应用代表性差异分析法筛选人癌候选抑癌基因

运用cDNA代表性差异分析法(cDNA representational difference analysis,cDNA RDA),以正常人鼻咽上皮细胞及鼻咽癌HNE1细胞作为比较的样品来源,分离了四个在鼻咽癌中缺失的cDNA片段.以此四个片段作探针,分别进行DNA杂交、RNA杂交,结果显示,这些差异性的cDNA序列确实来自正常人鼻咽上皮且只在其中表达和/或在鼻咽癌HNE1中表达降低,并在鼻咽癌病人中存在不同程度的缺失.序列分析结果表明这些差异性表达的基因为具有相当抑癌基因功能的已知基因和可能与鼻咽癌相关的抑癌基因的新基因.从而说明cDNA RDA是一种高效、敏感、假阳性低的克隆抑癌基因的有效方法.     

用RDA技术寻找肝再生相关基因的初步研究

利用表达性差异显示分析 (RDA)技术 ,研究大鼠 2 /3肝部分切除后 1h肝组织中基因的选择性表达 ,建立了大鼠 2 /3肝部分切除术后 1h再生肝组织选择性表达基因EST库。该库约含 3× 10 4 个独立克隆 ,其中 95 %以上的克隆含有插入片段 ,长度约 2 0 0～ 70 0bp不等 ,对随机挑出的 5 2个克隆的序列分析表明其中大多数基因与肝再生调控相关 (38/5 2 )。 10株未报道序列经RNA杂交证实 ,其中 6株与肝再生相关。     

The use of nuclear and mitochondrial single nucleotide polymorphisms to identify cryptic species

There is growing interest in the use of single nucleotide polymorphisms for evolutionary and population genetics. We tested the efficacy of one of the available single nucleotide polymorphism techniques, single-base extension, in distinguishing four cryptic species of Microtus. Sequence data were available for these species at nuclear and mitochondrial loci and their identity could be independently confirmed using karyotypes. We found that the development and optimization of single nucleotide polymorphisms required extensive effort, and that the method accurately identified the correct nucleotide at single nucleotide polymorphism sites approximately 90% of the time at the conserved nuclear locus. Correct identification rates were much lower at the highly variable mitochondrial locus.     

隐种及其在两栖动物中的研究进展

近年来,由于分子生物学的迅猛发展,物种内隐藏的生物多样性越来越多地被发现,由此引发的隐种问题也备受关注。隐种的存在对现有的生物多样性评估提出了挑战。在实践上,隐种的界定对于自然资源的保护和管理具有重要意义。两栖动物由于迁移能力较弱,通常具有较强的遗传分化,因而可能包含较丰富的隐种多样性。简述了隐种的概念和形成机制,归纳了国内外两栖类动物中隐种的研究进展,并着重介绍了界定两栖动物隐种的研究方法,最后,对今后两栖动物隐种的研究作了简要展望。我国两栖动物资源丰富,并具有独特的地形和气候特点,对这些物种遗传多样性和系统地理学的研究亟待开展。     

Targeting of marker loci to chicken chromosome 16 by representational difference analysis

Representational difference analysis (RDA) was initially used to identify differences between two inbred lines of chickens, line N and line 15I, on which the Compton mapping reference population is based. RDA was subsequently used to identify marker loci targeted specifically to chicken chromosome 16. Chromosome 16 contains the major histocompatibility complex (MHC), nucleolar organiser region (NOR) and Rfp-Y complex. To generate markers specific for this chromosome a bird was selected from the Compton mapping reference population which had inherited N line alleles for the MHC, NOR and Rfp-Y regions on this chromosome. DNA from this bird was compared with pooled DNA from 16 of its siblings, all of which had inherited line 15I alleles for the MHC, NOR and Rfp-Y regions. Initially amplicons were derived from Bam HI digested samples, RDA products were cloned after the first round of hybridisation and 113 clones were investigated: 45 of these identified Bam HI polymorphisms in this population. Of the 45 polymorphic clones, 17 have been mapped in the reference population so far, and these have identified seven new loci on chromosome 16. Interestingly a group of 16 other loci were linked on chromosome 4. The same birds were also compared by RDA following digestion with Taq I. Again large numbers of clones were generated of which 65 were investigated. Of these 17 clones were polymorphic and of five clones mapped so far three lie on chromosome 16. Two of the loci mapped to chromosome 16 have been used to identify yeast artificial chromosome (YAC) clones (GenBank accession numbers: AF057302, AF057303, AF057304, AF063218, AF06347, AF06348, AF06349, AF06350, AF063#51, AF06353, AF06354, AF06355, AF06356).     

