十字花科植物PEBP基因家族的分子进化

近年来植物基因组测序物种数量的指数增长, 为我们对植物环境适应性状的遗传和变异的全面理解提供了保障。磷脂酰乙醇胺结合蛋白(phosphatidylethanolamine-binding protein, PEBP)在植物的开花转变和株型建立中起着重要作用, 一直是植物生物学研究关注的热点领域之一。然而对该家族并没有利用新近测序的基因组数据进行比较基因组分析, 制约了对其在分子水平上的进化研究。为了确定PEBP基因家族的分子进化机制, 本研究利用生物信息学方法开展了7种十字花科植物拟南芥(Arabidopsis thaliana)、琴叶拟南芥(A. lyrata)、小鼠耳芥(A. pumila)、亚麻荠(Camelina sativa)、甘蓝(Brassica oleracea)、白菜(B. rapa)和油菜(B. napus)的PEBP基因家族成员的全基因组鉴定、结构特征和比较进化分析。从7个物种中共鉴定出91个PEBP基因, 系统进化分析表明它们分属5个亚家族: MFT、FT/TSF、TFL1、CEN和BFT。基因结构分析发现甘蓝、白菜和油菜的CEN基因内含子明显比其余4个物种的内含子长。蛋白结构域分析表明MFT比其他4个亚家族成员少了一个motif 2, TFL1比其他亚家族多了motif 8。选择压力分析发现7个物种PEBP同源基因均受到较强的纯化选择, 其中TFL1亚家族受到的纯化选择最弱。共线性分析表明十字花科植物PEBP基因家族随古代多倍体事件发生不同程度的扩张, TSF在甘蓝、白菜和油菜中丢失。非生物胁迫下, 在拟南芥中过量表达小鼠耳芥的一个MFT基因, 转基因拟南芥种子的萌发率明显低于野生型, 暗示MFT基因在调控种子萌发上的功能保守。本研究为深入研究十字花科植物PEBP基因的进化特征和生物学功能奠定了基础。     

Anatomy of the stimulative sequences flanking the ARS consensus sequence of chromosome VI in Saccharomyces cerevisiae

We have analyzed the relationship between autonomously replicating sequence (ARS) structure and function for three ARS (ARS605, ARS607 and ARS609) from chromosome VI of Saccharomyces cerevisiae by systematic XhoI-linker mutation in the ARS consensus sequence (ACS) and flanking sequences. All mutations that encroached upon the ACS destroyed ARS activity. DNA sequences stimulative for ARS function were identified on either side of the ACS of ARS605 and only on the 3'-side of the ACS of ARS607. In ARS609, however, no such stimulative sequences were observed. Base substitutions complementary to the wild-type sequence of those stimulative regions, in ARS605 and ARS607, that did not change the AG of unwinding nor affected ARS activity suggests that these regions have, at least, a function as DNA-unwinding elements (DUE). ARS605, ARS607 and ARS609 DNA are of low AG value and showed hypersensitivity to single-strand-specific nuclease when inserted in negatively supercoiled plasmid. Linker mutations inhibitory for ARS activity (5L11 and 7L14) also caused significant changes in local nucleotide (nt) sensitivity within the ACS and its adjoining regions. Complementary base substitutions, however, did not affect these changes in local nt sensitivity. These results imply that the stimulative regions flanking the ACS are necessary to produce an optimum conformation around the ACS which may be important for full ARS activity.     

Otx2 can activate the isthmic organizer genetic network in the Xenopus embryo

Development and differentiation of the vertebrate caudal midbrain and anterior hindbrain are dependent on the isthmic organizer signals at the midbrain/hindbrain boundary (MHB). The future MHB forms at the boundary between the Otx2 and Gbx2 expression domains. Recent studies in mice and chick suggested that the apposition of Otx2- and Gbx2-expressing cells is instrumental for the positioning and early induction of the MHB genetic cascade. We show that Otx2 and Gbx2 perform different roles in this process. We find that ectopically expressed Otx2 on its own can induce a substantial part of the MHB genetic network, namely En2, Wnt1, Pax-2, Fgf8 and Gbx2, in a concentration-dependent manner. This induction does not require protein synthesis and ends during neurulation. In contrast, Gbx2 is a negative regulator of Otx2 and the MHB genes. Based on the temporal patterns of expression of the genes involved, we propose that Otx2 might be the early inducer of the isthmic organizer genetic network while Gbx2 restricts Otx2 expression along the anterior-posterior axis and establishes an Otx2 gradient.     

