桉蝙蛾幼虫实时荧光定量PCR内参基因的筛选

本研究通过筛选桉蝙蛾Endoclita signifer Walker幼虫不同龄期与不同体节中稳定表达的内参基因,为桉蝙蛾基因表达研究提供参考。通过实时荧光定量PCR技术测定5个候选内参基因（ACTIN、GAPDH、TUB、RIB、EF）在桉蝙蛾幼虫不同龄期（3龄、5龄、9龄、12龄）与不同体节（头部、胸部、腹部）及全样品（由所有样品组成）处理中的表达量,后利用GeNorm、NormFinder、BestKeeper对5个候选基因的稳定性进行评估,最后由RefFinder综合分析结果,选出最佳的内参基因。基于4种分析方法的评估可知,桉蝙蛾5龄和9龄幼虫的不同体节、不同龄期幼虫的头部和胸部中内参基因的最佳数目为2,而3龄和12龄幼虫的不同体节、不同龄期幼虫的腹部及全样本中内参基因的最佳数目为3。3龄、5龄、9龄和12龄幼虫的不同体节中可分别选择ACTIN+RIB+GAPDH、EF+RIB、GAPDH+EF和ACTIN+RIB+GAPDH作为最稳定的内参基因组合;EF+RIB、RIB+GAPDH和EF+GAPDH+ACTIN可分别作为不同龄期幼虫头部、胸部和腹部的最佳内参基因组合;综合考虑桉蝙蛾幼虫不同龄期与不同体节的影响时,可选择RIB、ACTIN和GAPDH作为内参基因。     

Population structure and cryptic replacement of local populations in the endangered bitterling Acheilognathus cyanostigma

Ichthyological Research - Genetic structure and its artificial disturbance in the endangered Japanese bitterling Acheilognathus cyanostigma were examined based on mitochondrial cytochrome b gene...     

A comparison of mosquito predation by the fish Pseudomugil signifier Kner and Gambusia holbrooki (Girard) in laboratory trials.

Using larval Cx. annulirostris mosquitoes, this study compared the predation rate of Pseudomugil signifer and Gambusia holbrooki for four larval instars, three prey densities, and three vegetation densities. There was no significant difference in the quantity of first, second, and third instar larvae consumed by the two fish species after 24 h. There was a significant negative relationship between the predation rate and both larval instar and prey density for both fish species. In simulated vegetation trials, P. signifer performed marginally better than G. holbrooki in medium to high density vegetation (0.3 stems/cm 2 and 0.6 stems/cm2, respectively).     

Evolution of plant mitochondrial genomes via substoichiometric intermediates

Comparison of the modern fertile maize mitochondrial genome (N) with an ancestral maize mitochondrial genome (RU) reveals a 12 kb duplication (containing the atpA gene) in the modern genome that is absent from the ancestor. Cloning, mapping, and sequencing of the relevant portions of the ancestral genome shows that this duplication probably arose via a three-stage recombination process involving substoichiometric intermediates. Comparison with analogous observations on yeast mitochondrial genomes suggests that this three-stage model of genome reorganization can be generally applied to plant mitochondrial genomes to explain both deletions and the creation of novel repeats, common features of plant mitochondrial genome evolution.     

Structure and organization of the mitochondrial genome of the unicellular red alga Cyanidioschyzon merolae deduced from the complete nucleotide sequence.

The complete nucleotide sequence of the mitochondrial genome of a very primitive unicellular red alga, Cyanidioschyzon merolae , has been determined. The mitochondrial genome of C.merolae contains 34 genes for proteins including unidentified open reading frames (ORFs) (three subunits of cytochrome c oxidase, apocytochrome b protein, three subunits of F1F0-ATPase, seven subunits of NADH ubiquinone oxidoreductase, three subunits of succinate dehydrogenase, four proteins implicated in c-type cytochrome biogenesis, 11 ribosomal subunits and two unidentified open reading frames), three genes for rRNAs and 25 genes for tRNAs. The G+C content of this mitochondrial genome is 27.2%. The genes are encoded on both strands. The genome size is comparatively small for a plant mitochondrial genome (32 211 bp). The mitochondrial genome resembles those of plants in its gene content because it contains several ribosomal protein genes and ORFs shared by other plant mitochondrial genomes. In contrast, it resembles those of animals in the genome organization, because it has very short intergenic regions and no introns. The gene set in this mitochondrial genome is a subset of that of Reclinomonas americana , an amoeboid protozoan. The results suggest that plant mitochondria originate from the same ancestor as other mitochondria and that most genes were lost from the mitochondrial genome at a fairly early stage of the evolution of the plants.     

