基于12S,16S和28S rRNA基因序列的中国小毛瓢虫属系统发育分析

【目的】小毛瓢虫属Scymnus Kugelann昆虫主要捕食蚜虫、蚧虫等害虫,是一类经济上重要的天敌昆虫。目前针对小毛瓢虫属的系统发育研究尚属空白,亚属之间的系统演化关系尚不明确,为了建立合理的分类系统,亟需对小毛瓢虫属的亲缘关系进行研究和探讨。【方法】以华南农业大学馆藏的小毛瓢虫属5亚属共44种为研究对象,采用PCR技术对12S, 16S和28S rRNA基因的部分序列进行扩增;运用MEGA 7.0分析了小毛瓢虫属内12S, 16S和28S rRNA基因的碱基组成,基于K2P模型计算了小毛瓢虫属44种的种间遗传距离;采用最大似然法(maximum-likelihood, ML)和贝叶斯推断法(Bayesian-inference, BI)构建该属的系统发育树。【结果】扩增获得小毛瓢虫属44种的12S rRNA基因序列平均长度为356 bp, 16S rRNA基因序列平均长度为351 bp, 28S rRNA基因序列平均长度为315 bp;序列分析表明,12S rRNA基因的A, T, G和C平均含量分别为38.8%, 43.5%, 11.9%和5.8%, 16S rRNA基因的A, T, G和C平均含量分别为37.6%, 40.3%, 14.4%和7.7%, 28S rRNA基因的A, T, G和C平均含量分别为26.7%, 18.3%, 31.4%和23.5%;基于联合序列分析的种间遗传距离为0.004～0.276,平均遗传距离为0.115。系统发育分析结果表明,小毛瓢虫属为单系起源,而小毛瓢虫亚属Scymnus(Scymnus) Kugelann、毛瓢虫亚属Scymnus(Neopullus) Sasaji、小瓢虫亚属Scymnus(Pullus) Mulsant和拟小瓢虫亚属Scymnus(Parapullus) Yang均为并系起源。【结论】基于12S, 16S和28S rRNA基因序列的小毛瓢虫属系统发育分析显示传统的形态学分类体系与基于分子数据分析的结果部分不一致,这表明应该对该属内各亚属的鉴别特征进行全面检视,筛选并确立各亚属的形态指标,同时也表明该属内的亚属分类单元需重新厘定。     

尖塘鳢属鱼类线粒体12SrRNA基因序列分析

利用PCR技术扩增和测序了线纹尖塘鳢、云斑尖塘鳢和海丰沙塘鳢线粒体12SrRNA基因,结合从GenBank中下载的部分同源序列,共分析了5种鱼类的系统发育关系。在Kimura2-parameter模型构建的邻接树中,原产泰国的云斑尖塘鳢与原产澳州线纹尖塘鳢均为单系类群,二者为亲缘关系最为密切的姐妹群,海丰沙塘鳢与其它群体的亲缘关系较远,支持将尖塘鳢属从塘鳢属中分出的传统分类处理。尖塘鳢属内云斑尖塘鳢和线纹尖塘鳢鱼类种内DNA序列无差异,而种间差异明显,表明线粒体12SrRNA基因可作为塘鳢科鱼类种类鉴定的良好分子标记。     

Geminicoccus roseus gen. nov., sp. nov., an aerobic phototrophic Alphaproteobacterium isolated from a marine aquaculture biofilter

A Gram-negative, strictly aerobic, diplococcoid bacterium (strain D2-3^T) was isolated from the biofilter of a recirculating marine aquaculture system. Phylogenetic analysis of the 16S rRNA gene sequence of D2-3^T indicated that the new organism occupied a novel lineage within the -1 subclass of Proteobacteria and was related to the genera Rhodothalassium, Azospirillum, Craurococcus, Acidiphilium, and Tistrella. The highest sequence similarity (90.8%) of the 16S rRNA gene sequence of D2-3^T was to that of Candidatus “Alysiosphaera europaea”. D2-3^T was mesophilic, heterotrophic, required sea salt, and had a pH optimum of 8.0. Growth in the presence of light resulted in the formation of pink colonies, a 25% increased cell yield, and a slightly increased growth rate. D2-3^T contained carotenoids and low amounts of bacteriochlorophyll a. Membranes of D2-3^T contained b-type cytochromes. The G+C content of the DNA was 60.3±0.1 mol%. Phylogenetic, morphological, physiological, and biochemical analyses demonstrated that D2-3^T represented a new aerobic phototrophic genus, for which the name Geminicoccus roseus gen. nov., sp. nov. is proposed for the type species (D2-3^T=DSM 18922^T=ATCC BAA-1445^T).     

