Red重组系统用于大肠杆菌基因修饰的研究进展

Red重组作为一种新的重组系统已经被广泛用于大肠杆菌的基因敲除、基因替换。与传统的Rec重组相比,Red重组具有同源臂短,重组效率高等优点。本文分别详细介绍了Red重组系统中Exo、Beta、Gam三种蛋白质的功能,Red重组系统运用在大肠杆菌基因敲除中的三种质粒及其功能,同时概括了Red重组的技术要点及技术难点,分析了文献报道的阿拉伯糖诱导浓度和诱导时间、转化后的复苏温度及时间、引物同源臂长度对于重组率的影响,总结出了Red重组的最佳条件。     

重组痘苗病毒在基因工程中的应用及展望

1982年以来,美国Moss及Panletti实验室相继报道了以痘苗病毒为载体,把单纯疱疹病毒TK基因、乙肝病毒表面抗原基因,流感病毒血凝素基因等重组至痘苗病毒基因组的特定部位,获得能表达外源基因的重组痘苗病毒。由于重组痘苗病毒具有一些特殊的特点和优点。     

Red重组系统用于大肠杆菌基因修饰研究进展

Red重组作为一种新的重组系统已经被广泛用于大肠杆菌的基因敲除、基因替换.与传统的Rec重组相比,Red重组具有同源臂短,重组效率高等优点.分别详细介绍了Red重组系统中Exo、Beta、Gam 3种蛋白质的功能,Red重组系统运用于大肠杆菌基因敲除的3种质粒及其功能,同时概括了Red重组的技术要点及技术难点,分析了文献报道的阿拉伯糖诱导浓度和诱导时间、转化后的复苏温度及时间、引物同源臂长度对于重组率的影响,总结出了Red重组的最佳条件.     

改造的HPV16型E7基因重组穿梭质粒的构建与鉴定

分别以卡介苗(BCG)基因组DNA及宫颈癌组织提取DNA为模板,通过PCR扩增得到19kD抗原的胞壁区及其上游调控元件(19ss)基因序列和HPV16型E7基因序列。先将19ss基因与大肠杆菌-分枝杆菌穿梭质粒pMV261重组,得到重组质粒pMCW。再将E7基因克隆至pMCW,得到重组质粒pMCW-E7。最后用PCR引物定点诱变法突变E7基因与转化有关的位点,得到重组质粒pMCW-mE7。对重组质粒pMCW-mE7用PCR扩增、双酶切及测序鉴定证实,19ss基因及突变的E7基因正确插入穿梭质粒pMV261。成功构建了重组穿梭质粒pMCW-mE7。     

利用细菌人工染色体同源重组对小鼠基因进行条件型敲除

目的:探索通过细菌人工染色体(BAC)同源重组系统构建条件基因敲除载体的高效率方法,提高条件基因敲除小鼠(Flox小鼠)的构建效率。方法:利用作者自己构建的噬菌体重组酶系统,通过BAC同源重组进行条件型基因敲除载体构建工作。首先通过亚克隆构建了一系列载体含有同源臂的靶向质粒,线性化后,打靶片段经电穿孔法转入大肠杆菌内,与相应的BAC同源重组,再经过三步同源重组和一步位点特异性重组,构建小鼠条件型基因敲除载体。结果:高效率构建了小鼠基因的最终条件基因敲除载体。结论:通过BAC同源重组高效构建条件基因敲除载体,为条件基因敲除载体的构建提供了全新思路,并为FLox小鼠的建立,及相应基因在发育、生理、致病机制等方面的功能研究奠定了基础。     

格特隐球菌核基因、交配型位点和线粒体基因的多位点序列分型及重组分析

研究格特隐球菌VGI和VGII基因型菌株间线粒体基因重组与核基因、交配型位点重组间的关系。采用分子鉴定区分受试格特隐球菌的血清型、基因型和交配型;选取包含核基因和线粒体基因10个位点的多位点序列分型（ MLST）进行基因型和系统发育分析;采用同质性检验评价基因系谱间的一致性。多位点的序列和系统发育分析结合同质性检验表明,受试位点中,仅ATP6位点发现VGI和VGII菌株间基因的杂交和重组,该基因系谱与其余位点的基因系谱呈现不一致性。结果显示,受试的部分VGI菌株中的ATP6位点含有VGII菌株的基因序列,表明VGI和VGII菌株间线粒体基因的重组;没有发现核基因及交配型位点中两者间的重组,提示VGI和VGII菌株间线粒体基因的重组现象与交配行为无关。     

