冻结速率对血小板冷冻干燥保存的影响

冷冻干燥法是使血小板能够长期保存的一种理想方法。冻结过程对血小板的冻干保存至关重要。采用梯度降温、搁板预冷、液氮冻结等三种冻结方式,研究了冻结速率对血小板冷冻干燥保存恢复率的影响。实验结果表明用搁板预冷的方式冻结并干燥的血小板复水后的恢复率最高,达到(93.0.2)%,此时的冻结速度约为10℃/min。扫描电镜照片显示冻干复水后的血小板保持了完整的细胞结构,但与新鲜血小板相比略呈球形。冻干复水后的血小板对1U/ml凝血酶的最大聚集率接近于新鲜血小板,但聚集速度比新鲜血小板慢。     

羊栖菜多糖通过激活Caspase途径诱导Lovo细胞凋亡

研究了羊栖菜多糖（Sargassum Fusiforme Polysaccharides,SFPS）诱导人大肠癌lovo细胞凋亡及凋亡过程中caspase-3、caspase-8、caspase-9的活性变化。MTT法检测SFPS对lovo细胞增殖的抑制率;通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡;应用Western印迹法测定caspase-3酶原和caspase-9的变化;RToPCR检测caspase-3 mRNA表达;caspase-3,caspase-8、caspase-9活性检测试剂盒观察caspase-3、caspase-8、caspase-9的活性改变。结果显示,SFPS对lovo细胞增殖有显著抑制作用,经形态变化、DNA条带和流式细胞分析,可见明显的细胞凋亡特征。SFPS处理lovo细胞后,发现caspase-3酶原蛋白表达降低,caspase-3 mRNA高表达,并具有剂量和时间的依赖性。而在检测蛋白中,也发现caspase-9被激活进而形成具有活性的片段。另外,caspase的活性检测也进一步发现caspase-3、caspase-9的活性逐步增高。实验结果提示SFPS在体外诱导lovo胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一,并且是通过激活启动caspase-9,进而激活下游效应caspase-3的级联反应来实现的。     

李叶绣线菊叶片和叶柄再生体系的建立

1植物名称李叶绣线菊（Spiraea prunifolia Sieb．et Zucc．）。 2材料类别带叶柄的幼嫩叶片。     

Cytokinin affects circadian-clock oscillation in a phytochrome B- and Arabidopsis response regulator 4-dependent manner

In higher plants, many developmental processes, such as photomorphogenesis and flowering, are coregulated by light and the phytohormone cytokinin. Interactions between light and cytokinin pathways are presumably mediated by common signaling intermediates. However, the molecular mechanism of these interactions remains unclear. Here, we report that cytokinin specifically induces the expression of the Arabidopsis circadian oscillator genes LATE ELONGATED HYPOCOTYL ( LHY ) and CIRCADIAN CLOCK-ASSOCIATED 1 ( CCA1 ) but represses the expression of TIMING OF CAB EXPRESSION 1 in a light-dependent manner. Consistent with these observations, cytokinin causes a shifted phase of the circadian clock. Mutant studies showed that the altered clock oscillation modulated by cytokinin is dependent on phytochrome B ( PHYB ) and Arabidopsis RESPONSE REGULATOR 4 ( ARR4 ). Whereas overexpression of LHY or CCA1 renders plants slightly more sensitive to cytokinin, phyB and a lhy/cca1 double mutant are less sensitive to the hormone. These results suggest that cytokinin affects the circadian clock oscillation in a PHYB - and ARR4 -dependent manner and that cytokinin signaling is also regulated by light-signaling components, including PHYB , LHY and CCA1 . Therefore, phyB, ARR4 and the circadian oscillator may function as signaling intermediates to integrate light and cytokinin pathways.     

Molecular evolution of adomet synthetase by DNA recombination with a novel Separate-Mixing method

We describe a new approach to in vitro DNA recombination termed the Separate-Mixing method in this study. The reaction process of this method consists of two stages: at the first stage the reaction was implemented in two parallel teams, which generated random recombination by template-switching of growing poly-nucleotides from primers in the presence of unidirectional single-stranded DNA fragments used as templates, and then both teams were mixed together for further extension and recombination of DNA sequences at the second stage. Due to this particular strategy, the reaction process was also accompanied by two other processes of DNA shuffling and StEP simultaneously. Two AdoMet synthetase genes, sam2 from Saccharomyces cerevisiae and metK from Escherichia coli, which have only 56% homology on the DNA level, were used for recombination with the Separate-Mixing method. DNA recombination was available after a single round of reaction. When 10 randomly selected recombinants were sequenced, an unshuffled parental clone was not found, nor was unexpected insertion, deletion, or rearrangement detected. An evolved gene, sam’, was obtained after screening and selection, which could obviously increase the accumulation of AdoMet in S. cerevisiae. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 3, pp. 546–553. This article was submitted by the authors in English.     