Nocardioides halotolerans sp. nov., isolated from soil on Bigeum Island, Korea

A strictly aerobic, Gram-positive, motile, coccoid-shaped, halotolerant actinobacterium (10% NaCl, w/v), designated MSL-23^T, was isolated from a soil sample on Bigeum Island, Korea. Results of 16S rRNA gene sequence analysis indicated that the isolate belonged to the genus Nocardioides, with the highest sequence similarity (95.63%) being to Nocardioides kribbensis KCTC 19038^T. The major menaquinone was MK-8(H₄), and the predominant cellular fatty acids were i-C_16:0, ai-C_17:0, C_18:1 ω9c and 10-methyl-C_16:0. The DNA G+C content was 69.7 mol%. The 16S rRNA gene sequence of strain MSL 23^T and its chemotaxonomic properties showed it to be unique in the genus Nocardioides. Phenotypic characteristics distinguished strain MSL-23^T from other Nocardioides species. On the basis of the phenotypic, chemotaxonomic and phylogenetic data strain MSL-23^T represents a novel species, for which the name Nocardioides halotolerans sp. nov. is proposed, with MSL-23^T (=KCTC 19274^T=DSM 19273^T) as the type strain.     

黑木耳实时荧光定量PCR内参基因的筛选

黑木耳Auricularia heimuer是我国主要栽培的食用菌之一,具有较高的经济价值和生态价值。本研究以黑木耳不同菌株（A14、A137和A12）和不同生育期的样品（菌丝体、原基和子实体）为实验材料,提取RNA,反转录成cDNA,在全基因组注释结果的基础上,选择12个候选内参基因（APRTase、β-TUB、RPL2、EF-1a、EF-2、PGI、PGM、H ⁺-ATPase、Tspd、TUB-1a、18S rRNA和28S rRNA）并设计跨内含子的引物,采用实时荧光定量PCR（qRT-PCR）技术进行扩增,利用geNorm、NormFinder、BestKeeper和ΔCt算法以及综合评价软件RefFinder,筛选适宜的内参基因。结果表明18S rRNA、β-TUB、EF1-a和28S rRNA适宜作为不同菌株的内参基因,APRTase、18S rRNA和28S rRNA适宜作为不同生育期的内参基因。     

台湾地区芽胞杆菌物种多样性

了解芽胞杆菌资源多样性, 可为芽胞杆菌功能资源挖掘和菌剂开发提供基础。从台湾地区8个市(县)采集土壤样本, 从20份土壤样品中分离获得了136株芽胞杆菌, 采用16S rRNA基因同源性将其鉴定为芽胞杆菌科的2个属、20个种。分别属于芽胞杆菌属(Bacillus)的16个种和赖氨酸芽胞杆菌属(Lysinibacillus)的4个种。根据分离频度分析得知, 台湾地区土壤中的芽胞杆菌优势菌群为阿氏芽胞杆菌(Bacillus aryabhattai)、苏云金芽胞杆菌(B. thuringiensis)和蜡样芽胞杆菌(B. cereus), 其他种类分布极其不均匀。芽胞杆菌Shannon多样性指数为1.2925-2.5850, 最高的为台中市和嘉义市(2.5850), 最低的为桃园县(1.2925)。根据分离频度对芽胞杆菌种类的聚类分析显示, 当欧式距离λ = 20时, 芽胞杆菌种类可分为高频度分布类型如阿氏芽胞杆菌(B. aryabhattai), 低频度分布类型如简单芽胞杆菌(B. simplex)。依据分离频度对8个采样点间的聚类分析未发现采样点间的芽胞杆菌种类分布的相关性。本研究认为台湾地区土壤中蕴藏着丰富芽胞杆菌种类多样性高, 具有很大的开发潜力。     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

