线粒体调控细胞凋亡的研究进展

线粒体与细胞凋亡有着密切的关系。细胞凋亡过程中线粒体释放膜间隙中与凋亡相关的活性物质,如细胞色素C、A IF、Smac/D IABLO等, 促进活性氧的产生,线粒体上MPTP孔道的开放,削弱了线粒体膜两侧的质子梯度,导致ATP合成下降;释放的活性物质激活Caspase,引起细胞凋亡。     

利用微卫星和线粒体DNA标记研究山羊群体间的遗传关系

本研究利用微卫星和线粒体DNA分析山羊的遗传多样性及系统进化关系,用25个微卫星位点分析了安哥拉山羊、山东莱芜黑山羊、罕山白绒山羊、太行山羊及乌珠穆沁绒山羊这5个山羊品种的遗传多样性,共祖遗传距离的UPGMA聚类表明遗传距离和地理距离是一致的,如内蒙的罕山白绒山羊和乌珠穆沁绒山羊聚到一起。利用线粒体DNA分析安哥拉山羊、山东莱芜黑山羊、鲁北白山羊、太行山羊及乌珠穆沁绒山羊,揭示这5个山羊品种分成A和C 2个支系,并进行了群体结构和群体扩张分析。通过比较2种分子标记的分析结果,发现利用微卫星来研究群体的遗传多样性及品种间的关系具有较高的准确性,而线粒体在研究群体系统进化具有一定的优势,在分析品种间关系方面可能不是理想标记。     

Multiple changes in tobacco mitochondrial DNA populations during tissue culture

Changes in the composition of mitochondrial DNA (mtDNA) in tobacco during the processes of long-term tissue culture and regeneration were detected by DNA gel blot analyses using several mtDNA fragments as probes. The changes in mtDNA were identified by differential accumulations of mtDNA fragments in cultured cells that were hybridized with probes atp6, cob, and nad3-rpsl2. The changes detected by each probe did not appear to be linked, since they were different with each probe and in different subcultures of calli. Based on the current results and previous detailed analyses of the changes detected by atp6, a possible model that explains the alterations in mtDNA during tissue culture is presented. In this model, the changes are attributed to multiple shifts in the equilibrium that was maintained between different subgenomic mtDNA molecules.     

Changes in sugar beet mitochondrial DNA induced during callus stage

The organization of mitochondrial DNA was studied in seed‐derived plants of sugar beet, Beta vulgaris L., and compared with the structure of DNA isolated from calli propagated in vitro. Tissues representing three genotypes (diploid male sterile, diploid maintainer and diploid fertile) were maintained under in vitro conditions for different periods before the DNA was analysed. Restriction fragment length polymorphism with BamHI and XhoI enzymes, and Southern hybridization with atpA and atp6 homologous probes were used. Hybridization experiments showed conspicuous differences in the organization of both genes in long‐term callus cultures (2 years or older). The novel organization of mitochondrial DNA in the calli of the male sterile genotype showed an additional 3.9 kb fragment after hybridization with the atp6, and 7.9 kb and 3.5 kb bands after hybridization with atpA. In the fertile genotype, changes in mitochondrial DNA structure were manifested in the disappearance of a 2.1 kb fragment after probing with atpA. Alterations induced in the mitochondrial genome of the male sterile line showed unique features in mtDNA rearrangements.     

基于线粒体RNAs序列分析雁形目家禽的分歧时间

雁形目鸟类具有重要的社会经济意义和科学研究价值,部分种类已被驯化成为重要的家禽,然而雁形目内部物种之间的关系仍存在许多争议。本研究利用线粒体RNAs序列标定雁形目部分鸟类的分歧时间,旨在进一步查明该类群系统进化关系。首先, 采用酚仿法分别从太湖鹅和朗徳鹅血液中提取基因组DNA,PCR 扩增获得线粒体RNAs基因序列。 然后,从GenBank 中提取10 种鸟类线粒体DNA的同源序列,以鸵鸟为外群,采用贝叶斯法结合RNA结构信息重建系统发生关系;最后,以黑雁属与雁属的分歧时间为锚定点,利用r8s软件分析了雁形目物种间的分歧时间。 结果表明,中国鹅与欧洲鹅的分歧时间为0.61百万年,番鸭与家鸭的分歧时间为15.5百万年。依据家鹅祖先分歧时间,联系当时发生的地学事件,作者认为家鹅祖先物种分化与更新世冰川运动有关。研究结果有助于我国水禽遗传资源的保护与利用。     

Absence of a correlation between mitochondrial complementation and root weight heterosis in sugarbeets

