携带红色荧光蛋白报告基因的重组腺病毒载体的构建

目的采用体外连接技术构建含红色荧光蛋白(RFP)报告基因的重组腺病毒载体,为基因治疗与基因疫苗研究提供重要工具。方法将pTurbo RFP-N上的含红色荧光蛋白基因片段,经酶切连接定向克隆至转移载体pShuttle上,构成重组质粒pShuttle-TurboRFP-N。用I-CeuI/PI-SceI双酶切重组质粒pShuttle-TurboRFP-N和骨架质粒pH5′040 pkGFP-II,回收目的片段,连接,获得重组腺病毒载体pH5.′040.CMV.RFP-N。将其线性化后,经脂质体介导转染至AD293细胞内包装出重组腺病毒颗粒。通过荧光显微镜观察其在AD293细胞内的包装效率和RFP表达情况。扩增并再次感染AD293细胞检测病毒颗粒感染能力,并测定其生物活性及滴度。结果经酶切鉴定,证明已正确构建重组腺病毒载体AdH5.′040.CMV.RFP-N;经AD293细胞包装成具有感染性的病毒颗粒,并能在真核细胞中高效表达目的基因;扩增纯化后获得重组腺病毒颗粒数为3.6×1012vp/mL,滴度达1×1010pfu/mL。结论成功构建了携带红色荧光蛋白报告基因的重组腺病毒载体,并能够在AD293细胞中高效地表达,为基因治疗与基因疫苗研究提供重要工具。     

Non-destructive assessment of flesh firmness and dietary antioxidants of greenhouse-grown tomato (Solanum lycopersicum L.) at different fruit maturity stages

Non-destructive methods have been widely recognized for evaluating fruit quality traits of many horticultural crops and food processing industry. Destructive (analytical) test, and non-destructive evaluation of the quality traits were investigated and compared for ‘Red Rose’ tomato (Solanum lycopersicum L.) fruit grown under protected environment. Fresh tomato fruit at five distinctive maturity stages namely; breaker (BK), turning (TG), pink (PK), light-red (LR), and red (RD) were labeled and scanned using the handheld near infra-red (NIR) enhanced spectrometer at a wavelength range of 285–1200 nm. The labeled tomato samples were then measured analytically for flesh firmness, lycopene, β-carotene, total phenolic content (TPC) and total flavonoids content (TFC). The results revealed that quality traits could be estimated using NIR spectroscopy with a relatively high coefficient of determination (R²): 0.834 for total phenolic content, 0.864 for lycopene, 0.790 for total flavonoid content, 0.708 for β-carotene; and 0.679 for flesh firmness. The accumulation of Lyco and β-Car rapidly increased in tomatoes harvested between the TG and the LR maturity stages. Harvesting tomatoes at BK maturity stage resulted in significantly higher flesh firmness than harvesting at the later maturity stages. Tomato fruits had the lowest TPC and TFC contents at the earliest maturity stage (BK), while they had intermediate TPC and TFC levels at LR and RD maturity stages. NIR spectroscopic measurements of fruit firmness and lipophilic antioxidants in tomato fruit at various maturity stages were partially in accordance with those estimated by destructive (analytical) methods. Based on these findings, we recommend using non-destructive NIR spectroscopy as an effective tool for predicting tomato fruit quality during harvest stage and postharvest processing.     

适用于种群遗传管理的小熊猫微卫星亲子鉴定体系的建立

由于标记缺失、生产记录不详、亲权关系不明及部分个体来源不清等历史问题,中国小熊猫圈养种群存在谱系错漏、近亲繁殖等风险。近年来,随着小熊猫种群规模不断扩大,管理者们对谱系的准确性提出了更高的需求,亲子鉴定工作也成为了研究的重点。本文采用26个微卫星标记,对国内3个小熊猫圈养种群进行了亲缘关系运算,完成了相关谱系的查错与整理。26个位点多态性与稳定性良好,联合非亲排除概率达到0.9999以上,可解决国内小熊猫圈养种群的各类亲子鉴定需求。在单亲未知或双亲未知的情况下,8或11个位点组合可实现亲子鉴定。5个位点组合可进行个体识别。在小熊猫圈养管理过程中,应用一套亲子鉴定体系对小熊猫圈养的谱系进行查漏补缺,有利于制定科学的配对计划、避免近亲繁殖,对小熊猫种群保护有着重要意义。     

乳链菌肽细胞分子传感器的构建与应用

【背景】乳链菌肽主要是由乳酸乳球菌生产的一类多肽,对革兰氏阳性菌有抑菌作用,是目前联合国粮食及农业组织/世界卫生组织唯一批准使用的天然食品防腐剂。但是其产量低、缺乏简便高效的检测方法,限制了其研究和应用。【目的】构建一种可输出肉眼可见红色荧光的细胞分子传感器,以期能简单方便地检测样品中的乳链菌肽,同时应用该传感器筛选乳链菌肽生产菌株。【方法】用Golden-Gate克隆方法构建含乳链菌肽诱导启动子和下游红色荧光蛋白基因(两种)的载体,转入Lactococcus lactis中。用细胞传感器筛选可能的乳链菌肽生产菌株。【结果】构建的两种乳链菌肽细胞分子传感器都能对2?200 ng/mL乳链菌肽有灵敏的响应,可用于定量测定。两种传感器的最大荧光强度和表型也有所不同。利用细胞传感器确定了Lactococcus lactis ATCC 11454乳链菌肽的产生,同时排除了一个能产其他抗菌化合物的菌株。【结论】构建的细胞分子传感器能特异性地响应乳链菌肽,并能简单快速地筛选乳链菌肽菌株。     