宁波港压载水的细菌多样性研究

为揭示宁波港压载水的微生物群落结构在人类活动影响下的分布差异及对环境因子变化的响应趋势。该课题从压载水中分离细菌,通过微生物自动鉴定系统(Sherlock MIS)、分子生物学16S rRNA基因的PCR扩增及RFLP技术,对宁波港压载水的细菌结构特征进行比对和分析,并通过16S rRNA基因文库解析研究压载水中微生物群落多样性。结果表明印度洋水样中存在假交替单胞菌属(Pseudoalteromonas sp.)、交替单胞菌属(Alteromonas sp.)、海杆菌属(Marinobacter sp.)和玫瑰杆菌属(Roseobacter sp.)、肠杆菌属(Enterobacter sp.);新加坡水样中存在人苍白杆菌属(Ochrobactrum sp.)、甲基杆菌属(Methylobacterium sp.)、鞘鞍醇单胞菌(Sphingomonas sp.)、嗜冷杆菌属(Psychrobacter sp.)、假单胞菌(Pseudomonas sp.)、伯克霍尔德氏菌(Burkholderia sp.)和葡萄球菌属(Staphylococcus sp.)假单胞菌属(Pseudomonas sp.);而美洲水样中存在弧菌属(Vibrio sp.)、军团菌属(Legionella sp.)、莫拉氏菌属(Moraxella sp.)和不动杆菌属(Acinetobacter sp.)。压载水中含有大量的潜在致病菌。     

DNA条形码技术在海洋贝类鉴定中的实践: 以大亚湾生态监控区为例

为提高物种鉴定的准确性, 本研究采用DNA条形码技术对大亚湾生态监控区冬季采集的贝类样品进行了种类鉴定。结果表明, 26个形态种中, 有15个可以通过线粒体COI和16S rRNA基因的系统发育分析鉴定到种的水平。部分形态上难以鉴定的种类, 如线缝摺塔螺(Ptychobela suturalis)和区系螺(Funa sp.)可以通过条形码实现有效鉴定。锯齿巴非蛤(Paphia gallus)、西格织纹螺(Nassarius siquijorensis)、爪哇拟塔螺(Turricula javana)等种类存在相当大的种内遗传距离, 有存在隐存种的可能性。尽管基于线粒体COI和16S rRNA基因的种内遗传距离和属内种间的遗传距离发生重合, 无明显的条形码间隙, 但通过系统树的方法仍能有效鉴定物种。可见, DNA条形码技术能有效提高海洋贝类物种鉴定的准确性并发现隐存种。     

Similarities between a predicted secondary structure for the M1 RNA ribozyme and the tRNA binding center of 16 S rRNA from E. coli

We propose a new model for the secondary structure of the M1 RNA component of E. coli RNase P which is based on significant sequence homologies with parts of the E. coli 16 S rRNA. A large domain of the new model resembles closely the secondary structure of the tRNA binding center of 16 S rRNA. We suggest that this domain of M1 RNA when functioning as a ribozyme binds the mature part of the precursor tRNA.     

N4‐methylcytidine ribosomal RNA methylation in chloroplasts is crucial for chloroplast function,development, and abscisic acid response in Arabidopsis

Although the essential role of messenger RNA methylation in the nucleus is increasingly understood, the nature of ribosomal RNA (rRNA) methyltransferases and the role of rRNA methylation in chloroplasts remain largely unknown. A recent study revealed that CMAL (for Chloroplast mr aW‐ Like) is a chloroplast‐localized rRNA methyltransferase that is responsible for N4‐methylcytidine (m⁴C) in 16S chloroplast rRNA in Arabidopsis thaliana. In this study, we further examined the role of CMAL in chloroplast biogenesis and function, development, and hormone response. The cmal mutant showed reduced chlorophyll biosynthesis, photosynthetic activity, and growth‐defect phenotypes, including severely stunted stems, fewer siliques, and lower seed yield. The cmal mutant was hypersensitive to chloroplast translation inhibitors, such as lincomycin and erythromycin, indicating that the m⁴C‐methylation defect in the 16S rRNA leads to a reduced translational activity in chloroplasts. Importantly, the stunted stem of the cmal mutant was partially rescued by exogenous gibberellic acid or auxin. The cmal mutant grew poorer than wild type, whereas the CMAL‐overexpressing transgenic Arabidopsis plants grew better than wild type in the presence of abscisic acid. Altogether, these results indicate that CMAL is an indispensable rRNA methyltransferase in chloroplasts and is crucial for chloroplast biogenesis and function, photosynthesis, and hormone response during plant growth and development.     