Summary Oxidative phosphorylation of sugarbeet root mitochondria isolated from 11 hybrids and their parental inbreds, and of mixtures of mitochondria from the parental inbreds, were measured at four different sampling stages, (Aug. 1–7, Sept. 1–7, roots stored at 5°C, and regrowth of stored roots). Mitochondrial complementation (the increased efficiency or ADP:0 ratio of mixtures of inbred mitochondria) was calculated for each hybrid at each stage. When beets were growing rapidly (Aug. 1–7) it was small but at the other stages it was absent. Only at the Aug. 1–7 sampling stage was there a significant correlation (0.75) between mitochondrial complementation and root weight heterosis.The small complementation effects made it difficult to detect differences in complementation, and thus difficult to predict root yield heterosis. Thus, this technique is not recommended as a tool in sugarbeet breeding.     

Significance of mitochondrial complementation for plant breeding: Negative evidence from a study on maize

Summary Mitochondrial activity was assessed in two double-cross varieties of Zea mays, in their single-cross parents and in their inbred lines. Mitochondria were isolated from shoots of dark-grown seedlings. An improved isolation procedure yielding stable mitochondria is described. A strong positive correlation was found between the adenosine-5-diphosphate to oxygen ration (ADP:O) and the respiratory control (RC). No mitochondrial complementation in terms of ADP:O, RC, and oxygen uptake could be detected. The results are discussed in relation to the possible role of mitochondria in hybrid vigour.     

胡萝卜叶片线粒体DNA提取方法研究

本实验以胡萝卜叶片为材料,通过高盐差速离心和蔗糖沉淀差速离心两种方法提取线粒体DNA。通过改变分离和沉淀线粒体离心力大小、使用DNaseⅠ处理得到了无核DNA污染的线粒体。用SDS和蛋白酶K 裂解线粒体,经酚/氯仿/异戊醇抽提除去蛋白,并用RNase A 消化从而得到单纯线粒体DNA。本实验还特别设计了叶绿体特异性引物检测线粒体DNA纯度。结果表明：利用优化后的蔗糖沉淀差速离心法提取胡萝卜线粒体DNA,不仅操作简单,而且所得线粒体DNA纯度高、产量高,每克叶片能够得到34.46 μg 线粒体DNA。     

The possible use of mitochondrial complementation as an indicator of yield heterosis in breeding hybrid wheat

Summary Nine hybrid wheats and their six parental lines were grown in a field trial at two seed densities. The ADP:0 ratios for mitochondria extracted from the six parental lines and for the appropriate 1:1 mixtures of mitochondria from these parental lines were measured. The percentage additional mitochondrial efficiency of the mixtures over their more efficient parental line was found to be significantly correlated with the percentage yield heterosis of fully restored relatively disease-free hybrids grown at the lower seed density. These results indicate that measurements of mitochondrial complementation may have some value as a first screening of potential parental lines and may thus facilitate the rational choice of suitable material for producing hybrid wheat.     

Restriction analysis of the mitochondrial DNA of male-sterile and maintainer lines of pearl millet, Pennisetum americanum L.

Mitochondrial DNA from two pairs of cytoplasmic male-sterile (cms) and maintainer lines of pearl millet was investigated by restriction-enzyme analysis and Southern-blot hybridization using three mitochondrial gene probes. Each pair of male-sterile and maintainer lines was of a different nuclear origin. The objective was to distinguish differences in the DNA base-sequence organization of the mitochondrial genomes of cms and maintainer lines from the two sources. Restriction-enzyme analysis revealed differences between the different cms and maintainer lines. Southern-blot hybridization experiments using cloned mitochondrial gene probes further distinguished differences between different lines. It is expected that the restriction-fragment-length polymorphisms revealed in the Southern-blot-hybridization experiments will be useful in distinguishing and classifying cms and maintainer lines obtained from different nuclear backgrounds.     

Genetic variation of mitochondrial and nuclear genomes in carrots revealed by random amplified polymorphic DNA (RAPD)

Mitochondrial and nuclear genomic diversities of 8 carrot (Daucus carota ssp. sativus) varieties, including 6 pure lines and 2 cytoplasmic male sterile (cms) lines, were taxonomically identified using PCR with 19 RAPD primers. Dendrograms based on polymorphisms of both mitochondrial and nuclear genomes were constructed. According to the dendrogram of the mitochondrial genome revealed by RAPD, 4 differentiated clusters formed, in good accordance with the classification based on analyses with restriction enzyme digestion. Two cms lines were grouped into the same cluster, as genetically separated from the others. Thus, the cytoplasm donors of these male sterile lines were thought to be wild carrots. Conversely, RAPD analysis of the nuclear genome for these eight cultivars revealed no evident clusters although some cultivars were of a similar origin or place of cultivation. A correlation between nuclear and mitochondrial dendrograms was absent. RAPD has proved to be a useful tool for identifying mitochondrial and nuclear genomes. This technique will greatly aid in promoting efficient improvement of carrots. This revised version was published online in July 2006 with corrections to the Cover Date.     