小菜蛾对溴氰菊酯抗性相关的DNA片段的克隆与分析

采用代表性差异分析（representational difference analysis, RDA ）方法,以小菜蛾Plutella xylostella (L.)的敏感品系作为对照组,溴氰菊酯抗性近等基因系（near isogenic line）作为测试组,通过三轮消减杂交得到大小为 150～300 bp的差异扩增带,经亚克隆测序并与GenBank等数据库同源比较,发现差异片段与已知抗性相关基因无同源性;以随机选取的差异片段作探针进行Southern blot分析,在测试组扩增子和三轮差异产物中都获得了阳性结果,而在对照组扩增子中的结果是阴性的。这些结果表明测序片段可能是与抗性相关的新基因序列或调控序列。     

cDNA representational difference analysis of the deltamelthrin-resistant and -susceptible populations in diamondback moth (Plutella xylostella L.)

Abstract:  The improved cDNA representational difference analysis was preliminarily used to analyse the genetic differences between the deltamethrin-susceptible and -resistant strain of the diamondback moth. The driver amplicon is the cDNA from the susceptible strain while the tester amplicon is from the resistant strain. After four rounds of subtractive hybridization, we obtained one different product, the size of which was about 200 bp. Comparisons between our sequencing results and data in GenBank show that there is one sequence which has a relatively high homology to the ubiquitin gene, but there are no reports indicating that its upregulated expression is correlated with insecticide resistance. Hence further studies are warranted.     

Analysis of sex-linked sequences supports a new mammal species in Europe

European mammals have been the focus of particularly detailed taxonomic studies by traditional morphological methods. However, DNA analyses have the potential to reveal additional, cryptic species. We describe two highly divergent evolutionary lineages within a small Eurasian mammal, the field vole (Microtus agrestis). We show that the two lineages can be detected not only with maternally (mitochondrial DNA), but also with paternally (Y chromosome) and biparentally (X chromosome) inherited DNA sequences. Reciprocal monophyly of all genealogies and their congruent geographical distributions is consistent with reproductive isolation. Our results suggest that the field vole should be reclassified as two separate species.     

TF-1细胞凋亡相关基因的研究

利用近年来发展起来的代表差异分析（cDNA representational differences analysis, cDNA-RDA）技术研究了在人红白血病细胞株TF-1细胞撤除细胞因子后进入凋亡时诱导表达的基因.发现了6个新基因片段.其中有三个经与GenBank nr和dbEST查询均没有发现同源性,已经向GenBank进行登记,登记号分别为U83208,U83279,U83397.此外还发现一批已知基因的表达与凋亡相关,其中包括Hou和人硫氧还原蛋白等, 提示它们在凋亡中可能起作用.这项工作为进一步研究凋亡相关基因打下了良好基础.通过RDA的研究结果,有可能发现人白血病细胞凋亡的特异标记蛋白或发挥作用的重要蛋白,以期为白血病治疗提供理论基础.     