Mycobacterium fluoranthenivorans sp. nov., a fluoranthene and aflatoxin B1 degrading bacterium from contaminated soil of a former coal gas plant

Mycobacterium strain FA4^T was isolated with fluoranthene as the single carbon source from soil of a former coal gas plant, polluted with polycyclic aromatic hydrocarbons. The physiological properties, fatty acid pattern, and the 16S ribosomal RNA gene sequence indicated membership to the genus Mycobacterium, but were different from all type strains of Mycobacterium species. Based on comparative 16S rRNA gene sequence analyses strain FA4^T could be assigned to the Mycobacterium neoaurum taxon showing 98% sequence similarity to M. diernhoferi as its closest neighbour. The occurrence of epoxymycolate in the cell wall differentiates FA4 from all members of this taxon which synthesize wax-ester mycolates in addition to alpha-mycolates. Strain FA4^T is able to degrade aflatoxin B₁. This biological attribute might be useful in biological detoxification processes of foods and feeds. From the investigated characteristics it is concluded that strain FA4^T represents a new species, for which we propose the name Mycobacterium fluoranthenivorans sp. nov. The type strain of Mycobacterium fluoranthenivorans is FA4^T (DSM 44556^T = CIP 108203^T).     

Deinococcus misasensis and Deinococcus roseus, novel members of the genus Deinococcus, isolated from a radioactive site in Japan

Two gamma- and UV-radiation resistant, Gram-positive, red- or pink-pigmented, rod-shaped, strictly aerobic, oxidase- and catalase-positive bacterial strains, TDMA-25^T and TDMA-uv51^T, were isolated from fresh water collected at Misasa, a radioactive site in Japan. Phylogenetic analysis based on 16S rRNA gene sequences placed both in a distinct lineage in the family Deinococcaceae, and the highest degrees of sequence similarity determined belonged to Deinococcus maricopensis LB-34^T (88.8–89.3%), Deinococcus pimensis KR-235^T (86.4–86.7%) and Deinococcus yavapaiensis KR-236^T (86.1%). The DNA G+C content of the strains was 53–58 mol%. The major respiratory quinone was MK-8. The predominant fatty acids were C_15:0 iso, C_16:0 iso, C_13:0 iso, C_17:0 iso, C_16:0, C_13:0 anteiso, C_15:0 and C_12:0 iso. The strains degraded gelatin, casein, starch and Tween 80. Unique physiological characteristics, differences in their fatty acid profiles, and genotypic and phylogenetic features, differentiated strains TDMA-25^T and TDMA-uv51^T from closely related Deinococcus species. Hence, the two strains are described as novel species of the genus Deinococcus. The names Deinococcus misasensis sp. nov. (type strain TDMA-25^T=JCM 14369=NBRC 102116=CCUG 53610) and Deinococcus roseus sp. nov. (type strain TDMA-uv51^T=JCM 14370=NBRC 102117=CCUG 53611) are proposed.     

Modulation of 16S rRNA function by ribosomal protein S12

Ribosomal protein S12 is a critical component of the decoding center of the 30S ribosomal subunit and is involved in both tRNA selection and the response to streptomycin. We have investigated the interplay between S12 and some of the surrounding 16S rRNA residues by examining the phenotypes of double-mutant ribosomes in strains of Escherichia coli carrying deletions in all chromosomal rrn operons and expressing total rRNA from a single plasmid-borne rrn operon. We show that the combination of S12 and otherwise benign mutations at positions C1409-G1491 in 16S rRNA severely compromises cell growth while the level and range of aminoglycoside resistances conferred by the G1491U/C substitutions is markedly increased by a mutant S12 protein. The G1491U/C mutations in addition confer resistance to the unrelated antibiotic, capreomycin. S12 also interacts with the 912 region of 16S rRNA. Genetic selection of suppressors of streptomycin dependence caused by mutations at proline 90 in S12 yielded a C912U substitution in 16S rRNA. The C912U mutation on its own confers resistance to streptomycin and restricts miscoding, properties that distinguish it from a majority of the previously described error-promoting ram mutants that also reverse streptomycin dependence.     