The use of mitochondrial DNA polymorphisms to distinguish commercial cultivars of the broad bean, Vicia faba L.

Summary Restriction fragment length polymorphisms (RFLPs) have been used to detect mitochondrial DNA (mtDNA) variation among 9 commercial cultivars of Vicia faba L. The mitochondrial DNA was initially digested with 8 restriction endonucleases revealing complex banding patterns on ethidium bromide (EtBr) stained gels. However, no RFLPs were visualised from these complex profiles. Southern hybridisation using total digested mtDNA as a probe against mtDNA digested with the same restriction enzymes revealed a limited number of RFLPs which allowed the 9 cultivars to be consistently distinguished into two main cytoplasmic types. Southern hybridisation with 23 random mitochondrial clones covering 56 kb of the mitochondrial genome revealed considerable levels of polymorphisms. Of the 23 clones analysed, 12 detected at least 22 polymorphisms using 3 restriction enzymes among the cultivars analysed. These RFLPs allowed the 9 commercial cultivars analysed in this study to be distinguished into at least 6 separate groups. Most polymorphisms distinguished the cultivars into two main cytoplasmic groups.     

Assessment of cytoplasmic polymorphisms by PCR-RFLP of the mitochondrial orfB region in wild and cultivated radishes (Raphanus)

In order to determine the genetic relationship between wild and cultivated radish species, and those among the cultivated species, structural and sequence variations in the mitochondrial orfB gene region were studied in one cultivated and two wild species of Raphanus. Using PCR amplification patterns and RFLP of a PCR product of the region, 232 wild and 420 cultivated radish plants were classified into one of three types of orfB variation. The wild radish (especially the Japanese one) showed large polymorphism in each population with eight of 13 Japanese populations studied containing all three types, whereas cultivars were generally monomorphic. Although type 1 having Ogura male sterile cytoplasm was present with the highest frequency in Japanese wild radish, most cultivars were divided into type 2 or 3 with normal cytoplasm. Type 2 was widely distributed in European, Chinese and major Japanese varieties, while some Chinese varieties and several Japanese local radishes had type 3 cytoplasm. The comparison provides valuable information about the origin and differentiation of cultivated radishes and the relationship between cultivated and wild radishes.     

PCR-based markers to differentiate the mitochondrial genomes of petaloid and male fertile carrot (Daucus carota L.)

Cytoplasmic male sterility (CMS) is essential for the development of highly adapted and uniform hybrid varieties of carrot and other crops. The most widely used type of CMS in carrot is petaloidy, in which the stamens are replaced by petals or bract-like structures. We have developed a series of mitochondria-specific PCR markers to distinguish cytoplasms inducing petaloidy (Sp) and male-fertility (N). The markers target the atp1,atp6, atp9, orfB (atp8), nad6 and cob loci from the mitochondrial genomes of a diverse collection of male fertile and petaloid carrots. We report 14 primer pairs that amplify marker fragments from either Sp or N cytoplasms and three primer pairs that amplify fragments with length polymorphism. The amplification products span sites of insertions, deletions or recombinations adjacent to or within the coding regions of the targeted genes. The markers reported here are useful tools to identify the type of cytoplasm in cultivated carrot and to evaluate variation in the mitochondrial genomes within the genus Daucus. This revised version was published online in August 2006 with corrections to the Cover Date.     

RFLP analysis of mitochondrial DNA in two cytoplasmic male sterility systems (CMS-D2 and CMS-D8) of cotton