Pre-MBT patterning of early gene regulation in Xenopus: the role of the cortical rotation and mesoderm induction

Patterning events that occur before the mid-blastula transition (MBT) and that organize the spatial pattern of gene expression in the animal hemisphere have been analyzed in Xenopus embryos. We present evidence that genes that play a role in dorsoventral specification display different modes of activation. Using early blastomere explants (16–128-cell stage) cultured until gastrula stages, we demonstrate by RT-PCR analysis that the expression of goosecoid (gsc), wnt-8 and brachyury (bra) is dependent on mesoderm induction. In contrast, nodal-related 3 (nr3) and siamois (sia) are expressed in a manner that is independent of mesoderm induction, however their spatially correct activation does require cortical rotation. The pattern of sia and nr3 expression reveals that the animal half of the 16-cell embryo is already distinctly polarized along the dorsoventral axis as a result of rearrangement of the egg structure during cortical rotation. Similar to the antagonistic activity between the ventral and the dorsal mesoderm, the ventral animal blastomeres can attenuate the expression of nr3 and sia in dorsal animal blastomeres. Our data suggest that no Nieuwkoop center activity at the blastula stage is required for the activation of nr3 and sia in vivo.     

Nucleotide sequence of the FokI restriction-modification system: separate strand-specificity domains in the methyltransferase

The genes for FokI, a type-IIS restriction-modification system from Flavobacterium okeanokoites (asymmetric recognition sequence: 5'-GGATG/3'-CCTAC), were cloned into Escherichia coli. Recombinants carrying the fokIR and fokIM genes were found to modify their DNA completely, and to restrict lambdoid phages weakly. The nt sequences of the genes were determined, and the probable start codons were confirmed by aa sequencing. The FokI endonuclease (R · FokI) and methyltransferase (M · FokI are encoded by single, adjacent genes, aligned in the same orientation, in the order M then R. The genes are large by the standards of type-II systems, 1.9 kb for the M gene, and 1.7 kb for the R gene. Preceding each gene is a pair of FokI recognition sites; it is conceivable that interactions between the sites and the FokI proteins could regulate expression of the genes. The aa sequences of the N- and C-terminal halves of M · FokI are similar to one another, and to certain other DNA-adenine methyltransferases, suggesting that the enzyme has a ‘tandem’ structure, such as could have arisen by the fusion of a pair of adjacent, ancestral M genes. Truncated derivatives of M · FokI were constructed by deleting the 5'- or 3' -ends of the fokIM gene. Deleting most of the C-terminus of M · FokI produced derivatives that methylated only the top (GGATG) strand of the recognition sequence. Conversely, deleting most of the N-terminus produced derivatives that methylated only the bottom (CATCC) strand of the recognition sequence. These results indicate that the domains in M · FokI for methylating the two strands of the recognition sequence are largely separate.     

深海白色侧齿霉遗传转化体系建立及渗透压调控相关的EaSHO1基因功能

海洋中具有丰富的动植物及微生物资源,海洋真菌是其重要组成之一。我们前期的研究发现一株深海真菌白色侧齿霉Engyodontium album能产生具有抑菌活性的次级代谢产物engyodontiumin A,该化合物能抑制黑曲霉、金黄色葡萄球菌及创伤弧菌等病原菌的生长,是一种潜在的海洋源抗菌药物。目前,该菌遗传转化体系尚未建立,不利于开展次级代谢产物合成调控机制及其他功能基因研究。本研究成功制备了深海白色侧齿霉菌的原生质体,建立了借助聚乙二醇3350介导的原生质体转化体系,并将pCT74-sGFP载体成功导入白色侧齿霉的原生质体中,结果显示外源GFP能稳定表达。此外,为了明确白色侧齿霉菌是否能够开展基因敲除研究,通过氨基酸序列同源比对,我们选取酵母高渗甘油信号途径中的同源基因EaSHO1进行初步探究。利用同源重组的方法成功将目的基因EaSHO1的开放阅读框(ORF)替换成潮霉素磷酸转移酶基因(HPH),由此获得EaSHO1基因敲除突变体,并对突变体进行Southern杂交验证及初步的表型分析。结果表明,EaSHO1缺失不影响白色侧齿霉菌的营养生长及对高盐胁迫的响应,亚细胞定位结果显示EaS...     