Red同源重组在大肠杆菌基因组修饰中的应用

Red同源重组技术发展迅速,已广泛应用于大肠杆菌基因组修饰,在点突变、基因敲除、序列整合等方面发挥着重要作用。简要综述了Red同源重组的重组机制和操作策略等研究进展,并介绍了Red同源重组在大肠杆菌基因组减小及多基因代谢途径优化方面的应用情况。     

细菌重组系统及其应用研究进展

利用重组酶和辅助蛋白共同作用于DNA片段上,使不同基因重新组合以完成基因重组的现象在细菌中广泛存在,基因重组对于细菌的遗传多样性、进化等具有重要意义。目前,细菌基因重组主要分为同源重组、位点特异性重组和转座重组3种类型。本文主要对细菌重组系统重组酶的种类、作用机制及其在细菌遗传操作中的应用策略进行阐述。     

重组病毒杀虫剂应用研究进展

应用分子生物学技术可以将昆虫特异性的毒素基因、某些酶基因等外源基因插入昆虫病毒基因组,或通过改造昆虫病毒基因组等方法构建重组病毒杀虫剂,提高杀虫效果。温室及田间释放实验证实,重组病毒杀虫剂可以显著提高现场防治效果。连续多代抗性筛选实验表明,宿主被诱导产生对重组病毒杀虫剂抗性的速度低于野生型病毒杀虫剂。采用在剂型中添加光增白剂等保护剂、在基因组中插入具有增效作用的基因、应用病毒增强蛋白等技术可以提高重组病毒杀虫效果。随着基因工程技术的发展和安全性研究的深入,以重组杆状病毒为主的重组昆虫病毒杀虫剂的应用研究正面临着突破。     

博尔纳病病毒pEGFP-p24基因重组质粒的构建及鉴定

构建博尔纳病病毒pEGFP-p24基因重组表达质粒。通过PCR方法扩增获得博尔纳病痛毒p24基因的完整序列,将此片段定向克隆到pEGFP-N1载体多克隆位点区,筛选重组阳性菌株,提取重组质粒,利用PCR方法和核酸序列测定验证重组质粒构建的正确性。PCR及核酸序列测定证明博尔纳病病毒pEGFP-p24基因重组表达质粒构建成功。构建的重组质粒将为研究博尔纳病病毒p24基因在真核细胞中的功能和作用提供实验依据。     

Seasonal patterns in feeding ecology of three small fishes in the Biandantang Lake, China

Feeding ecology of three small fish species, Hypseleotris swinhonis, Ctenogobius giurinus and Pseudorasbora parva was studied seasonally in the Biandantang Lake, a small, shallow lake in central China. Gut length, adjusted for total body length, was significantly higher in spring than in other seasons for all the three species. Seasonal changes in gut length were not associated with changes in food quality. Weight of fore-gut contents, adjusted for body weight, was significantly higher in winter and spring than in summer and autumn in H. swinhonis and C. giurinus , and significantly higher in autumn than in spring and summer for P. parva. Percentage of empty fore-guts was highest in summer and lowest in spring for H. swinhonis and C. giurinus , and highest in winter and lowest in autumn for P. parva. Diet of the three small fishes showed apparent seasonal changes, and these changes reflected partly the seasonal fluctuations of food resources in environment. Diet breadth was high in winter and low in autumn for H. swinhonis , high in winter and low in spring and summer for C. giurinus , and high in autumn and low in spring for P. parva. Diet overlaps between pairs of species were biologically significant in most cases, except between H. swinhonis and P. parva in summer and autumn and between C. giurinus and P. parva in autumn.     

安蕨属的分类与分布

1849年,捷克人Presl根据菲律宾产的一种蕨类植物Anisocampium cumingia-num Presl,建立了安蕨属Anisocapium Presl,几十年来未被各国植物学家(如Baker、Beddom、Makino、C.Christensen、Christ、Nakai、秦仁昌、Devol、Ohwi和Tagawa)所承认。1940年,Tard.-Blot et C.Chr.编写《印度支那植物通志》(Fl.Gen.Indo-Chine)时接受了安蕨属的概念。1947年,美国人Copeland研究东南亚蕨类植物时,也肯定了安蕨属是一个自然分类群。以后,     

