激活标签法及其在番茄功能基因组的应用

激活标签法是近年来新发展起来的一种研究基因功能有价值的补充方法。该文综述了激活标签法的研究进展及其在番茄功能基因组研究中的应用。     

The ‘pole-hooking’ method: A novel and economical technique for in situ tagging small to medium-sized fishes

We describe a new manual procedure for catching and tagging fish underwater, the ‘pole-hooking’ method, and demonstrate and discuss its advantages over standard tagging techniques (i.e. hook-and-line fishing). The new technique was designed for tagging Acanthistius brasilianus [Cuvier, M., Valenciennes, L.B., 1828. Histoire Naturelle des Poissons. Museau de Histoire Naturelle, Paris], a small to medium-sized fish inhabiting rocky reefs of the Argentine coast. The pole-hooking method minimizes injuries, avoids fast depressurization of fish and allows registration of the precise location of capture and release. In addition, the equipment needed is very simple and economical. The mean number of fish tagged per minute using this methodology on shallow rocky reefs (up to 25-m depth) was similar to that obtained during hook-and-line fishing sessions (ca. one tagged fish every 3 min), given similar densities of A. brasilianus in the fished reefs.     

棉铃虫膨胀线粒体基因组的鉴定与分析

以已公布的棉铃虫线粒体DNA序列对来自4头棉铃虫雄蛹的DNA的三代测序数据进行筛选,获得了11条与线粒体DNA有同源性的三代read序列,并根据其中的read 66003鉴定出了一种膨胀的线粒体基因组。该线粒体基因组大小为27 113 bp,其保守区域包含13个蛋白编码基因、2个rRNA基因、22个tRNA基因以及1个AT富集区,与已公布的棉铃虫线粒体基因组的结构相似。膨胀区域位于cox1基因编码区内部,大小为11 467 bp,经预测含有一个完整的真核基因(依赖ATP的RNA解旋酶)以及多种转座元件的片段,但与线粒体DNA无同源性,也无I类或Ⅱ类内含子存在的证据。对田间和室内棉铃虫DNA样品的PCR扩增未能检测到膨胀线粒体基因组的存在。以上结果表明膨胀片段可能是细胞核DNA序列通过偶然的水平转移事件而整合到线粒体基因组中的,且该种膨胀方式的发生概率极低。本文报道的膨胀线粒体基因组为日后动物线粒体基因组学的研究提示了一种独特的变异方式。     

甘蓝黑腐病菌rPf基因簇对胞外蛋白酶Ⅰ基因表达的调控

甘蓝黑腐病黄单胞菌（ＸａｎｔｈｏｍｏｎａｓｃａｍｐｅｓｔｒｉｓＰｖ．ｃａｍｐｅｓｔｒｉｓ）产生的胞外蛋白酶Ⅰ在致病的早期阶段起重要作用，该酶以及其它胞外酶和胞外多糖的合成受一致病因子调控基因簇（ｒＰｆ基因簇）的正向调控。本研究利用带有β－半乳糖苷酶报道基因（ｌａｃＺ）的转座子Ｔｎ５－Ｂ２０诱变蛋白酶Ⅰ基因克隆，获得了ｌａｃＺ在蛋白酶Ⅰ基因启动子控制下表达的Ｔｎ５－Ｂ２０插入突变质粒。通过将这种突变质粒导入野生型和各ｒｐｆ基因突变体菌株后，测定ｌａｃＺ基因在细胞生长周期中的表达水平，不仅进一步证实了这些ｒｐｆ基因对蛋白酶Ⅰ基因的正向调控作用，而且明确了它们的调控水平．发现ｒｐｆＡ、ｒｐｆＣ、ｒｐｆＥ、ｒｐｆＧ或ｒｐｆＨ突变后，蛋白酶Ⅰ基因的转录会降低９０％左右，而ｒｐｆＢ突变后，蛋白酶Ⅰ基因的转录只降低４８％。     

转座子mini-Tn10介导的运动突变对溶藻弧菌毒力的影响

采用转座子mini-Tn10/Km构建了致病性溶藻弧菌的突变库,采用半固体双层平板初筛到73株运动缺陷突变株,初筛的突变株纯化及重复筛选后获得运动性缺陷表型稳定的突变株ND-01MM,Southern鉴定确定其为单位点插入。野生型菌株ND-01和运动缺陷突变株ND-01MM对致病性溶藻弧菌的天然宿主大黄鱼粘液的趋化、粘附以及在大黄鱼吞噬细胞内存活能力等生理功能的比较研究发现,溶藻弧菌运动缺陷后趋化及粘附能力均极显著下降(P<0.01),但在吞噬细胞内的存活能力则与野生型菌株差异不显著(P>0.05)。采用腹腔注射和浸泡两种方式感染大黄鱼,结果发现运动缺陷对浸泡感染的影响较为明显,但对腹腔注射的感染方式影响不大。     

Habitat utilization and vertical movements of white marlin (Tetrapturus albidus) released from commercial and recreational fishing gears in the western North Atlantic Ocean: inferences from short duration pop-up archival satellite tags