Involvement of recQ in the ultraviolet damage repair pathway in Deinococcus radiodurans

Deinococcus radiodurans is a bacterium which can survive extremely DNA damage. To investigate the relationship between recQ and the ultraviolet radiation (UV) damage repair pathway, we created a four mutant strain by constructing recQ knockout mutants in uvrA1, uvrA2, and uvsE backgrounds. Using the rpoB/Rif^r system, we measured the mutation frequencies and rates in wild type, recQ (MQ), uvsE uvrA1 uvrA2 (TNK006), and uvsE uvrA1 uvrA2 recQ (TQ). We then isolated Rif^r mutants of these strains and sequenced the rpoB gene. The mutation frequency of TQ was 6.4, 10.1, and 2.43 times that of wild type, MQ, and TNK006, respectively, and resulted in rates of 4.7, 6.71, and 2.15 folds higher than that of wild type, MQ, and TNK006, respectively. All the strains demonstrated specific mutational hotspots. Furthermore, the TQ strain showed a transversion bias that was different from the other three strains. The results indicate that recQ is involved in the ultraviolet damage repair pathway via the interaction between recQ and uvrA1, uvrA2, and uvsE in D. radiodurans.     

入侵豚草与本地植物竞争对丛枝菌根真菌多样性的影响

外来植物入侵常引起入侵地丛枝菌根真菌（arbuscular mycorrhizal fungi,AMF）多样性的变化,但AMF多样性的变化与入侵域本地植物种类相关性目前尚不清楚。本研究采用野外同质园试验研究豚草与不同本地植物竞争时对AMF多样性的影响。本试验共设入侵豚草单种、本地植物狗尾草Setaria viridis单种、藜 Chenopodium album单种、黄香草木犀Melilotus officinalis单种、豚草和狗尾草混种、豚草和藜混种、豚草和黄香草木犀混种以及裸土（无任何植物生长）对照8个处理。采集根区土样后分离、鉴定AMF,分析植株生物量和AMF物种多样性变化间的关系。结果表明,对豚草而言,单种处理中生物量均高于混种处理;豚草分别与3种本地植物竞争时,与黄香草木犀混种中豚草竞争能力最强,而与藜混种中竞争能力最弱。豚草分别与3种本地植物混种时土壤中AMF种丰度和香农-威纳指数分别显著高于豚草单种和本地植物单种,其中豚草与藜混种中AMF的种丰度和香农-威纳指数最高,与狗尾草混种中AMF的种丰度和香农-威纳指数最低,而豚草分别与3种本地植物混种处理中孢子密度显著低于本地植物单种（豚草与黄香草木犀混种除外）。豚草单种、狗尾草单种、藜单种、豚草分别与狗尾草和藜混种4个处理中AMF优势种均为网状球囊霉Glomus reticulatum和缩隔球囊霉Septoglomus constrictum;黄香草木犀单种中优势种是网状球囊霉、缩隔球囊霉和根内根孢囊霉Rhizophagus Intraradices,豚草与黄香草木犀混种中优势种是网状球囊霉和根内根孢囊霉;狗尾草单种、豚草与狗尾草混种和豚草与藜混种3个处理的特有种分别是木薯根孢囊霉Rhizoglomus manihotis、近明球囊霉Claroideoglomus claroideum和美丽盾巨孢囊霉Scutellospora calospora。狗尾草单种生物量与孢子密度呈显著正相关,豚草与狗尾草混种中豚草的生物量与均匀度呈显著负相关,豚草与黄香草木犀混种中豚草的生物量与孢子密度呈显著正相关,与香农威纳指数呈显著负相关。综上可知,豚草与本地植物竞争对AMF多样性的影响具有异质性,这与本地植物种类密切相关。     