Differential responses of cryptic bat species to the urban landscape

Urbanization is a key global driver in the modification of land use and has been linked to population declines even in widespread and relatively common species. Cities comprise a complex assortment of habitat types yet we know relatively little about the effects of their composition and spatial configuration on species distribution. Although many bat species exploit human resources, the majority of species are negatively impacted by urbanization. Here, we use data from the National Bat Monitoring Programme, a long‐running citizen science scheme, to assess how two cryptic European bat species respond to the urban landscape. A total of 124 × 1 km² sites throughout Britain were surveyed. The landscape surrounding each site was mapped and classified into discrete biotope types (e.g., woodland). Generalized linear models were used to assess differences in the response to the urban environment between the two species, and which landscape factors were associated with the distributions of P. pipistrellus and P. pygmaeus. The relative prevalence of P. pygmaeus compared to P. pipistrellus was greater in urban landscapes with a higher density of rivers and lakes, whereas P. pipistrellus was frequently detected in landscapes comprising a high proportion of green space (e.g., parklands). Although P. pipistrellus is thought to be well adapted to the urban landscape, we found a strong negative response to urbanization at a relatively local scale (1 km), whilst P. pygmaeus was detected more regularly in wooded urban landscapes containing freshwater. These results show differential habitat use at a landscape scale of two morphologically similar species, indicating that cryptic species may respond differently to anthropogenic disturbance. Even species considered relatively common and well adapted to the urban landscape may respond negatively to the built environment highlighting the future challenges involved in maintaining biodiversity within an increasingly urbanized world.     

Genetic variation,population structure and cryptic species within the black mudfish,Neochanna diversus,an endemic galaxiid from New Zealand

To investigate the phylogenetic relationships and geographical structure among landlocked populations of the black mudfish, Neochanna diversus, mitochondrial DNA nucleotide sequence data were sampled from seven populations from the Waikato and Northland regions of New Zealand. The complete D-loop region was sequenced from 70 individuals, with 913 bp from the tRNA-pro end used in population and phylogenetic analysis. A tandem repeat array, which ranged in size up to 200 bp, was found in most populations at the 3′ end of the D-loop that was not able to be aligned for analysis. Of the seven sites sampled, two from Northland exhibited significant sequence divergence from all other sites. There was also a clear distinction among remaining Northland sites and those from the Waikato. An additional 518 bp segment of the 16S region was sequenced from all sites and compared with the other New Zealand mudfish species, N. apoda, N. burrowsius and the Tasmanian mudfish Galaxias (Neochanna) cleaveri using Galaxias maculatus as an outgroup. Both D-loop and 16S sequence data provided strong evidence for a cryptic species of mudfish present in Northland. The significant genetic structure apparent in the black mudfish appears most probably to be attributed to geological conditions during the Pliocene, where peat wetlands became apparent in the Waikato while Northland consisted of disjunct ‘islands’. Conservation and management of these populations must take into account the historical processes that have shaped these patterns of genetic diversity.     

Trophic resource partitioning drives fine‐scale coexistence in cryptic bat species

Understanding the processes that enable species coexistence has important implications for assessing how ecological systems will respond to global change. Morphology and functional similarity increase the potential for competition, and therefore, co‐occurring morphologically similar but genetically unique species are a good model system for testing coexistence mechanisms. We used DNA metabarcoding and high‐throughput sequencing to characterize for the first time the trophic ecology of two recently described cryptic bat species with parapatric ranges, Myotis escalerai and Myotis crypticus. We collected fecal samples from allopatric and sympatric regions and from syntopic and allotopic locations within the sympatric region to describe the diets both taxonomically and functionally and compare prey consumption with prey availability. The two bat species had highly similar diets characterized by high arthropod diversity, particularly Lepidoptera, Diptera and Araneae, and a high proportion of prey that is not volant at night, which points to extensive use of gleaning. Diet overlap at the prey item level was lower in syntopic populations, supporting trophic shift under fine‐scale co‐occurrence. Furthermore, the diet of M. escalerai had a marginally lower proportion of not nocturnally volant prey in syntopic populations, suggesting that the shift in diet may be driven by a change in foraging mode. Our findings suggest that fine‐scale coexistence mechanisms can have implications for maintaining broad‐scale diversity patterns. This study highlights the importance of including both allopatric and sympatric populations and choosing meaningful spatial scales for detecting ecological patterns. We conclude that a combination of high taxonomic resolution with a functional approach helps identify patterns of niche shift.     

