普通小麦5DL染色体分选及染色体BIBAC文库构建技术体系优化

 【目的】优化小麦染色体流式分选及特定染色体BAC文库构建技术体系,为加速小麦基因组学研究提供技术支撑。【方法】采用双阻断法对中国春5DL端体根尖分生组织进行细胞周期同步化处理,结合机械匀浆法制备染色体悬浮液进行流式核型分析,分选染色体5DL及PCR检测,对小麦染色体分选技术体系进行优化。对复合峰分选产物Bam HI酶切制备高分子量（high molecular weight,HMW）DNA,按10﹕1质量比与双元细菌人工染色体（binary-bacterial artificial chromosome,BIBAC）连接,电激转化感受态细胞DH10B,随机挑取100个重组子,经NotI完全酶切后脉冲电泳检测插入片段大小,优化小麦染色体BAC文库构建的技术体系。【结果】经1.25 mmol•L-1 羟基脲（hydroxyurea,HU）18 h,恢复6 h和1 µmol•L-1 氟乐灵（trifluralin）2.5 h的顺序处理的小麦根尖分生组织,细胞有丝分裂指数可超过60%。流式核型分析表明,5DL端体的流式核型图由清晰的三个复合峰和两个单峰组成。PCR鉴定表明,其中一单峰确由5DL染色体组成。部分酶切复合峰染色体3～10 min,可以获得50～300 kb 的HMW DNA。连接产物检测显示平均插入片段可达100 kb左右,根据荧光通道值,估算5DL端体的大小约为482 Mb,构建库容为19 000个克隆的5DL端体特异文库即可达到约4倍的覆盖率。【结论】上述两项优化技术适用于小麦染色体的分选及染色体特异文库的构建。     

流式细胞仪在猪性别控制中的应用初探

在国内首次报道使用流式细胞仪对猪进行性别控制研究。猪精液通过流式细胞仪进行X、Y精子分离,分离精子经过数小时保存后,用于陆川母猪输卵管授精,最后产出健康仔猪。试验结果表明:①第1次(2005-10-14)精子分离的纯度为:X88%、Y83%;活力为X35%、Y21%。第2次(2006-01-13)纯度为:X70%,Y68%;活力为:X52%,Y36%。②该试验中母猪受孕率为25%,未受孕者仍然可在生产条件下继续繁殖。③受孕母猪(1头)产出健康仔猪5头,出生成活率100%,大小均匀,活力良好,哺乳期(28 d断奶)平均日增重与生产群极为接近(151.43 g1、48.93 g),但产活仔数低(生产群为8.4头/窝)。④仔猪雌性率为60%。试验结果初步表明:在该实验室现有条件下,使用流式细胞仪分离的X、Y精子,母猪通过输卵管受精可以产出健康仔猪;精子分离对仔猪哺乳期生长无明显影响;对性别比例有一定调控作用。     

Direct visualization of horizontal gene transfer

Conjugation allows bacteria to acquire genes for antibiotic resistance, novel virulence attributes, and alternative metabolic pathways. Using a fluorescent protein fusion, SeqA-YFP, we have visualized this process in real time and in single cells of Escherichia coli. We found that the F pilus mediates DNA transfer at considerable cell-to-cell distances. Integration of transferred DNA by recombination occurred in up to 96% of recipients; in the remaining cells, the transferred DNA was fully degraded by the RecBCD helicase/nuclease. The acquired integrated DNA was tracked through successive replication rounds and was found to occasionally split and segregate with different chromosomes, leading to the inheritance of different gene clusters within the cell lineage. The incidence of DNA splitting corresponds to about one crossover per cell generation.     

Molecular sorting in the secretory pathway

Proteins can be secreted from animal cells by either a constitutive or a regulated pathway; those destined for regulated secretion are actively sorted into dense-core secretory granules. Although sorting is generally assumed to be accomplished by specific carriers, the nature of these carriers remains elusive. In this study, peptide hormones were used as affinity ligands to purify a set of 25-kilodalton proteins from canine pancreatic tissue. Their ligand specificities and patterns of expression have the characteristics of sorting carriers.     