Pleiotropic effects of heterozygosity at the mating-type locus of the yeast Saccharomyces cerevisiae on repair, recombination and transformation

Sexual (MAT a/) and sexual (MAT a/a) strains of the yeast Saccharomyces cerevisiae, which are completely isogenic except at the MAT locus, were compared in their response to ultraviolet radiation. The effects of UV on survival, mitotic intragenic recombination, photoreactivation, and transformation efficiency with UV-irradiated plasmid DNA were examined. The sexual strain had enhanced survival and higher rates of mitotic intragenic recombination compared with the asexual strain. Exposure to visible light subsequent to irradiation increased the survival of both sexual and asexual strains, and decreased their rates of mitotic intragenic recombination. Similar results were obtained by Haladus and Zuk (1980) in their examination of sexual strains homozygous for rad6-1, and wild-type sexuals.

Our sexual strain was also consistently more proficient at transforming plasmid DNA, whether that DNA had been irradiated or not. When pre-irradiated with 25 J/m² of UV, MAT a/ cells transformed more efficiently than MAT a/a cells. When subsequently exposed to light, the ability of these pre-irradiated cells to transform decreased for both strains with increasing irradiation of the plasmid. A smaller decrease in transformation efficiency occurred when cells of both strains were kept in the dark.

When pre-irradiated with 100 J/m², the MAT a/ cells showed a 2-fold increase in their transformation efficiency of both irradiated and unirradiated plasmids by up to 2-fold, a phenomenon not seen in the MAT a/a cells even when pre-irradiated with much higher doses of UV. This increase in transformation efficiency was not, however, seen in the MAT a/ cells when they were exposed to visible light after UV irradiation. These results suggest that cells with the MAT a genotype have a UV-inducible system that increases the efficiency of transformation in the absence of visible light. This increase in transformation is not an induced increase in the repair of plasmid DNA, but rather an increase in the ability of pre-irradiated MAT a/ cells to take up exogenous DNA. MAT a/a cells do not appear to have a similarity inducible system. To the best of our knowledge, this phenomenon has not been previously reported.     

A phylogenetic study of the free-living amoeba

Immunological distances were determined for four strains of the free-living amoeba classified as Amoeba proteus, two strains classified as Polychaos dubia and a single strain classified as Chaos carolinensis. The data show that the _ShP strain does not belong to the proteus group; that A. proteus is more closely related to C. carolinensis and is derived from Chaos as is the _ShP strain; that P. dubia and C. carolinensis are the more distantly related species and appear to be the first of the above to have diverged from a common ancestor; and that the amoebae have a long evolutionary history. The accuracy of the phylogenetic tree and the distance Wagner network was discussed. Since the amoebae may be polyphyletic in origin, the latter was assumed to be a more accurate representation of the immunological distance data.     

褐环乳牛肝菌对樟子松生长的调控——影响激素和代谢产物含量

为了进一步探究褐环乳牛肝菌对樟子松苗木生长的促进机制,利用生物化学和植物生理学等研究方法,在前期研究基础上对2年生沙地樟子松苗木施加褐环乳牛肝菌液体菌剂,并在2个月后对苗木各部分生长状态、生长相关激素、抗逆性代谢产物进行检测。结果表明：褐环乳牛肝菌可以通过升高樟子松IAA和GA含量促进樟子松苗木的生长。其中,对樟子松根系中IAA含量影响最为显著,接菌苗木根中IAA含量比未接菌苗木根中IAA含量高85%以上;与此结果相反,褐环乳牛肝菌可以降低樟子松苗木ABA含量,通过ABA含量变化调控IAA含量,进而调控根系的生长;除此之外,褐环乳牛肝菌可以调控樟子松苗木甜菜碱和黄酮化合物的含量。未接菌苗木的甜菜碱在根、茎、叶中的含量分别比接菌苗木高46%、26%和23%,说明接菌苗木在受环境的影响方面相较于未接菌苗木低。接菌处理可以显著提高樟子松苗木根部黄酮化合物含量。本研究结果可以为今后进一步研究提供数据借鉴,同时也可以为辽西半干旱地区森林健康调控与褐环乳牛肝菌科学应用于当地造林提供科学依据。     