Cytoplasmic male sterility (CMS) in higher plants is a maternally inherited trait and CMS-associated genes are known to be located in the mitochondrial genome. However, CMS-inducing genes in CMS-D2 and CMS-D8 of Upland cotton (Gossypium hirsutum L., AD1) are currently unknown. The objective of this study was to identify potential candidate DNA or gene sequences for CMS-D2 and CMS-D8 through restriction fragment length polymorphism (RFLP) analysis. Seven mtDNA gene probes and five restriction enzymes were first used to compare D2 (from G. harknessii Brandegee) and AD1 cytoplasms. With cox1, cox2, and atp1 as probes, RFLP polymorphisms were detected with one or more restriction enzyme digestions. The most notable difference was an additional fragment in the normal AD1 cytoplasm detected by cox2 in digests of three enzymes, and by cox1 and atp1 in digests with PstI. The RFLP analysis was then conducted among CMS-D2, CMS-D8 (from G. trilobum (DC.) Skovst.), and AD1 cytoplasms. Two probes from maize, atp1 and atp6, detected polymorphism among the different cytoplasmic lines. However, no difference in RFLP patterns was noted between male sterile (A) and restorer (R) lines with the D2 or D8 cytoplasm, indicating that the presence of the D2 or D8 restorer gene does not affect mtDNA organization in Upland cotton. The results demonstrate that RFLP using atp1 and atp6 as probes can distinguish the three cytoplasms. The atp1 and atp6 in CMS-D8 and these two genes together with cox1 and cox2 in CMS-D2 could be the candidates of CMS-associated genes in the mitochondrial genome, providing information for further molecular studies and developing PCR-based markers for the CMS cytoplasms in breeding. This research represents the first work using RFLP to analyze the genetic basis of CMS in cotton.     

Phylogeny and relationships in Daucus based on restriction fragment length polymorphisms (RFLPs) of the chloroplast and mitochondrial genomes

Chloroplast DNA (cpDNA) restriction site analysis of nine species of Daucus (covering four of five sections) and nine carrot accessions was done using 14 Petunia cpDNA clones as probes and 10 restriction enzymes. Cladistic analysis yielded a phylogeny generally concordant with a recent morphological classification. A cpDNA insertion-deletion mutation was found in the cultivated carrots (probe P10– Bgl II digest). Mitochondrial DNA (mtDNA) restriction site analysis of the same material using four maize mtDNA clones as probes and 10 enzymes exhibited extensive mitochondrial diversity in Daucus, both within and between sections. This revised version was published online in July 2006 with corrections to the Cover Date.     

基于线粒体控制区全序列的鄱阳湖水系鲢增殖放流群体与野生群体的遗传多样性分析

采用PCR和DNA测序技术研究鄱阳湖水系增殖放流群体与野生群体白鲢的遗传结构和群体遗传学特征,测定了瑞昌、赣江、增殖放流3个群体54尾鲢线粒体控制区全序列935bp,缺失或插入3个碱基,发现41个变异位点,均为简约信息位点,共检出24个单倍型。转换／颠换比为14.344。在邻接树上来自不同地点的鲢混杂分布于各分支,没有出现明显的谱系结构和地理聚群。AMOVA 分析表明变异主要来自种群内,种群间出现显著分化。FST=0.22388也表明遗传变异主要来自种群内。3个群体间的遗传距离为0.01035～0.01634,Fst值为0.08252～0.39171,表明3个群体间存在显著遗传分化。赣江、瑞昌和增殖放流群体的单倍型多样性分别为0.727、0.978、0.947,核苷酸多样性分别为0.00897、0.01135、0.00978,其中瑞昌群体的单倍型多样性与核苷酸多态性在3个群体中最丰富,其次是增殖放流群体,赣江群体单倍型多样性及核苷酸多样性最低,应加以保护。     

In silico-selection of Brassica rapa organelle genome-derived BACs using their end sequences and sequence level comparative analysis of the 124 kb mitochondrial genome sequences in the family Brassicaceae

We identified BAC clones which harbor DNAs derived from the B. rapa organelle genomes by in silico mapping of 80,292 B. rapa BAC end sequences on the Arabidopsis organelle genomes and subsequent insert size estimation and fingerprinting. A total of 1,048 putative chloroplast genome-derived BAC clones (2.6%) were identified. Fingerprinting and sequencing revealed that many of them represented the entire chloroplast genome (about 150 kb). Meanwhile, only 59 putative mitochondrial genome-derived BACs (0.15%) were identified and most of them showed rare agreement between the in silico map and fingerprinting. We sequenced BAC clone KBrB042G11 (42G11) and compared it to the mitochondrial genome of B. napus and A. thaliana which showed dynamic rearrangement events. The order of 33 orthologous genes was collinear between the 42G11 BAC and its counterpart in B. napus. Five distinctive rearrangements and two InDels were identified between these two closely related species and the rearrangements were related to the occurrence of small tandem repeat sequences. Sequences of the 33 orthologous genes in the homoeologous regions of B. napus and B. rapa were almost 100% identical. Gene orders showed no colinearity between Arabidopsis and Brassica even though 31 orthologous genes shared high sequence similarity with p-values over 1E-32. FISH analysis using the identified BAC revealed a large chloroplast genome insertion in the pericentromeric region of chromosome (chr.) 4 of B. rapa.