Polymorphism of SARS-CoV Genomes

In this work, severe acute respiratory syndrome associated coronavirus (SARS-CoV) genome BJ202 (AY864806) was completely sequenced. The genome was directly accessed from the stool sample of a patient in Beijing. Comparative genomics methods were used to analyze the sequence variations of 116 SARS-CoV genomes (including BJ202) available in the NCBI Gen-Bank. With the genome sequence of GZ02 as the reference, there were 41 polymorphic sites identified in BJ202 and a total of 278 polymorphic sites present in at least two of the 116 genomes. The distribution of the polymorphic sites was biased over the whole genome. Nearly half of the variations (50.4%, 140/278) clustered in the one third of the whole genome at the 3′ end (19.0 kb-29.7 kb). Regions encoding Orf10-11, Orf3/4, E, M and S protein had the highest mutation rates. A total of 15 PCR products (about 6.0 kb of the genome) including 11 fragments containing 12 known polymorphic sites and 4 fragments without identified polymorphic sites were cloned and sequenced. Results showed that 3 unique polymorphic sites of BJ202 (positions 13 804, 15 031 and 20 792) along with 3 other polymorphic sites (26 428, 26 477 and 27 243) all contained 2 kinds of nucleotides. It is interesting to find that position 18379 which has not been identified to be polymorphic in any of the other 115 published SARS-CoV genomes is actually a polymorphic site. The nucleotide composition of this site is A (8) to G (6). Among 116 SARS-CoV genomes, 18 types of deletions and 2 insertions were identified. Most of them were related to a 300 bp region (27 700-28 000) which encodes parts of the putative ORF9 and ORF10-11. A phylogenetic tree illustrating the divergence of whole BJ202 genome from 115 other completely sequenced SARS-CoVs was also constructed. BJ202 was phylogeneticly closer to BJ01 and LLJ-2004.     

白花花楸的组织培养和快速繁殖

1植物名称 白花花楸[Sorbusaria（L．）Crantz．]。 2材料类别当年生种子。     

大叶黑桫椤孢子超低温保存

大叶黑桫椤孢子在液氮中长期保存的结果表明：（1）大叶黑桫椤孢子可以保存于液氮中;（2）液氮保存后的孢子萌发率比未用液氮保存的普遍下降,保存90d的用室温慢速化冻的孢子萌发率最高（65.3％）,相对保持率最高可达79.3％;（3）采用室温（22～25℃）慢速化冻的效果优于37℃温水浴快速化冻的。据此认为,液氮超低温长期保存大叶黑桫椤孢子是可行的。     

水稻化感品种对土壤微生物的影响

通过盆栽实验研究三叶期水稻化感品种对土壤微生物种群数量的影响。结果显示,土壤细菌、放线菌、真菌、氨化细菌和好气性自生固氮菌数量在水稻化感品种P1312777和水稻非化感品种“辽粳九”、“秋光”的土壤中存在显著差异,土壤中大多数微生物被水稻化感品种所抑制,但化感品种土壤中的放线菌和好气性纤维素分解菌数量则介于两种非化感品种之间。这一研究表明,水稻化感品种能显著地影响土壤微生物种群数量。     

不同农业土地利用方式和管理对土壤有机碳的影响 ——以北京市延庆盆地为例

自20世纪80年代以来,我国农业土地利用方式和农田管理发生了巨大变化,由此引起的土壤有机碳（SOC）含量、密度及其垂直分布发生了相应的变化,研究不同土地利用方式和管理对土壤有机碳的影响对于探讨农田生态系统的固碳作用具有重要的意义.以北京市延怀盆地为典型研究地区,选择6种农业土地利用和管理模式,共计42块样地,在1m深土体内分层采集197个土壤样品.研究结果表明：（1） 不同农业土地利用和管理方式对SOC含量的影响主要发生在0～25 cm土层中,剖面中SOC含量自上向下明显降低.（2） 通过对6种土地利用和管理方式下土壤SOC含量进行比较,结果发现果园和高投入的玉米地土壤在0～100 cm土层中SOC含量均较高,变化范围分别为4.16～10.00 g kg^-1和4.73～9.31 g kg^-1; 菜地土壤在0～40 cm土层中SOC含量较高,变化范围为6.42～9.67 g kg^-1;大豆地、中、低投入玉米地土壤在0～100 cm土层中SOC含量较低,变化范围分别为3.27～7.73 g kg^-1、3.14～8.33 g kg^-1和1.83～7.67 g kg^-1. （3） 不同农业土地利用方式对SOC密度影响的趋势与对SOC含量影响的趋势基本一致,在0～100 cm土壤中,SOC密度的顺序为果园＞菜地＞高投入玉米地＞中投入玉米地＞大豆地＞低投入玉米地,变化范围为4.15～8.22 kg m^-2.