Researchers have applied numerous techniques to improve billfish stock assessments, including habitat‐based models that incorporate behavioral and oceanographic parameters to standardize historical catch‐per‐unit‐effort time‐series data. These methods have allowed researchers to account for significant changes in the depths of pelagic longline (PLL) gear deployments over time. This study presents habitat‐use data recovered from high‐resolution 5‐ and 10‐day pop‐up satellite archival tags (PSATs) attached to 47 surviving white marlin released from commercial and recreational fishing gears offshore of the U.S. East Coast, the northern Caribbean, and Venezuela between 2002 and 2004. Data recovered from transmitting tags indicated that white marlin spent nearly half of their time associated with warm, near‐surface waters (<10 m). All fish displayed frequent short duration (mean: 39.8 min) vertical excursions from surface waters to depths averaging 51 m. Qualitative and multivariate classifications of data from completely transmitted movements of surviving white marlin revealed two major types of descents: one pattern was characterized by deep ‘V’‐shaped excursions of relatively short duration (mean: 23.4 min) while the other featured descents that were more broadly ‘U’‐shaped and confined to a specific depth range for an extended period of time (mean: 75.8 min). Based on the frequency, persistence, and patterns of these vertical movements, white marlin appear to direct a considerable proportion of foraging effort well below surface waters, a behavior that may account for relatively high catch rates of white marlin on some deep‐set PLL deployments.     

转基因植物中删除选择标记基因的研究进展

选择标记基因广泛应用于植物转基因工程中,然而这些基于除草剂和抗生素类抗性选择标记基因的利用,在转基因筛选和鉴定完成后功能多余。当前转基因植物的标记基因及相关产物对环境污染和食用安全的潜在影响,已经引起了人们的广泛关注。随着转基因技术的不断发展和人们对转基因植物和食品安全性的关注,删除转基因植物中的选择标记基因非常重要。文章着重讨论了植物转基因过程中,选择标记基因的利用,删除选择标记基因的几种方法,包括共转化法、位点特异性重组、染色体内重组、转座子介导的重组、删除叶绿体转化中选择标记基因,以及无选择标记基因植株的直接再生等。同时,还对这些技术的发展作了展望。     

Parentage assignment of stocked black sea bream <Emphasis Type="Italic">Acanthopagrus schlegelii</Emphasis> in Hiroshima Bay using microsatellite DNA markers

The genetic contribution of 51 broodstock, comprising 29 females and 22 males, reared at Hiroshima City Marine Products Promotion Center for the production of stocked black sea bream was monitored during two consecutive years using seven microsatellite DNA loci. The high discrimination ability of these markers was reflected in the polymorphic identification content (PIC=0.831), the exclusion probability (Q≈1), and the low probability of identity index (I=3.635⁻¹⁰). The total number of breeders contributing to the mating process was estimated at 32 (62.7%) in 2000 and 30 (58.8%) in 2001. On pedigree reconstruction, 69.3% of the offspring were successfully assigned to a single broodstock pair. Loss of alleles accounted for 16.9% during seed production; nevertheless, 90.9% of males and 69.0% of females participated in the mating process. Based on microsatellite genetic tagging, 58.9% of the fish sampled during the two months after release were identified as hatchery stock, presenting no significant differences from wild conspecifics in either fork length or body weight.     

Effect of habitat fragmentation on spawning migration of brown trout (Salmo trutta L.)

Abstract –  Human-induced habitat alteration is one of the main causes of the decline of freshwater fish populations. The watershed of the River Bidasoa (Spain) is an example of heavily fragmented habitat. The local brown trout ( Salmo trutta L.) population is disturbed, with evidence of poor recruitment as well as low adult densities in the main stem. Forty male and female adult migratory trout were tagged with external or internal radio transmitters and released. Fixed stations with data loggers and mobile antennae were used with daily surveys to track fish movements during the migration and spawning period (3 months). Migration distances did not exceed 10 km, and half of the fish never entered a tributary in the study area. Fragmentation because of weirs on the main stem apparently prevented fish from reaching their spawning destination. Fish that entered the tributaries were first confronted with an accessibility problem because of low discharge. However, each fish chose one tributary, without making attempts to run up in other tributaries. Once in the tributary, fish were restrained in their upstream movements by dams. The study area appeared to be isolated from the vast upper part of the watershed. Within the study area, upper parts of tributaries also seemed strongly disconnected from the main stem. This study illustrates the negative impact of river fragmentation on S. trutta migration pattern. Population sustainability can be directly affected through the low availability of spawning grounds for migratory fish. Long-term effects of fragmentation may cause reproductive isolation within watersheds, which in the case of trout also means isolated phenotypic population units.     

玉米Mutator转座子结构特征和基因克隆的研究进展

Mutator（Mu）转座子是已发现的植物中转座活性最强的转座子,其较高的转座频率以及趋向于插入单拷贝功能基因转座的特性,使其成为构建玉米突变体库的一个主要方法。当前玉米B73品系基因组测序已经基本完成,利用反向遗传学方法,研究玉米基因的功能更为方便。文中简要介绍Mu转座子的结构特征以及Mu的分类,介绍3种克隆Mu转座子标签插入位点侧翼序列的技术,对Mu研究所存在的问题进行分析,展望Mu的应用。