啮齿动物作用下退耕地山杏种子扩散与贮藏的季节变化

啮齿动物对植物种子的取食和扩散影响种子的时空分布,继而影响种子的萌发和幼苗建成,因而在森林更新中起着重要作用.在国有济源市愚公林场,选择退耕地生境,于春季、夏季、秋季分别释放人工标记的山杏种子,观察啮齿动物扩散与埋藏山杏种子的季节性差异.结果表明：1）退耕地中的啮齿动物主要包括大林姬鼠、社鼠、黑线姬鼠;2）山杏种子扩散速率在春季显著慢于夏季,夏季显著慢于秋季;3）种子搬运量受季节和种子状态交互作用影响,春季显著少于夏季,夏季显著少于秋季;4）不同季节种子平均搬运距离不同,秋季不同状态种子的搬运距离均大于春季和夏季;5）啮齿动物对山杏种子的贮藏点大小多为1粒种子,少量为2、3粒种子,且贮藏点大小与季节间存在显著的交互作用,春季单粒种子的贮藏点数量显著少于夏季和秋季,而夏季与秋季的贮藏点则倾向于多粒种子;6）在夏季和秋季各有5枚（共释放1800枚）被啮齿动物分散贮藏的山杏种子建成幼苗.     

Species and individual differences in juvenile female alloparental care are associated with oxytocin receptor density in the striatum and the lateral septum

The neuropeptide oxytocin has been implicated in the regulation of affiliative behavior and maternal responsiveness in several mammalian species. Rodent species vary considerably in the expression of juvenile alloparental behavior. For example, alloparental behavior is spontaneous in juvenile female prairie voles (approximately 20 days of age), takes 1-3 days of pup exposure to develop in juvenile rats, and is nearly absent in juvenile mice and meadow voles. Here, we tested the hypothesis that species differences in pup responsiveness in juvenile rodents are associated with oxytocin receptor (OTR) density in specific brain regions. We found that OTR density in the nucleus accumbens (NA) is highest in juvenile prairie voles, intermediate in juvenile rats, and lowest in juvenile mice and meadow voles. In the caudate putamen (CP), OTR binding was highest in prairie voles, intermediate in rats and meadow voles, and lowest in mice. In contrast, the lateral septum (LS) shows an opposite pattern, with OTR binding being high in mice and meadow voles and low in prairie voles and rats. Thus, alloparental responsiveness in juvenile rodents is positively correlated with OTR density in the NA and CP and negatively correlated with OTR density in the LS. We then investigated whether a similar receptor-behavior relationship exists among juvenile female prairie voles by correlating individual variation in alloparental behavior with variation in OTR density. The time spent adopting crouching postures, the most distinctive component of alloparental behavior in juveniles, was positively correlated with OTR density in the NA (r = 0.47) and CP (r = 0.45) and negatively correlated with OTR density in the lateral septum (r = -0.53). Thus, variation in OTR density in the NA, CP, and LS may underlie both species and individual differences in alloparental care in rodents.     

世界栽培水杉的调查(1947～2007年)

自第一批种子于1947年12月底走出中国国门,著名‘活化石’水杉已在世界上栽培了60年。过去的5年间通过各种调查方式获得了6大洲近50个国家的2535份资料。调查结果显示,水杉除在欧美及东亚等国家栽培非常普遍外,阿根廷、澳大利亚、智利、印度、墨西哥、尼泊尔、新西兰、南非、泰国、土耳其、津巴布韦等国也有成功的栽培。生长最好的是北美植物耐寒带5-9及欧洲和东亚相似气候地区的栽培水杉,植株最高已达38m,最大胸围已达6.2m。调查内容包括引种的时间、栽培地点、株数、高度及胸围,调查单位不仅包括公立的单位,还包括不对外开放的私人住宅。另外,还讨论了栽培用途、限制因子以及原生种群的资源保护等。     

Development of microsatellite markers in the Australasian snake-necked turtle Chelodina rugusa and cross-species amplification

Seventeen microsatellite loci were developed for the snake-necked turtle, Chelodina rugosa (Ogilby, 1890). Sixteen of the loci were polymorphic but three of these loci had null alleles. One locus displayed linkage disequilibrium. These 17 markers were tested for amplification in eight congeneric species with varying success; 98% amplification in Chelodina burrungandjii, 72% in C. canni, 38% in C. expansa, 58% in C. longicollis, 67% in C. mccordi, 73% in C. oblonga, 81% in C. parkeri, and 68% in C. pritchardi. These microsatellite markers will be useful for population assignment, gene flow, mating systems and hybridization studies in the genus Chelodina.     