Centric fusion, satellite DNA, and DNA polarity in mouse chromosomes

A fluorescent staining technique has demonstrated a contralateral arrangement of fluorescent spots in the centromeric region of mouse metacentric chromosomes which have resulted from centric fusion. The results suggest that centric fusion involves the maintenance of DNA polaritv through the centromere and that the thymidine-rich chain of satellite DNA in the centromeric region is associated with the same DNA chain in every mouse autosome.     

High-resolution chromosome sorting and DNA spot-blot analysis assign McArdle's syndrome to chromosome 11

A rapid gene-mapping system uses a high-resolution, dual-laser sorter to identify genes from separate human chromosomes prepared with a new stain combination. This system was used to sort 21 unique chromosome types onto nitrocellulose filter papers. Several labeled gene probes hybridized to the sorted chromosomal DNA types predicted by their previous chromosome assignments. The skeletal muscle glycogen phosphorylase gene was then mapped to a portion of chromosome 11 by spot blotting normal and translocated chromosomes.     

利用流式细胞仪分选土壤中发生自然转化的阳性转化子

为确定土壤中的DNA通过自然转化的方式发生水平基因转移的频率,将1个带有egfp和卡那霉素抗性基因Kanr的质粒DNA添加到自然土壤中,分离菌悬液,上流式细胞仪分选能发出绿色荧光且具有卡那霉素抗性的阳性转化子,确定土壤中发生自然转化的频率小于10~(-7)。分离的菌悬液通过培养基的富集,从39 736 426个细胞中分选获得了82个细胞,最终得到了纯化的阳性转化子,证实土壤中的DNA通过自然转化发生了水平基因转移。     

Somatic cell hybrid between the established human line D98 (presumptive HeLa) and 3T3

Somatic cell hybrids have been made between an established human cell line with a long culture history and established mouse fibroblast line. When first analyzed, the hybrid cells contained nearly twice as many mouse chromosomes as the mouse parent line and a human chromosome complemnent of about half that of the human parent. There was further loss of human chromosomes on continued cultivation. This behavior resembles that of other human mouse hybrids and appears to be characteristic of the human-mouse combination. However, the number of human chromosomes is greater than in hybrids made from human diploid fibroblasts. Some clones contain more than a haptoid quantity of human DNA per cell and should synthesize a much greater number of human gene products.     

遗传转移的MADI过程

单体附加在小麦细胞中的黑麦染色体,在减数分裂过程中高频率地断裂,破碎和丢失。它们同时引起小麦染色体的断裂和丢失。断裂的黑麦和小麦染色体,以较高频率重新融合形成罗伯逊易位。破碎的黑麦染色体的DNA片段,可以整合在小麦染色体上,完成遗传的转移。本研究发现的这种通过染色体的“单体附加一破碎一整合”过程实现遗传转移的方式,简称为“MADI”(美代)过程,为把外源种质导入栽培植物的研究提供了一种有效的方法。     

DNA content and DNA-based centromeric index of the 24 human chromosomes

The chromosomes of two human males were identified by fluorescent banding, restained, and measured by scanning microscopy and computer analysis. The two variables, DNA content and DNA-based centromeric index, provided almost complete discrimination of chromosome types. Some chromosomes showed significant differences in DNA content between the men, and for one man two pairs of chromosomes showed significant differences between homologs.     

一个适用于整数的快速排序程序

本文就待排序数据是整数的情形给出了一种快速的排序方法及程序。     

植物流式细胞遗传学及其应用研究进展*

 流式细胞遗传学是利用流式细胞术对植物有丝分裂细胞的染色体进行分离、纯化和分选等操作的细胞遗传学分支学科,已在许多领域广泛的应用,如与DNA 分子标记结合进行物理作图,利用FISH和PRINS进行细胞遗传作图,重组DNA文库的构建,标记片段的定向分离以及蛋白质分析。利用流式细胞技术已对数十种植物物种进行了染色体分析,应用最多的是豆类作物和禾谷类作物。流式细胞术有许多潜在的利用价值,主要包括植物染色体和染色体臂特异BAC文库的构建,低拷贝序列的目标分离,ESTs和杂交DNA 序列的高产量作图以及对染色体蛋白的整体分析等。本文综述了植物流式细胞遗传学的研究方法及其在植物研究中的应用,并对其在植物基因组分析的潜在利用价值作了展望。     

Hemoglobin concentration in normal diploid and intersex triploid chickens: genetic inactivation or canalization?

