含铬重组液激活部分缺失金属原子簇的钼铁蛋白的研究

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白经邻菲口罗啉和Ｏ２ 处理后,变为部分缺失ＦｅＭｏｃｏ 和Ｐ－ｃｌｕｓｔｅｒ的失活蛋白。与由Ｋ２ＣｒＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,处理蛋白对乙炔和质子还原的活性都得以显著恢复;然而,它的吸收光谱和圆二色谱虽有明显恢复,但仍与还原钼铁蛋白有所不同。这表明,激活蛋白中也许存在功能与钼铁蛋白相似,而结构则有所差异的含铬（ＣｒＦｅ）蛋白     

Construction of a high-density genetic map of <Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. using genotyping by sequencing technology

The Chinese jujube (Ziziphus jujuba Mill., 2n = 2 × = 24), one of the most popular fruit trees in China, is widely cultivated and utilized in Asia. High-density genetic linkage maps are valuable resources for molecular breeding and functional genomics; however, they are still under-developed for the jujube. The genotyping by sequencing (GBS) strategy could be an efficient and cost-effective tool for single nucleotide polymorphism (SNP) discovery based on the sequenced jujube genome. Here, we report a new high-density genetic map constructed using GBS technology. An F1 population with 145 progenies and their parents (‘Dongzao’ × ‘Zhongningyuanzao’) were sequenced on the Illumina HiSeq 4000 platform. In total, 79.8 Gb of raw data containing 256,708,177 paired-end reads were generated. After data filtering and SNP genotyping, 40,372 polymorphic SNP markers were developed between the parents and 2540 (1756 non-redundant) markers were mapped onto the integrated genetic linkage map. The map spanned 1456.53 cM and was distributed among 12 linkage groups, which is consistent with the haploid chromosome number of the jujube. The average marker interval was 0.88 cM. The genetic map allowed us to anchor 224 Mb (63.7 %) of scaffolds from the sequenced ‘Junzao’ genome, containing 52 newly anchored scaffolds, which extended the genome assembly by 7 Mb. In conclusion, GBS technology was applied efficiently for SNP discovery in this study. The high-density genetic map will serve as a unique tool for molecular-assisted breeding and genomic studies, which will contribute to further research and improvement of the jujube in the near future.     

宁夏枸杞‘中科绿川1号’果实和叶片的主要成分分析

以国家林业局审定的宁夏枸杞新品种‘中科绿川1号’为实验材料,研究不同月份采集的枸杞叶片芦丁含量,以及果实总黄酮、总多糖、总类胡萝卜素、玉米黄素双棕榈酸酯的含量变化。结果显示,叶片芦丁含量在枸杞盛果期较低,抽条期较高;果实总黄酮、总类胡萝卜素、玉米黄素双棕榈酸酯的含量在头茬果中含量较高,盛果期果实含量较低;果实总多糖可能与果实量呈负相关;褐化果(俗称"油果")总黄酮含量与正常果差异不大,可用作提取枸杞黄酮类物质的原材料。研究结果可为枸杞果实分级、成分提取提供新思路。     

A series of novel directional cloning and expression vectors for blunt-end ligation of PCR products

Novel directional cloning and expression vectors were developed for blunt-end ligation of PCR products that are suitable for high-throughput cloning and simplifying the screening procedure. The PCR products, without further processing, are cloned into vectors digested with SchI and, following transformation, the desired recombinants give typical blue colonies on selectable plates. The principle of this selection strategy is that the construction also generates a full-length ideal lacO gene. To the best of our knowledge, this is the first time that this lacO reconstruction strategy has been applied in the selection of recombinants.     