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 22 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112 bp long intervening sequence (IVS) at position 542 and a conserved 121–123 bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.     

Purification and properties of a maltoheptaose- and maltohexaose-forming amylase produced by Bacillus subtilis US116

A new bacterial strain, identified as Bacillus subtilis US116, was isolated from Tunisian soil and selected for its potential production of an atypical amylase with an industrial interest. The identification was founded on physiological tests and molecular techniques related to the 16S rRNA, 23S rRNA genes and intergenic sequences showing the highest similarity of 98% with regions in the complete genome of Bacillus subtilis 168 (accession no. Z99104). This strain produces an atypical amylase that was purified to homogeneity by a combination of acetone precipitation, size exclusion and ion exchange chromatography. The molecular mass of the enzyme is about 60 kDa as determined by SDS–PAGE. Optimal conditions for the activity of the purified enzyme are pH 6 and 65 °C. The half-life duration is about 3 h at 70 °C and 5 h at 65 °C. This enzyme belongs to the endo-type amylases according to the hydrolytic mode study using Ceralpha and Betamyl methods. It is classified as a maltoheptaose- and maltohexaose-forming amylase since it generates about 30% maltohexaose (DP6) and 20% maltoheptaose (DP7) from starch. Moreover, the minimum length of maltosaccharide cleaved by this enzyme was maltoheptaose.     

Rhodococcus phenolicus sp. nov., a novel bioprocessor isolated actinomycete with the ability to degrade chlorobenzene, dichlorobenzene and phenol as sole carbon sources

The aerobic degradation of phenol, chlorobenzene and dichlorobenzene as a sole carbon source has been observed in bacterial Gram-positive strain G2P^T isolated from a wastewater bioprocessor. Cells display branching mycelia fragmenting into rod and coccoid elements when grown on TSA. Aerial hyphae formation occurs when grown on phenol and chlorinated aromatics as the sole carbon source. Growth was observed at up to 0.75% phenol as a sole carbon source, indicating a strong tolerance for the compound. The 16S rRNA gene sequence shares the greatest similarity with members of the Rhodococcus genus, with the closest shared nucleotide identity of 98% with the aromatic toxin degrading bacteria Rhodococcus zopfii DSM 44108^T. Neighbor-joining and parsimony analysis of Corynebacterineae 16S rRNA gene sequences consistently places strain G2P^T in a clade shared with R. zopfii within the Rhodococcus rhodochrous subclade. Based on a unique polyphasic profile involving phenotypic, ribosomal DNA sequence analysis, DNA–DNA hybridization, mol% DNA G+C content and fatty acid composition, G2P^T is proposed to represent a previously uncharacterized, novel species in the genus Rhodococcus. The name Rhodococcus phenolicus is proposed for the isolate with the type strain G2P^T (=DSM 44812) (=NRL B-24343).     

用传统分离培养法和高通量测序技术分析印度块菌子囊果内细菌的群落结构

块菌是重要的经济真菌, 在其生长发育过程中, 细菌扮演了重要角色。本文利用传统分离培养方法和高通量测序技术分析了印度块菌(Tuber indicum)子囊果内细菌的群落结构。共分离得到细菌532株, 根据物种累积曲线, 选取其中的112株细菌进行了16S rRNA基因序列分析, 共鉴定出4属40种, 其中假单胞菌属(Pseudomonas)菌株占所测菌株数的80%, 不动杆菌属(Acinetobacter)占12.5%, 链霉菌属(Streptomyces)占5%, 贪噬菌属(Variovorax)占2.5%。通过对印度块菌子囊果16S rRNA基因的V1-V3区高通量测序分析, 共获得细菌序列9,862条, 分属于7门43属220种, 其中变形菌门、拟杆菌门和放线菌门的物种占总物种数的99.7%, 是印度块菌子囊果内的优势细菌。黄杆菌属(Flavobacterium)、壤霉菌属(Agromyces)、微杆菌属(Microbacterium)、剑菌属(Ensifer)和寡养食单胞菌属(Stenotrophomonas)的物种数占总物种的86.3%, 是印度块菌子囊果内细菌的优势属。研究结果表明, 采用胰蛋白大豆培养基仅分离得到印度块菌子囊果内少数细菌物种, 而采用高通量测序技术分析发现, 印度块菌子囊果内细菌物种种类丰富, 群落结构复杂。