Phylogenetic relationships within Pyrenodesmia sensu lato and the role of pigments in its taxonomic interpretation

Most lichens of the family Teloschistaceae (Ascomycota) produce yellow-orange-red anthraquinone pigments. However, the genus Pyrenodesmia encompasses species in which anthraquinones are absent and replaced by a gray pigment Sedifolia-gray. It was shown recently that these species are related to taxa with both anthraquinones and Sedifolia-gray (Caloplaca xerica group, C. haematites group, and C. cretensis) and to species with a brown pigment instead of both anthraquinones and Sedifolia-gray (C. demissa, C. obscurella, and C. reptans). Nevertheless, relationships between mentioned anthraquinone-containing and anthraquinone-lacking species remained unclear. In total, 8 DNA loci from 41 species were used here to resolve these uncertainties. We concluded that C. demissa, C. obscurella, and C. reptans are rather distant from the core of Pyrenodesmia, and we place them outside of Pyrenodesmia sensu lato. Within Pyrenodesmia sensu lato, three lineages were revealed and recognized on a generic level: the genus Pyrenodesmia sensu stricto (21 species), the genus Kuettlingeria (14 species), which is resurrected here, and the genus Sanguineodiscus (4 species), which is newly described here. The genus Pyrenodesmia includes taxa that never contain anthraquinones, but Sedifolia-gray. It matches with the former C. variabilis group. Taxa of the genera Kuettlingeria and Sanguineodiscus have anthraquinones in their apothecia and Sedifolia-gray in their thalli. The genus Kuettlingeria includes the former C. xerica group plus C. cretensis and C. diphyodes. The genus Sanguineodiscus includes the former C. haematites group and C. bicolor. The identity of Kuettlingeria (Caloplaca) diphyodes was clarified and the name Pyrenodesmia helygeoides was resurrected. Twenty-four new combinations were proposed.     

西藏湖泊长刺溞复合种的系统进化关系

本文通过对西藏湖泊长刺溞复合种(Daphnia longispina complex)中分布最广的3个物种, 即长刺溞(D. longispina)、盔形溞(D. galeata)和颈齿溞(D. dentifera)线粒体COI基因序列以及GenBank中欧洲的长刺溞、加拿大的颈齿溞和我国东部低海拔地区的盔型溞COI基因序列的比较分析, 研究了西藏湖泊长刺溞复合种的系统进化关系, 发现西藏地区的盔型溞、颈齿溞和长刺溞均已出现较大分化。颈齿溞种群内遗传差异度为0.33-2.32%, 盔型溞为0.33-2.74%, 长刺溞的遗传差异度最高, 为1.31-5.50%。基于COI基因序列构建的最大似然树和贝叶斯系统树均表明, 长刺溞复合种由3个进化分支组成, 分别对应长刺溞、盔型溞和颈齿溞, 三者之间的遗传差异度为9.40-16.98%(Kimura 2-parameter双参数模型)。基于COI基因单倍型(haplotype)所构建的网络关系也支持上述3个分支的存在。早期记录虽然显示长刺溞在我国分布较广, 但本次调查只在班公错有发现, 相比之下, 盔形溞和颈齿溞则分布更广。我们的研究表明, 由于形态学鉴定上的局限性, 早期的长刺溞记录很可能混杂了容易引起混淆的盔型溞或颈齿溞。     

Isolation of the epidermal growth factor from the shrew submaxillary gland

Entomocidal crystals produced by Bacillus thuringiensis ssp. israelensis are formed by two proteins with molecular masses of 130 and 28 kDa, whereas the protein with a molecular mass of 70 kDa appears as a result of 130 kDa protein limited proteolysis by admixtures of bacterial proteinases in the course of its dissolution. The comparison of the N-terminal sequences of the protein with molecular mass of 70 kDa (Met-Glu-Asn-Xaa-Pro-Leu-Asp-Thr-Leu-Ser-Ile-Val-Asn-Glu-Thr-Asp) and that of 28 kDa (Met-Glu-Asn-Leu-Asn-[Phe]-[Trp]-Pro-Leu-Gln-Asp-Ile-Lys-Val-Asn-Pro) reveals only marginal similarity between them (only 4 identical residues among 16 aligned). Both B. thuringiensis israelensis crystal-forming proteins appear hardly related to those contained in the crystal produced by other B. thuringiensis subspecies, e.g. kurstaki. It might be concluded that at least some of the entomocidal proteins found in the crystalline inclusion bodies of various B. thuringiensis subspecies revealed rather strong variations in their primary structures that facilitate their adaptation to different hosts.