Combining molecular, morphological and ecological data to infer species boundaries in a cryptic tropical pitviper

Few operational methods exist for delimiting species boundaries, and these usually require sampling strategies that are unrealistic for widespread organisms that occur at low densities. Here we apply molecular, morphological and ecological species delimitation criteria to a wide-ranging, fragmented group of Asian green pitvipers, the Popeia popeiorum complex. A mitochondrial DNA phylogeny for the group indicates two well-differentiated clades, corresponding mainly to northern and southern parts of its range. Strong phylogeographical structure within each clade suggests isolation in forest refugia during the Pliocene and a southward colonization of the Sunda islands during the Pleistocene. Multivariate analysis of morphological characters reveals a generally conserved pattern of geographical variation, incongruent with the recovered phylogenetic history. We compare groups delineated by mtDNA variation to morphological and ecological divisions in the complex, and discuss the implications of these for the taxonomy of the group. Discordance between species boundaries inferred from different criteria suggests that combining independent sources of data provides the most reliable estimation of species boundaries in organisms that are difficult to sample in large numbers.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 343–364.     

Hemiboea guangdongensis comb. & stat. nov., a cryptic species segregated from H. subcapitata (Gesneriaceae) based on morphological and molecular data

The taxonomic problem of cryptic species has long been recognized. Hemiboea subcapitata C. B. Clarke is a widespread and morphologically diverse species including two varieties, H. subcapitata var. subcapitata C. B. Clarke and H. subcapitata var. guangdongensis (Z. Y. Li) Z. Y. Li. However, genetic distance and molecular phylogenetic analyses based on nuclear ITS and four plastid DNA sequences (atpB‐rbcL, matK, rbcL, rpS16 intron) revealed that H. subcapitata var. guangdongensis is sister to H. subacaulis, and separated from H. subcapitata var. subcapitata, suggesting that it should be raised to the rank of an independent species as H. guangdongensis (Z. Y. Li) X. Q. Li & X. G. Xiang, comb. & stat. nov. Morphologically, the new species can be distinguished from H. subacaulis by its calyx 5‐sect from base, segments narrowly oblong‐lanceolate (versus calyx 5‐sect from base or 2‐lipped and adaxial lip 2‐lobed from below middle, segments ovate), peduncle glabrous (versus pubescent), and vermiform sclereids dispersed in leaf mesophyll (versus without sclereids).     

Maize DNA enrichment by representational difference analysis in a maize chromosome addition line of oat

 The recent recovery of maize (Zea mays L.) single-chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses has provided novel source materials for the potential isolation of maize chromosome-specific sequences for use in genetic mapping and gene cloning. We report here the application of a technique, known as representational difference analysis (RDA), to selectively isolate maize sequences from a maize chromosome-3 addition line of oat. DNA fragments from the addition line and from the oat parent were prepared using BamHI digestion and primer ligation followed by PCR amplification. A subtractive hybridization technique using an excess of the oat parental DNA was employed to reduce the availability for amplification of DNA fragments from the addition lines that were in common with the ones from the oat parental line. After three rounds of hybridization and amplification, the resulting DNA fragments were cloned into a plasmid vector. A DNA library containing 400 clones was constructed by this method. In a test of 18 clones selected at random from this library, four (22%) detected maize-specific repetitive DNA sequences and nine (50%) showed strong hybridization to genomic DNA of maize but weak hybridization to genomic DNA of oat. Among these latter nine clones, three detected low-copy DNA sequences and two of them detected DNA sequences specific to chromosome 3 of maize, the chromosome retained in the source maize addition line of oat. The other eight out of the 13 clones that had strong hybridization to maize DNA detected repetitive DNA sequences or high-copy number sequences present on most or all maize chromosomes. We estimate that the maize DNA sequences were enriched from about 1.8% to over 72% of the total DNA by this procedure. Most of the isolated DNA fragments detected multiple or repeated DNA sequences in maize, indicating that the major part of the maize genome consists of repetitive DNA sequences that do not cross-hybridize to oat genomic sequences. Received: 18 November 1997 / Accepted: 12 March 1998     