A complete mitochondrial genome sequence of Ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (Raphanus sativus L.)

ABSTRACT: BACKGROUND: Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS) is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF) created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138. RESULTS: Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3kb, similarity >99.9%). It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size) that are non-syntenic to normal-type genome, and the gene orf138 was found to be located at the edge of the largest unique region. Blast analysis performed to assign the unique regions showed that about 80% of the region was covered by short homologous sequences to the mitochondrial sequences of normal-type radish or other reported Brassicaceae species, although no homology was found for the remaining 20% of sequences. CONCLUSIONS: Ogura-type mitochondrial genome was highly rearranged compared with the normal-type genome by recombination through one large repeat and multiple short repeats. The rearrangement has produced four unique regions in Ogura-type mitochondrial genome, and most of the unique regions are composed of known Brassicaceae mitochondrial sequences. This suggests that the regions unique to the Ogura-type genome were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of Brassicaceae, and novel genes such as orf138 could have been created by the shuffling process of mitochondrial genome.     

The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.Correspondence to: F. Huang     

Translocation of a 190-kb mitochondrial fragment into rice chromosome 12 followed by the integration of four retrotransposons

A 190-kb mitochondrial DNA sequence interrupted by seven foreign DNA segments was identified in rice chromosome 12. This fragment is the largest mitochondrial fragment translocated into the rice nuclear genome. The sequence is composed of a 190-kb segment of mitochondrial origin corresponding to 38.79% of the mitochondrial genome, 45 kb comprising four segments of retrotransposon origin, and 13 kb comprising three segments of unknown origin. The 190-kb sequence shows more than 99.68% similarity to the current mitochondrial sequence, suggesting that its integration into the nucleus was quite recent. Several sequences in the 190-kb segment have been rearranged relative to the current mitochondrial sequence, suggesting that the past and present arrangements of the mitochondrial genome differ. The four retrotransposons show no mutual sequence similarity and are integrated into different locations, suggesting that their integration events were independent, frequent, and quite recent. A fragment of the mitochondrial genome present in the nuclear genome, such as the 248-kb sequence characterized in this study, is a good relic with which to investigate the past mitochondrial genome structure and the behavior of independent retrotransposons during evolution.     

Nuclear genes that encode mitochondrial proteins for DNA and RNA metabolism are clustered in the Arabidopsis genome

The plant mitochondrial genome is complex in structure, owing to a high degree of recombination activity that subdivides the genome and increases genetic variation. The replication activity of various portions of the mitochondrial genome appears to be nonuniform, providing the plant with an ability to modulate its mitochondrial genotype during development. These and other interesting features of the plant mitochondrial genome suggest that adaptive changes have occurred in DNA maintenance and transmission that will provide insight into unique aspects of plant mitochondrial biology and mitochondrial-chloroplast coevolution. A search in the Arabidopsis genome for genes involved in the regulation of mitochondrial DNA metabolism revealed a region of chromosome III that is unusually rich in genes for mitochondrial DNA and RNA maintenance. An apparently similar genetic linkage was observed in the rice genome. Several of the genes identified within the chromosome III interval appear to target the plastid or to be targeted dually to the mitochondria and the plastid, suggesting that the process of endosymbiosis likely is accompanied by an intimate coevolution of these two organelles for their genome maintenance functions.     

一种棉花线粒体DNA的提取方法

线粒体是重要的细胞器,它有自身的基因组。其基因组DNA与细胞核基因组DNA相比,含量较低。棉花当中富含棉酚、丹宁等物质,这对提取DNA有很大的影响。因此我们根据棉花自身的特点,找到了一种提取棉花线粒体DNA经济有效的方法,其质量可以满足限制性酶切、PCR、分子杂交等实验的要求。     

The current landscape for the treatment of mitochondrial disorders

The mitochondrial organelle is crucial to the energy metabolism of the eukaryotic cell. Defects in mitochondrial function lie at the core of a wide range of disorders, including both rare primary mitochondrial disorders and more common conditions such as Parkinson's disease and diabetes. Inherited defects in mitochondrial function can be found in both the nuclear genome and the mitochondrial genome, with the latter creating unique challenges in the treatment and understanding of disease passed on through the mitochondrial genome. In this review, we will describe the limited treatment regimens currently used to alleviate primary mitochondrial disorders, as well as the potential for emerging technologies (in particular, those involving direct manipulation of the mitochondrial genome) to more decisively treat this class of disease. We will also emphasize the critical parallels between primary mitochondrial disorders and more common ailments such as cancer and diabetes.     