Bacillus thuringiensis ssp. israelensis δ-Endotoxin Entomocidal crystal Insecticide Mosquito     

Trichinella nativa and Trichinella T9 in the Hokkaido island, Japan

Trichinella sp. muscle larvae were isolated from the thigh muscle of two red foxes (Vulpes vulpes) captured in Sapporo and Otofuke, Hokkaido, Japan, in 2003. Multiplex PCR designed for genotyping the genus Trichinella revealed that the Sapporo isolate showed a specific pattern to T. britovi complex (T. britovi, Trichinella T8 and Trichinella T9) and the Otofuke isolate showed that to T. nativa. Nucleotide sequences of a part of the mitochondrial cytochrome oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) of the Sapporo isolate showed the highest similarity to those of Trichinella T9, a species detected in the mainland of Japan. This study shows that both T. nativa and Trichinella T9 are circulating in wildlife of the Hokkaido island.     

蝙蝠蛾鳞翅虫草Samsoniella hepiali模式菌株线粒体基因组系统发育分析

蝙蝠蛾鳞翅虫草Samsoniella hepiali是重要的药用虫生真菌,根据现代分类系统,隶属于虫草科Cordycipitaceae鳞翅虫草属Samsoniella。本研究对蝙蝠蛾鳞翅虫草模式菌株(ICMM 82-2)线粒体基因组进行测序、组装和注释,发现其线粒体基因组成闭合环状,大小为24 246 bp。基因区域占比为85.10%,共编码42个基因,包括15个蛋白质编码基因(PCGs)、2个rRNA基因和25个tRNA基因。15个PCGs均以ATG为起始密码子,以TAA为终止密码子,偏好密码子为UUA。各氨基酸使用频率具有较大差异,其中亮氨酸(Leu)出现频率最高。25个tRNA基因可转运全部20种氨基酸,其中19个tRNA基因二级结构为三叶草结构。本研究基于线粒体基因组中14个PCGs基因串联数据集,构建肉座菌目6科50个物种的系统发育树,进一步证实蝙蝠蛾鳞翅虫草模式菌株系统发育位置隶属于肉座菌目Hypocreales虫草科Cordycipitaceae鳞翅虫草属Samsoniella。通过虫草科线粒体基因组共线性分析,发现其中12个物种有5个同源区,3个物种存在6个同源区,同源区域A和B长度变异较大。与其他虫草科物种相比,蝙蝠蛾鳞翅虫草线粒体基因组较小,同源区域相对较短。蝙蝠蛾鳞翅虫草模式菌株线粒体基因组信息有助于鉴别其物种特异性,确定其系统发育关系,将为虫草系统发育研究提供新思路,为继续研究鳞翅虫草属物种药用价值提供系统发育依据。     

基因组数据揭示肺形侧耳X1野生菌株后代单孢的特异交配型位点

肺形侧耳味道鲜美,营养丰富,深受广大消费者喜爱。它属于四极性异宗结合担子菌,但其交配型位点结构仍未被解析。本研究利用二代测序技术对肺形侧耳的基因组进行测序,通过生物信息学方法找到了肺形侧耳野生菌株X1菌株后代单核菌株的交配型位点。结果显示肺形侧耳的A交配型位点较为特异,2株原生质体单核化菌株（X1-1和X1-15）的A交配型位点结构差异较大,X1-1含一对保守的HD1和HD2基因,而X1-15除了一对HD1和HD2基因外,还含有额外的2个HD2基因和1个HD1基因。肺形侧耳的B交配型位点与其他担子菌的交配型位点相似,含有8个信息素受体基因和1个信息素前体基因。本研究揭示的肺形侧耳特异的交配型位点结构为后期的遗传育种提供了理论依据。