Of 19 adult intersex chickens examined, 17 were triploid animals with ZZW sex chromosomes. Triploid erythrocytes were about 1.5 times the size of diploid erythrocytes, and were present in amounts that were about two-thirds the number of diploid erythrocytes. Both diploid and triploid animals had similar hematocrits and hemoglobin concentrations. Triploidy is reflected in a relative increase in RNA, as well as in DNA, per erythrocyte. This, in addition to the trisomic mode of inheritance observed in several genetically distinct loci determining isoantigens, indicates that all three sets of chromosomes in the triploid animals are genetically active. Apparently, there is a homeostatic mechanism maintaining the concentration of hemoglobin for the species in spite of triploidy and its effect on erythrocyte size.     

Downstream fining by selective deposition in a laboratory flume

There has long been debate about the relative importance of abrasion versus selective deposition of the coarsest clasts in causing downstream fining of sediment in river systems. Although high fining rates observed in many natural rivers seem to require strong selective deposition, the ability of selective deposition to produce downstream size sorting has never been measured under controlled conditions. In an experiment using a long flume and a poorly sorted, bimodal gravel feed, downstream fining was produced by a factor of 1.3 in median size and 1.8 in 90th percentile size, over a distance of 21 meters. The experimental conditions rule out abrasion effects. Selective deposition appears to be a natural consequence of the transport and deposition of sufficiently poorly sorted or bimodal gravels and appears to be capable of accounting for fining rates observed in natural gravel rivers.     

Radium-226 and radon-222: concentration in atlantic and pacific oceans

Measurements of radon-222 in seawater suggest the following. The radium-226 content of surface water in both the Atlantic and Pacific oceans is uniformly close to about 4 x 10(-14) gram per liter. The deep Pacific has a concentration of radium-226 that is four times higher and the deep Atlantic a concentration twice as high as that of the surface. These distribution profiles can be explained by the same particle-settling rate for radium-226 from surface to depth for the two oceans and by a threefold longer residence time of water in the deep Pacific than in the deep Atlantic. The vertical distribution of the deficiency of radon-222 in the surface water of the northwest Pacific Ocean suggests a coefficient of vertical eddy diffusion as high as 120 square centimeters per second and a gas-exchange rate for carbon dioxide in surface water between 14 and 60 moles per square meter per year. Vertical profiles of the excess of radon-222 in near-bottom water of the South Atlantic give coefficients of vertical eddy diffusion ranging from 1.5 to more than 50 square centimeters per second.     

Suppressing drosophila circadian rhythm with dim light

Drosophila larvae were reared and allowed to pupate in continuous bright white light. The pupae were then transferred to a much dimmer blue light. In continuous blue light of intensity below 0.001 erg per square centimeter per second, adult flies emerged in pulses 24.7 hours apart, each pulse occupying about 6 hours. But in continuous light of intensity exceeding 0.1 erg per square centimeter per second, they emerged at a steady rate. This intensity range from effective darkness to effective light is roughly from starlight to moonlight. Inside this range, the emergence peaks broaden for about a week with little change of period.     

Sea floor spreading, topography, and the second layer

Local sea floor topography and also the thickness of the second layer of the oceanic rise-ridge system appear related to the spreading rate in the region. Slow spreading, away from the ridge center at 1 to 2 centimeters per year, is associated with a thick second layer, a central rift, and adjacent rift mountains. Fast spreading, 3 to 4.5 centimeters per year, is associated with a thin second layer and subdued topography lacking any central rift. The volume of lava discharged in this layer per unit time and per unit length along the crest of the whole active system is relatively constant regardless of the spreading rate. Total second layer discharge of the system has been about 5 to 6 cubic kilometers per year during the last several million years.     