Mitochondrial DNA replication and repair defects: Clinical phenotypes and therapeutic interventions

Mitochondria is a unique cellular organelle involved in multiple cellular processes and is critical for maintaining cellular homeostasis. This semi-autonomous organelle contains its circular genome – mtDNA (mitochondrial DNA), that undergoes continuous cycles of replication and repair to maintain the mitochondrial genome integrity. The majority of the mitochondrial genes, including mitochondrial replisome and repair genes, are nuclear-encoded. Although the repair machinery of mitochondria is quite efficient, the mitochondrial genome is highly susceptible to oxidative damage and other types of exogenous and endogenous agent-induced DNA damage, due to the absence of protective histones and their proximity to the main ROS production sites. Mutations in replication and repair genes of mitochondria can result in mtDNA depletion and deletions subsequently leading to mitochondrial genome instability. The combined action of mutations and deletions can result in compromised mitochondrial genome maintenance and lead to various mitochondrial disorders. Here, we review the mechanism of mitochondrial DNA replication and repair process, key proteins involved, and their altered function in mitochondrial disorders. The focus of this review will be on the key genes of mitochondrial DNA replication and repair machinery and the clinical phenotypes associated with mutations in these genes.     

Complete mitochondrial genome of the red-spotted tokay gecko (Gekko gecko, Reptilia: Gekkonidae): comparison of red- and black-spotted tokay geckos

Here, we sequenced the complete mitochondrial genome of the red-spotted tokay gecko (Squamata: Gekkonidae). The genome is 16,590 bp in size. Its gene arrangement pattern was identical with that of black-spotted tokay gecko. We compared the mitochondrial genome of red-spotted tokay gecko with that of the black-spotted tokay gecko. Nucleotide sequence of the two whole mitochondrial genomes was 97.99% similar, and the relatively high similarity seems to indicate that they may be separated at the subspecies level. The information of mitochondrial genome comparison of the two morphological types of tokay gecko is discussed in detail.     

Complete mitochondrial genome of Kallima inachus (Lepidoptera: Nymphalidae: Nymphalinae): comparison of K. inachus and Argynnis hyperbius

In this paper, we sequenced the complete mitochondrial genome of Kallima inachus (Lepidoptera: Nymphalidae: Nymphalinae), which is considered a rare species in China. The genome is 15,183 bp in size. Its gene arrangement pattern was identical with those of Argynnis hyperbius. We compared the mitochondrial genome of K. inachus with that of A. hyperbius. Nucleotide sequence similarity between the two whole mitochondrial genomes was 85.92%, and the relatively low similarity seems to indicate that the two species are distinctly separated on the species level. The information on the mitochondrial genome comparison of the two species is discussed in detail in this paper.     

The mitochondrial genome of the soybean cyst nematode, Heterodera glycines

We sequenced the entire coding region of the mitochondrial genome of Heterodera glycines. The sequence obtained comprised 14.9 kb, with PCR evidence indicating that the entire genome comprised a single, circular molecule of approximately 21-22 kb. The genome is the most T-rich nematode mitochondrial genome reported to date, with T representing over half of all nucleotides on the coding strand. The genome also contains the highest number of poly(T) tracts so far reported (to our knowledge), with 60 poly(T) tracts ≥ 12 Ts. All genes are transcribed from the same mitochondrial strand. The organization of the mitochondrial genome of H. glycines shows a number of similarities compared with Radopholus similis, but fewer similarities when compared with Meloidogyne javanica. Very few gene boundaries are shared with Globodera pallida or Globodera rostochiensis. Partial mitochondrial genome sequences were also obtained for Heterodera cardiolata (5.3 kb) and Punctodera chalcoensis (6.8 kb), and these had identical organizations compared with H. glycines. We found PCR evidence of a minicircular mitochondrial genome in P. chalcoensis, but at low levels and lacking a noncoding region. Such circularised genome fragments may be present at low levels in a range of nematodes, with multipartite mitochondrial genomes representing a shift to a condition in which these subgenomic circles predominate.     

