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1.
Sci Rep ; 8(1): 11640, 2018 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-30076357

RESUMO

Deceptive pollination is key to the species richness of Orchidaceae. However, the genetic basis of species diversification is still under study. Section Trigonopedia is a monophyletic clade of genus Cypripedium distributed in the southwest of China. The species of this section are pollinated by different flies. Pollinator differentiation makes section Trigonopedia an ideal group for studying the genetic basis underlying species diversification. Here, we sequenced the transcriptomes of eight species of the genus Cypripedium, including six co-flowering species of section Trigonopedia and two species outside this section as an outgroup. We reconstructed the phylogeny of the section with the combined 1572 single-copy genes extracted from the eight species and produced a highly resolved tree of the section. Furthermore, we combined substitution rate estimation and differential expression analysis to identify candidate genes, including genes related to floral scent synthesis and environmental adaptation, involved in species differentiation. Field investigations showed that these species have adapted to different habitats. We propose that the species diversification in this section is initiated by floral scent differentiation, followed by habitat differentiation, finally leading to speciation. This study sheds novel light on the diversification of closely related orchid species in the Qinghai-Tibetan region.


Assuntos
Adaptação Fisiológica/genética , Orchidaceae/genética , Polinização/genética , Transcriptoma/genética , Animais , Dípteros/genética , Dípteros/fisiologia , Flores/genética , Flores/crescimento & desenvolvimento , Orchidaceae/crescimento & desenvolvimento , Especificidade da Espécie
2.
PLoS One ; 11(1): e0146880, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26752741

RESUMO

Orchidaceae are one of the largest families of flowering plants, with over 27,000 species described and all orchids are listed in CITES. Moreover, the seedlings of orchid species from the same genus are similar. The objective of DNA barcoding is rapid, accurate, and automated species identification, which may be used to identify illegally traded endangered species from vegetative specimens of Paphiopedilum (Venus slipper), a flagship group for plant conservation with high ornamental and commercial values. Here, we selected eight chloroplast barcodes and nrITS to evaluate their suitability in Venus slippers. The results indicate that all tested barcodes had no barcoding gap and the core plant barcodes showed low resolution for the identification of Venus slippers (18.86%). Of the single-locus barcodes, nrITS is the most efficient for the species identification of the genus (52.27%), whereas matK + atpF-atpH is the most efficient multi-locus combination (28.97%). Therefore, we recommend the combination of matK + atpF-atpH + ITS as a barcode for Venus slippers. Furthermore, there is an upper limit of resolution of the candidate barcodes, and only half of the taxa with multiple samples were identified successfully. The low efficiency of these candidate barcodes in Venus slippers may be caused by relatively recent speciation, the upper limit of the barcodes, and/or the sampling density. Although the discriminatory power is relatively low, DNA barcoding may be a promising tool to identify species involved in illegal trade, which has broad applications and is valuable for orchid conservation.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA de Cloroplastos/genética , DNA de Plantas/genética , Genes de Plantas , Orchidaceae/genética , DNA Intergênico/genética , Variação Genética , Haplótipos , Filogenia , Reprodutibilidade dos Testes , Especificidade da Espécie
4.
Nat Genet ; 47(1): 65-72, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25420146

RESUMO

Orchidaceae, renowned for its spectacular flowers and other reproductive and ecological adaptations, is one of the most diverse plant families. Here we present the genome sequence of the tropical epiphytic orchid Phalaenopsis equestris, a frequently used parent species for orchid breeding. P. equestris is the first plant with crassulacean acid metabolism (CAM) for which the genome has been sequenced. Our assembled genome contains 29,431 predicted protein-coding genes. We find that contigs likely to be underassembled, owing to heterozygosity, are enriched for genes that might be involved in self-incompatibility pathways. We find evidence for an orchid-specific paleopolyploidy event that preceded the radiation of most orchid clades, and our results suggest that gene duplication might have contributed to the evolution of CAM photosynthesis in P. equestris. Finally, we find expanded and diversified families of MADS-box C/D-class, B-class AP3 and AGL6-class genes, which might contribute to the highly specialized morphology of orchid flowers.