Analysis of variable sites between two complete South China tiger (Panthera tigris amoyensis) mitochondrial genomes

In order to investigate the mitochondrial genome of Panthera tigris amoyensis, two South China tigers (P25 and P27) were analyzed following 15 cymt-specific primer sets. The entire mtDNA sequence was found to be 16,957 bp and 17,001 bp long for P25 and P27 respectively, and this difference in length between P25 and P27 occurred in the number of tandem repeats in the RS-3 segment of the control region. The structural characteristics of complete P. t. amoyensis mitochondrial genomes were also highly similar to those of P. uncia. Additionally, the rate of point mutation was only 0.3% and a total of 59 variable sites between P25 and P27 were found. Out of the 59 variable sites, 6 were located in 6 different tRNA genes, 6 in the 2 rRNA genes, 7 in non-coding regions (one located between tRNA-Asn and tRNA-Tyr and six in the D-loop), and 40 in 10 protein-coding genes. COI held the largest amount of variable sites (9 sites) and Cytb contained the highest variable rate (0.7%) in the complete sequences. Moreover, out of the 40 variable sites located in 10 protein-coding genes, 12 sites were nonsynonymous.     

应急大鼠肾上腺髓质嗜铬细胞颗粒数目与其钙含量的关系

采用电镜细胞立体形态计量法及电镜Ｘ射线显微定量分析术,对制动应急大鼠的肾上腺髓质细胞内嗜铬颗粒数密度和颗粒内Ｃａ浓度变化进行测量。结果显示,两者在制动过程中均呈进行性下降,但颗粒内钙浓度的下降快于颗粒数目的减少（Ｐ〈０．０１）。该结果支持颗粒内钙释放入胞质,参与胞质内游离钙浓度的升高,进而激发颗粒胞吐的假设,为主宰嗜铬颗粒是一种细胞内钙库并参与细胞分泌提供了实验依据。     

人用狂犬病疫苗过敏性反应探讨

采用超滤浓缩技术生产人用狂犬病疫苗后,疫苗效力得到明显提高;胆在使用中,包括过敏反应等在内的不良反应等有所增多且严重程度明显增大。以豚鼠为实验动物模型,探讨人用狂犬病疫苗引起过敏反应的原因。结果显示,地鼠民分是引起过敏的重要物质,过敏的严重程度随制品浓缩倍数的增大而增大。     

水稻白叶枯病广谱抗性基因Xa21导入两用不育系培矮64S

以克隆的Xa21基因为外源基因,成熟胚愈伤组织为转化受体,应用农杆菌介导法对水稻两用型核不育系培矮64S进行转化,获46株转基因植株。PCR和Southern分析结果表明,Xa21已整合到受体基因组。用稻白叶枯病病原菌(Xanthomonasoryzaepv.oryzae)菲律宾小种6号接种鉴定,结果表明大多数转基因植株获得了抗病性。已整合的Xa21基因能够稳定地遗传,在所检测转基因株系的T1代中,Xa21基因显示3:1的分离。     

人骨保护素(OPG)重组腺病毒的制备及其生物活性研究

采用RT-PCR法得到人OPG的编码区cDNA,克隆至穿梭质粒pShuttle,构建重组有OPG编码区cDNA的腺病毒DNA,经Pac I 酶切线性化,在脂质体介导下转染HEK293细胞,制备重组腺病毒并测定病毒滴度约为5×10⁶～1.5×10⁷ pfu/mL。体外感染小鼠成肌细胞C2C12,Western blot及ELISA检测证实有OPG蛋白的表达,并可在细胞培养上清中持续表达6周。感染OPG重组腺病毒的C2C12细胞生长状态良好、细胞周期无明显变化。将重组腺病毒加入体外培养的小鼠骨髓细胞的培养基中,诱导形成的破骨细胞数量及在象牙片上形成的吸收陷窝的数量显著减少（P<0.01）。      

Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris

Tannase (tannin acyl hydrolase) is an industrially important enzyme produced by a large number of fungi, which hydrolyzes the ester and depside bonds of gallotannins and gallic acid esters. In the present work, a tannase from Aspergillus oryzae has been cloned and expressed in Pichia pastoris. The catalytic activity of the recombinant enzyme was assayed. A secretory form of enzyme was made with the aid of Saccharomyces cerevisiae alpha-factor, and a simple procedure purification protocol yielded tannase in pure form. The productivity of secreted tannase achieved 7000 IU/L by fed-batch culture. Recombinant tannase had a molecular mass of 90 kDa, which consisted of two kinds of subunits linked by a disulfide bond(s). Our study is the first report on the heterologous expression of tannase suggesting that the P. pastoris system represents an attractive means of generating large quantities of tannase for both research and industrial purpose.