNA with a mutant/normal allele ratio as low as 1:10000. This technique was applied to detect a paternally inherited CD17 mutation from 53 maternal plasma samples. The results were consistent with those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. In conclusion, by combining Gap-LCR and qPCR technology we successfully established a highly sensitive technique to detect low abundance point mutations. This technique can be applied to detect fetal DNA point mutation in maternal plasma.