基于荧光检测技术的小麦品种SSR鉴定体系的建立

【目的】建立基于SSR荧光标记的小麦品种DNA指纹鉴定体系,为中国小麦育成品种鉴定提供高通量技术手段。【方法】收集已定位到小麦21条染色体上的SSR标记引物,通过PCR扩增和变性聚丙烯酰胺凝胶电泳检测技术,筛选在中国小麦育成品种中多态性高的标记,对筛选出的引物5′末端利用6-FAM、HEX、ROX和TAMRA 4种荧光染料之一进行标记,利用DNA分析仪对扩增产物的峰型进行评价并检测不同等位变异的扩增片段大小,选择峰型简单易读、多态性高、较均匀分布到21条染色体上的标记,确定不同位点等位变异的大小及相应的参照品种,建立基于荧光SSR标记的高通量小麦品种鉴定体系。【结果】利用2 438对SSR标记对8份植物学性状差异大的小麦育成品种进行初步筛选,共筛选出260对多态性较高、扩增稳定的SSR引物。利用上述260对引物对48份小麦品种继续进行复筛,选出130对扩增稳定、多态性高、带型好的SSR引物。对这些引物的正向5′末端分别标记6-FAM、HEX、ROX和TAMRA荧光后,利用DNA分析仪进行检测评价,最终选择了42个PCR扩增稳定、峰图简单、多态性高、连锁群分布均匀的SSR荧光标记。根据DNA分析仪检测到的每个标记的不同等位变异大小,为相应位点的不同等位变异进行了命名,并为每个等位变异选取了相应参照品种。根据每个引物标记的荧光和扩增的片段大小范围对42对引物进行合理搭配,在同一毛细管内对多个荧光标记进行检测,提高了DNA指纹数据采集效率,降低了检测成本。利用该体系对1 625份小麦育成品种进行DNA指纹数据采集,在42个位点中共检测到434个等位变异,每个位点的等位变异个数在3-23,平均10.3个;每个位点的PIC值范围为0.240-0.829,平均0.610。利用1 625份小麦育成品种DNA指纹数据,构建了中国小麦育成品种的DNA指纹数据库。【结论】筛选出42对SSR标记引物,建立了基于荧光SSR标记的小麦品种鉴定体系,可用于高通量小麦品种DNA指纹鉴定。     

浙江省早籼稻种质资源的表型多样性分析与评价

为探究浙江省早籼稻种质资源遗传多样性,提高早籼稻育种中亲本选择的利用效率,通过变异系数、遗传多样性指数、主成分分析、二维排序分析、逐步回归分析和聚类分析等方法对100份早籼稻种质资源的14个主要表型性状的多样性水平进行分析。结果表明:14个表型性状的变异系数在0.96%(生育期)~41.07%(每穗瘪谷数);14个表型性状的遗传多样性指数在5.50(每穗瘪谷数)~5.61(生育期);基于主成分二维排序分析筛选到单穗重大、每穗瘪谷数少、长宽比小、倒二叶长度和生育期适中、穗长长且单株有效穗多的种质资源,根据二维排序结果,早籼稻材料4128、4209、4225和4278是在二维排序重叠种质,可以作为育种亲本或者中间材料。主成分分析和综合评价表明,100份早籼稻种质资源中材料4021综合性状排名第1,筛选到每穗实粒数、每穗瘪谷数、结实率、单株有效穗、谷粒长和谷粒宽这6个表型性状可以作为早籼稻资源综合评价的关键指标;同时基于离差平方和法进行层次聚类,将100份早籼稻材料分为4类,各类表型性状差异明显。     

Energy conservation through more efficient lighting

The efficiency of a mercury-rare gas electrical discharge, which forms the basis of a fluorescent lamp, can be increased about 5 percent simply by increasing the concentration of mercury-196 from 0.146 percent (natural) to about 3 percent. These findings can be implemented immediately without any significant change in the process of manufacturing of this widely used source of illumination, provided that mercury-196 can be obtained economically. The potential energy savings for the United States are estimated to be worth in excess of $200 million per year.