Assuntos
Genoma de Planta , Orchidaceae/genética , Evolução Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Íntrons/genética , Proteínas de Domínio MADS , Taxa de Mutação , Orchidaceae/classificação , Orchidaceae/metabolismo , Fotossíntese/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA de Plantas/biossíntese , RNA de Plantas/genética , Alinhamento de Sequência , Especificidade da Espécie
5.
PLoS One ; 8(4): e60097, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23577083

RESUMO

BACKGROUND: The Aerides-Vanda alliance is a complex group in the subtribe Aeridinae (subfamily Epidendroideae, Orchidaceae). Some phylogenetic systems of this alliance have been previously proposed based on molecular and morphological analyses. However, several taxonomic problems within this alliance as well as between it and its allies remain unsolved. METHODOLOGY/PRINCIPAL FINDINGS: We utilized ITS and five plastid DNA regions in this phylogenetic analysis. Consensus trees strongly indicate that the Aerides-Vanda alliance is monophyletic, and the 14 genera of this alliance can be grouped into the following clades with 14 subclades: 1. Aerides, comprising two subclades: Rhynchostylis and Aerides; 2. Ascocentropsis; 3. Papilionanthe; 4. Vanda, comprising five subclades: Neofinetia, Christensonia, Seidenfadenia, Ascocentrum, and Vanda-Trudelia, in which Vanda and Trudelia form a subclade; 5. Tsiorchis, comprising three subclades: Chenorchis, Tsiorchis, and two species of Ascocentrum; 6. Paraholcoglossum; and 7. Holcoglossum. Among the 14 genera, only Ascocentrum is triphyletic: two species of the Ascocentrum subclade, an independent subclade Ascocentrum subclade in the Tsiorchis clade; the Ascocentrum subclade in the Vanda clade; and one species in the Holcoglossum clade. The Vanda and Trudelia species belong to the same subclade. The molecular conclusion is consistent with their morphological characteristics. CONCLUSIONS: We elucidate the relationship among the 14 genera of the Aerides-Vanda alliance. Our phylogenetic results reveal that the Aerides-Vanda alliance is monophyletic, but it can be divided into 14 genera. The data prove that Ascocentrum is triphyletic. Plants with elongate-terete leaves and small flowers should be treated as a new genus, Pendulorchis. Saccolabium himalaicum (Ascocentrum himalaicum) should be transferred to Pendulorchis. Ascocentrum pumilum, endemic to Taiwan, should be transferred to Holcoglossum. A new combination, Holcoglossum pumilum, was also established. Trudelia should not be recognized as an independent genus. Two new species, Pendulorchis gaoligongensis and Holcoglossum singchianum, were described as well.


Assuntos
Orchidaceae/classificação , Filogenia , Cloroplastos/genética , DNA Intergênico/genética , DNA de Plantas/genética , Orchidaceae/citologia , Orchidaceae/genética
6.
PLoS One ; 8(1): e53695, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23308277

RESUMO

BACKGROUND: Outcrossing is known to carry genetic advantages in comparison with inbreeding. In many cases, flowering plants develop a self-incompatibility mechanism, along with a floral component adaptation mechanism, to avoid self-pollination and to promote outbreeding. Orchids commonly have a lip in their flower that functions as the a visiting plate for insect pollinators. Aside from the lip, however, many species (including Coelogyne rigida) have sheaths around the axis of inflorescence. The function of these sheaths remains unknown, and has long been a puzzle to researchers. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the function of these sheaths in relation to the lip and the pollinators, as well as their role in the modes of pollination and reproduction of Coelogyne rigida in 30 flowering populations of orchids in the limestone area of Southeast Yunnan, China. We found that self-incompatible C. rigida developed specialized bird perches around the basal axis of inflorescence to attract sunbirds and to complement their behavioral tendency to change foraging locations frequently. This self-incompatibility mechanism operates separately from the floral component adaptation mechanism. This mechanism thus prevents bees from repeatedly visiting the floral lip of the same plant which, in turn, results in autogamy. In this way, instead of preventing autogamy, C. rigida responds to these negative effects through a highly efficient cross-pollination method that successfully transfers pollen to different plants. CONCLUSIONS: The proposed method ensures reproductive success, while offsetting the infertile self-pollination by insects, thereby reducing mating costs and addressing the lack of cross-pollination. The adaptation provides a novel and striking example of structural adaptation that promotes cross-pollination in angiosperms.