The complete mitochondrial genome of Caprella scaura (Crustacea, Amphipoda, Caprellidea), with emphasis on the unique gene order pattern and duplicated control region

The nucleotide and amino acid sequences and the gene order of the mitochondrial genome are highly informative for studying phylogeny, population genetics, and phylogeography. This study determined the complete mitochondrial genome of the caprellid species Caprella scaura. The mitochondrial genome of C. scaura has a total length of 15,079 bp, with an AT content of 66.43%. The mitochondrial genome contains typical gene components, including 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes. In comparison with the mitochondrial genome of a gammarid, some distinct characteristics were found. For example, the order of the two conserved gene blocks is inverted between Gammaridea and C. scaura. In addition, two copies of almost identical control regions were found in the mitochondrial genome of C. scaura. These unique characteristics will be useful for determining the evolutionary history of the Caprellidea.     

Mitochondrial genome structure of rice suspension culture from cytoplasmic male-sterile line (A-58CMS): reappraisal of the master circle

Summary The mitochondrial DNA (mtDNA) from the cultured cells of a cytoplasmic male-sterile line (A-58CMS) of rice (Oryza sativa) was cloned and its physical map was constructed. There was structural alteration on the mitochondrial genome during the cell culture. Detailed restriction analysis of cosmid clones having mtDNA fragments suggested either that the master genome has a 100-kb duplication (the genome size becomes 450 kb) or that a master circle is not present in the genome (the net structural complexity becomes 350 kb). The physical map of plant mitochondrial genomes thus far reported is illustrated in a single circle, namely a master circle. However, no circular DNA molecule corresponding to a master circle has yet been proved. In the present report, representation of plant mitochondrial genomes and a possibility for mitochondrial genome without a master circle are discussed.     

Acheilognathus changtingensis sp. nov., a new species of the cyprinid genus Acheilognathus (Teleostei: Cyprinidae) from Southeastern China based on morphological and molecular evidence

A new species of the bitterling genus Acheilognathus, Acheilognathus changtingensis sp. nov., was recently discovered from Changting County in Hanjiang River, Fujian Province, Southeastern China. It can be diagnosed by the following combination of characters: dorsal fin with three simple and 15 (occasionally 14) branched fin rays, anal fin with three simple and 12 (occasionally 11) branched fin rays; dorsal fin pale and anal fin slightly pale; white spots on anal-fin rays forming a transverse band, and anal fin margined with white band in males. Its unique characters are the many dispersed black spots on the dorsal, anal, pelvic, caudal fins, and on the head. Using the mitochondrial cytochrome Jb gene as a molecular marker, we reconstructed phylogenetic trees of A. changtingensis sp. nov. and other species in Acheilognathus to confirm its taxonomic status and study its speciation. Analyses of both morphological and molecular data consistently indicated the taxonomic status of the present new species. The results also show that A. changtingensis sp. nov. and Acheilognathus macropterus are sister species that diverged about 14.50 MYA by geographical isolation.     

Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice

Summary The entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice (Oryza sativa cv. Nipponbare) was identified using clone banks that cover the chloroplast and mitochondrial genomes. The mitochondrial fragments that were homologous to chloroplast DNA were mapped and sequenced. The nucleotide sequences around the termini of integrated chloroplast sequences in the rice mtDNA revealed no common sequences or structures that might enhance the transfer of DNA. Sixteen chloroplast sequences, ranging from 32 bases to 6.8 kb in length, were found to be dispersed throughout the rice mitochondrial genome. The total length of these sequences is equal to approximately 6% (22 kb) of the rice mitochondrial genome and to 19% of the chloroplast genome. The transfer of segments of chloroplast DNA seems to have occurred at different times, both before and after the divergence of rice and maize. The mitochondrial genome appears to have been rearranged after the transfer of chloroplast sequences as a result of recombination at these sequences. The rice mitochondrial DNA contains nine intact tRNA genes and three tRNA pseudogenes derived from the chloroplast genome.