Biotic and abiotic factors driving the diversification dynamics of Crocodylia

Species diversity patterns are governed by complex interactions among biotic and abiotic factors over time and space, but are essentially the result of the diversification dynamics (differential speciation and extinction rates) over the long-term evolutionary history of a clade. Previous studies have suggested that temporal variation in global temperature drove long-term diversity changes in Crocodylia, a monophyletic group of large ectothermic organisms. We use a large database of crocodylian fossil occurrences (192 spp.) and body mass estimations, under a taxic approach, to characterize the global diversification dynamics of crocodylians since the Cretaceous, and their correlation with multiple biotic and abiotic factors in a Bayesian framework. The diversification dynamic of crocodylians, which appears to have originated in the Turonian (c. 92.5 Ma), is characterized by several phases with high extinction and speciation rates within a predominantly low long-term mean rate. Our results reveal long-term diversification dynamics of Crocodylia to be a highly complex process driven by a combination of biotic and abiotic factors which influenced the speciation and extinction rates in dissimilar ways. Higher crocodylian extinction rates are related to low body mass disparity, indicating selective extinctions of taxa at both ends of the body mass spectrum. Speciation rate slowdowns are noted when the diversity of the clade is high and the warm temperate climatic belt is reduced. Our finding supports the idea that temporal variations of body mass disparity, self-diversity, and the warm climate belt size provided more direct mechanistic explanations for crocodylian diversification than do proxies of global temperature.     

Genome-Wide Association Analysis Identifies a Genetic Basis of Infectivity in a Model Bacterial Pathogen

Knowledge of the genetic architecture of pathogen infectivity and host resistance is essential for a mechanistic understanding of coevolutionary processes, yet the genetic basis of these interacting traits remains unknown for most host–pathogen systems. We used a comparative genomic approach to explore the genetic basis of infectivity in Pasteuria ramosa, a Gram-positive bacterial pathogen of planktonic crustaceans that has been established as a model for studies of Red Queen host–pathogen coevolution. We sequenced the genomes of a geographically, phenotypically, and genetically diverse collection of P. ramosa strains and performed a genome-wide association study to identify genetic correlates of infection phenotype. We found multiple polymorphisms within a single gene, Pcl7, that correlate perfectly with one common and widespread infection phenotype. We then confirmed this perfect association via Sanger sequencing in a large and diverse sample set of P. ramosa clones. Pcl7 codes for a collagen-like protein, a class of adhesion proteins known or suspected to be involved in the infection mechanisms of a number of important bacterial pathogens. Consistent with expectations under Red Queen coevolution, sequence variation of Pcl7 shows evidence of balancing selection, including extraordinarily high diversity and absence of geographic structure. Based on structural homology with a collagen-like protein of Bacillus anthracis, we propose a hypothesis for the structure of Pcl7 and the physical location of the phenotype-associated polymorphisms. Our results offer strong evidence for a gene governing infectivity and provide a molecular basis for further study of Red Queen dynamics in this model host–pathogen system.     

Rheological study on coagulation of blood with special reference to the triggering mechanism of venous thrombus formation

A markedly reduced blood flow, an elevation of hematocrit and an increased aggregability of erythrocytes [red blood cells (RBCs)] are risk factors for venous thrombus formation (intravascular blood coagulation). However, these risk factors alone seem to be insufficient to stimulate the coagulation cascade in the absence of a primary triggering mechanism. In this paper, our rheological and biochemical studies on blood coagulation, especially focusing on procoagulant activity of RBCs, are summarized. It is shown that the intrinsic coagulation pathway is triggered by the activation of factor IX (F-IX) by RBCs. The F-IX-activating enzyme in normal human erythrocyte (RBC) membranes was purified, identified and characterized. The activation of F-IX by RBCs was enhanced by a decrease in flow shear rate and an elevation in hematocrit. The procoagulant ability of RBCs and coagulation of blood obtained from individuals with a relatively high level of hypercoagulability were enhanced compared with those for normals. The studies demonstrated a new triggering mechanism for coagulation or thrombus formation that may occur under stagnant flow conditions.     

Bioactivity of the compounds in genus Dysoxylum

The genus Dysoxylum is a large genus comprising of about a dozen species, distributed in India. This is the genus which is rich in compounds like terpenoids and alkaloids. The isolated compounds from the genus were reported to have interesting biological activities. This review contains the chemical structures, along with their biological activities reported in the literature.