代表性差异分析比较两株来自不同地区的猪源Lactobacillus菌株

[目的]对分离自猪肠道的乳酸杆菌S1菌株进行鉴定,并比较该菌株与同种的001T菌株的基因差异.[方法]对S1菌株进行16S rRNA基因序列分析和种特异PCR检测,并且对S1菌株和Lactobacillus sobrius 001T进行代表性差异分析(Representational difference analysis,RDA).[结果]16S rRNA基因序列分析表明,与S1菌株最相似的已知菌为L.sobrius.变性梯度凝胶电泳分析显示,仔猪空、回肠细菌图谱中有一与S1菌株有相同迁移位置的优势条带,克隆、测序鉴定表明,与该条带相匹配的16S rRNA基因克隆(Clone S)的最相似已知菌也为L.sobrius.16S rRNA基因系统进化分析表明,S1菌株与Clone S和L.sobrius 16S rRNA基因序列同源性分别为99.8%和99.6%.L.sobrius特异性引物也可以扩增S1株菌的16S rRNA基因的特定片段.因此S1菌株可被确定为Lsobrius.RDA对菌株S1和同种的猪源L.sobrius 001T菌株的基因差异进行分析,未发现这两株菌的基因组差异.[结论]猪肠道乳杆酸菌S1菌株属于L.sobrius,其与猪源L sobrius 001T菌株为相似菌株.     

External diversity is restrained by internal conservatism: New nudibranch mollusc contributes to the cryptic species problem

Cryptic species are an important concept of modern biodiversity studies and a potential basis for immensely increasing the amount of biological diversity worldwide; however, the conceptual grounds of cryptic species still need to be refined. Here, using a remarkable example from marine invertebrates, the case of a species complex of common large‐sized nudibranchs of the genus Dendronotus in European waters is presented, which is relevant for the ongoing cryptic species discussion. Based on extensive sampling in the seas of Northern Hemisphere from United Kingdom to the Sea of Japan, a morphological and molecular framework is constructed for understanding the diversity of European Dendronotus nudibranchs. The present study discovered notable multilevel character diversity within the common Dendronotus “frondosus” species complex. This implies the highest degree of variation of external characters with parallel colour patterns among different species and significantly constrained conservatism of internal characters. The molecular data are congruent with the stable differences in the internal features and confirm the presence of three distinct species in this complex. While two species were already known, the third species is evidently new, based on morphological and molecular evidence and is described here as Dendronotus europaeus sp. n. In the light of multilevel character diversity, the general problem of plausibility of the distinction and practical usage of the terms “cryptic” and “pseudocryptic” species is discussed.     

Parallel evolution in ecological and reproductive traits to produce cryptic damselfly species across the holarctic

The damselfly genus Enallagma originated in the Nearctic, and two Nearctic lineages recently underwent radiations partly associated with multiple independent habitat shifts from lakes dominated by fish predators into lakes dominated by dragonfly predators. A previous molecular study of four Palearctic morphospecies and all representative Nearctic species identified the presence of two cryptic species sets, with each set having Palearctic and Nearctic representatives. However, the cryptic species within each set are not sibling species. Here, we present quantitative data on ecologically important larval morphologies and behaviors involved in predator avoidance and on adult male morphological structures involved in mate recognition to quantify the phenotypic relationships among these cryptic species sets. For the adult stage, our data indicate strong parallel evolution of the structures involved in specific mate recognition-the male cerci. For the larval stage, morphometric analyses show that the Palearctic species evolved a nearly identical morphology to the sibling-clade members in the Nearctic that live in waters where dragonflies are the top predators. This implicates the importance of dragonfly predation in the history of the Palearctic clade. Behavioral analyses suggest population differentiation in response to the actual predator environment in the Palearctic clade, consistent with the species differentiation seen in the Nearctic. Our results suggest parallel evolution of adult traits that influence specific mate choice and larval traits that influence ecological performance underlie the striking similarity of Enallagma species across continents. This concurrent parallel evolution in both stages of a complex life cycle, especially when both stages do not share the same selective environment, may be a very unusual mechanism generating cryptic species.