Assuntos
Adaptação Fisiológica , Aptidão Genética/fisiologia , Inflorescência/anatomia & histologia , Orchidaceae/anatomia & histologia , Reprodução/fisiologia , Animais , Abelhas , Aves/fisiologia , China , Comportamento Alimentar , Feminino , Inflorescência/fisiologia , Masculino , Orchidaceae/fisiologia , Pólen/fisiologia , Polinização , Vespas
7.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 25(7): 592-5, 599, 2009 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-19737475

RESUMO

AIM: To construct the recombinant plasmid of pGPC3-EGFP containing human AFP(542-550) gene, EGFP gene and GPC3 gene to express fusion protein GPC3-hAFP(542-550)-EGFP and to discover its localization on cytoplasmic membrane. METHODS: GPC3 gene was obtained from total RNA of human placental tissues by RT-PCR; After the enhanced green fluorescent protein (EGFP) gene was amplified from pEGFP-N1 plasmid and the gene segment of-KOZAK-GPCN + afp(542-550)-was chemically synthesized, the recombinant plasmid pcDNA3.1(+)/GPCN+afp(542-550)-EGFP-GPCC (pGPC3-EGFP) containing three chimeric genes of strong epitope hAFP(542-550), GPI-anchored protein GPC3 and EGFP was constructed. The fusion protein (GPC3-hAFP(542-550)-EGFP) was detected on RNA and protein levels at 24 h and 48 h after pGPC3-EGFP was transfected into HepG2 (GPC3(+)AFP(+)) via lipofectamine 2000. EGFP expression was observed by fluorescent microscopy after pGPC3-EGFP was transfected into HepG2 using pEGFP-N1 plasmid transfection as a positive control. The fusion protein in both membrane proteins and soluble proteins extracted from the transfected 293 cells (GPC3(-)AFP(-)) was detected by Western blot using GPC3 monoclonal antibody as primary antibody. RESULTS: The recombinant plasmid pGPC3-EGFP was successfully constructed through restriction endonuclease digestion and sequencing; pGPC3-EGFP expression in HepG2 cells was detected not only by RT-PCR using specific primers (GPCN-F and EGFP-r) but also by Western blot using GFP polyclonal antibody and GPC3 monoclonal antibody. Green fluoresce was mainly found around pGPC3-EGFP transfected HepG2 cell periphery beside sporadic distribution in cytoplasm, but that of pEGFP-N1 transfected HepG2 cell was evenly distributed in the whole cell. Moreover, the fusion protein was not detected in soluble proteins but membrane proteins extracted from transfected 293 cells. CONCLUSION: The recombinant plasmid of pGPC3-EGFP based on protein engineering theory can express fusion protein (GPC3-hAFP(542-550)-EGFP) in eukaryotic cells. Furthermore, the fusion protein is still located on cytoplasmic membrane, which is a characteristic of GPI-anchored membrane protein, and is a new GPI-reanchored protein.


Assuntos
Membrana Celular/metabolismo , Epitopos/genética , Glipicanas/genética , Fragmentos de Peptídeos/genética , Proteínas Recombinantes/biossíntese , alfa-Fetoproteínas/genética , Proteínas de Fluorescência Verde/genética , Células Hep G2 , Humanos , Plasmídeos , Transfecção , alfa-Fetoproteínas/imunologia
8.
J Dig Dis ; 8(2): 89-95, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17532821