社鼠组织器官同工酶的研究

用聚丙烯酰胺凝胶等电聚焦电泳方法,分析了社鼠的肝、肾、心肌、骨骼肌、肺、脾和脑等多种组织器官的ＬＤＨ、ＡＤＨ、ＥＳＴ、和ＳＯＤ４种同工酶,对各组织器官的酶带数目和分布,以及酶活性进行了比较研究。结果表明,社鼠的ＬＤＨ同工酶、ＡＤＨ同工酶和ＥＳＴ同工酶具有比较明显的组织特异性,而ＳＯＤ同工酶的组织特异性较低。肺和脾除ＥＳＴ同工酶活性较高外,脑除ＬＤＨ同工酶活性较高外,其它３种同工酶的活性均较低;而肝和肾中４种同工酶的活性普遍很高。心肌和骨骼肌因氧张力不同而使ＬＤＨ同工酶酶谱存在明显差异,但其它３种同工酶酶谱却非常相似。同工酶的组织特异性与各组织器官所执行的生理功能是相一致的     

Slowmo is required for Drosophila germline proliferation

Null mutations in the Drosophila gene, slowmo (slmo), result in reduced mobility and lethality in first-instar larvae. Slowmo encodes a mitochondrial protein of unknown function, as do the two other homologs found in Drosophila. Here, we have studied a hypomorphic P-element allele of slmo demonstrating its effects on germline divisions in both testes and ovaries. Using in situ studies, enhancer-trap activity, and promoter fusions, we have shown that slmo expression in testes is found in the somatic cyst cells (SCC). The hypomorphic allele for Slmo revealed apoptotic loss of germline cells in the larval germline, culminating in a complete absence of the germline in adult flies. In females, a similar degeneration of the germarium is observed, while reporter gene expression is found in both germline and somatic cells. Using a null mutation in female germline clones, we find slmo is dispensable from the germline cells. Our results suggest that Slowmo is not required in germline cells directly, but is required in SCCs responsible for maintaining germline survival in both sexes.     

Influence of glucocorticoids on time-of-day-dependent variations in IgE-, histamine-, and platelet-activating factor-mediated systemic anaphylaxis in different mouse strains

A time-of-day-dependent variation in IgE-mediated passive systemic anaphylaxis was previously reported in ICR mice. In the present study, we investigated time-of-day-dependent variations in IgE-, histamine-, and platelet-activating factor (PAF)-mediated systemic anaphylaxis in C57BL/6, BALB/c, and NC/Nga mice at 9:00?h and 21:00?h, and evaluated the potential influence of glucocorticoids (GCs) on these variations. We found significant time-of-day-dependent variations in IgE-mediated systemic anaphylaxis in C57BL/6 mice, and in histamine- and PAF-mediated systemic anaphylaxis in BALB/c mice. Significant daily variations in IgE-, histamine-, and PAF-mediated systemic anaphylaxis were not observed in NC/Nga mice. Pretreatment with dexamethasone and adrenalectomy abolished the daily variations in IgE-mediated systemic anaphylaxis in C57BL/6 mice and in PAF-mediated systemic anaphylaxis in BALB/c mice, suggesting that GCs from adrenal glands are pivotal in regulating these variations. In contrast, pretreatment with dexamethasone and adrenalectomy did not abolish the daily variation in histamine-mediated systemic anaphylaxis in BALB/c mice, suggesting that GC-independent and adrenal gland-independent mechanisms are important for the variation. The present study demonstrated that time-of-day-dependent variations in systemic anaphylaxis differed among inbred mouse strains and with anaphylaxis-inducing substances. Thus, mouse strains, time of experiment, and anaphylaxis-inducing substances used must be considered to obtain appropriate experimental results.     

The Distribution of Different Clades of Seneca Valley Viruses in Guangdong Province,China

Seneca Valley virus (SVV), a newly determined etiological agent of vesicular disease in swine, causes porcine idiopathic disease and occasional acute death in piglets. Recently, an increased number of SVV infection cases have been reported in the United States (US) and China, resulting in significant economic losses to the swine industry. The first identification of SVV in China was reported in Guangdong Province, a major swine producing province. The cases of SVV were continuously reported in Guangdong in 2015 and 2016. However, the spread of SVV in Guangdong in 2017 remains unknown.In this study, we determined two new SVV strains, CH-GD-2017-1 and CH-GD-2017-2, from Guangdong. The genetic analysis suggested that the two Guangdong strains showed different characteristics to previous Guangdong strains. They showed lower nucleotide similarity with strains isolated in 2015 and 2016, and were more similar to the US strains.Phylogenetic analyses indicated that the new strains were clustered in a different clade with previous Guangdong strains.We found 28 mutated amino acids in the new strains, compared with the first Guangdong strain, SVV CH-01-2015. In the geographic analysis, we found that the US and China reported more SVV cases than other countries, and most of the SVV cases were reported in east and central China—of which, Guangdong Province is one of the major epidemic regions. In conclusion, our findings indicate that SVV continued to spread in Guangdong Province in 2017, and two different clades of SVVs have emerged in this region.