RESUMO

OBJECTIVE: To testify that the asialoorosomucoid (ASOR) prepared by us has liver-targeting specificity and to investigate its pharmacokinetic characteristics. METHODS: The distribution of 125I-ASOR in vivo was determined by single photon emission computed tomography (SPECT) and immunohistochemical technique after 125I-ASOR was injected into Sprague-Dawley (S-D) rats through their caudal veins. In vitro, different doses of pEGFP-N1 plasmid were transfected into both HepG2 cells and HT1080 cells with the use of ASOR-poly-L-lysine. At 24 and 48 h after transfection, the expression of green fluorescent protein (GFP) was determined under fluorescent microscope. Pharmacokinetic parameters were calculated according to two-compartment open system model with first-order kinetics. RESULTS: SPECT images showed that 125I-ASOR was located only in liver/stomach and root of caudal vein/bladder at 10 min after injection. The 125I-ASOR radioactivities of organs taken out from S-D rats were different at different times, and about 63% of 125I-ASOR was located in the liver at 10 min after injection. At 30 min after injection a peak of radioactivity was seen in stomach. The times of these two radioactivity peaks were different. Immunohistochemical study of liver frozen sections showed that ASOR was combined mainly with hepatocyte membrane, especially in areas with rich blood flow. In vitro study showed that ASOR targeted specifically cells with asialoglycoprotein receptor (ASGr). GFP expression was detected in HepG2 cells but not in HT1080 cells. Furthermore, the more quantity of pEGFP-N1 transfected and the longer expression time, the higher GFP expression level was in HepG2 cells. The 125I-ASOR pharmacokinetics equation for liver was Ct=662216e-3.362t+8896e-2343t. 125I-ASOR was excreted from liver slowly after an initial rapid decrease. The pharmacokinetic equation for stomach was Ct=-114815e-1.7t+1148153e-15t and the half-life of 125I-ASOR in stomach was 4.62 h. CONCLUSIONS: ASOR prepared by us could be an efficient gene transfer vector, ASOR was distributed mainly in the liver and stomach and had high targeting specificity to hepatocytes or hepatic originating cells.


Assuntos
Receptor de Asialoglicoproteína/efeitos dos fármacos , Assialoglicoproteínas/farmacocinética , Glicoproteínas/farmacocinética , Fígado/metabolismo , Orosomucoide/análogos & derivados , Estômago/efeitos dos fármacos , Animais , Assialoglicoproteínas/química , Técnicas de Transferência de Genes , Vetores Genéticos , Glicoproteínas/química , Hepatócitos/metabolismo , Injeções Intravenosas , Radioisótopos do Iodo , Orosomucoide/química , Orosomucoide/farmacocinética , Ratos , Ratos Sprague-Dawley , Tomografia Computadorizada de Emissão de Fóton Único
9.
Wei Sheng Wu Xue Bao ; 46(5): 802-6, 2006 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-17172032

RESUMO

The function of NS4B is incompletely understood. The aim of the study is to understand the influence of NS4B on anti-viral response. After cell line stably expressing NS4B established, the influence of IFN-alpha of different concentration on VSV was studied using plaque assay; cell expression profiling caused by NS4B was studied using DNA microarray, and the IFNGR1 fluorescence intensity was analyzed. Our data showed that HCV-NS4B could suppress immuno-associated gene expression, in particular, IFN-gamma receptor signal transduction-related genes. Taken together, NS4B could play some roles in HCV resistance to IFN therapy.


Assuntos
Receptores de Interferon/antagonistas & inibidores , Transdução de Sinais/fisiologia , Proteínas não Estruturais Virais/fisiologia , Farmacorresistência Viral , Regulação da Expressão Gênica , Células HeLa , Hepatite C/tratamento farmacológico , Humanos , Interferon gama/uso terapêutico , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Interferon/fisiologia
11.
Nature ; 441(7096): 945-6, 2006 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-16791185

RESUMO

Mating in flowering plants normally relies on animals, wind, gravity or secretion to convey pollen grains from the male (anther) to the female (stigma) organ. Here we describe a new type of self-pollination mechanism in the tree-living orchid Holcoglossum amesianum, in which the bisexual flower turns its anther against gravity through 360 degrees in order to insert pollen into its own stigma cavity - without the aid of any pollinating agent or medium. This mode of self-pollination, which occurs under windless, drought conditions when insects are scarce, adds to the variety of mechanisms that have evolved in angiosperms to ensure their reproductive success.


Assuntos
Fertilização , Flores , Orchidaceae/fisiologia , Pólen , China , Sementes
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