Xenopus tropicalis 免疫球蛋白轻链重组多样性的研究

热带爪蟾(Xenopus tropicalis)是两栖类动物免疫球蛋白研究中常用的模式生物之一,其免疫球蛋白轻链的类型和基因结构已经研究清楚,3种轻链类型分别为：ρ、σ和typeⅢ．通过克隆测序,并对3种轻链重组cDNA克隆的VJ基因片段的连接方式进行了分析,发现在表达的3种轻链中,仅有少量的N和P核苷酸的插入．σ轻链基因的体细胞VJ重组主要依靠微同源重组．而在ρ和typeⅢ 轻链中这种方式不是主要的,ρ和typeⅢ轻链或是通过VJ基因片段的直接连接或是部分删除后连接．研究结果为研究免疫球蛋白轻链多样性产生的机制提供了重要的线索．     

代谢工程改造热带假丝酵母生产1,2,4-丁三醇

【背景】 1,2,4-丁三醇属于手性多羟基醇,是一种重要的有机合成的化学中间体,以木糖为原料经四步酶反应是目前研究最多的生物合成路线。然而大肠杆菌的鲁棒性较弱,对发酵液中一些抑制剂的耐受性不是很好,同时存在严重的碳代谢抑制。近年来,鲁棒性较好的酵母菌成为更有吸引力的宿主,其中热带假丝酵母具有天然的木糖代谢途径,可以更好地利用木糖。【目的】在热带假丝酵母中构建从木糖到1,2,4-丁三醇的代谢途径。【方法】在热带假丝酵母中敲除木糖还原酶基因GRE3,从而阻断自身的木糖代谢途径。将来源于Caulobacter crescentus的木糖脱氢酶基因(xylB)和木糖酸脱水酶基因(xylD)及来源于Lactococcus lactis的酮酸脱羧酶基因(kdcA)克隆至C. tropicalis 207中,得到重组菌C. tropicalis BT,在此基础上考察重组菌代谢木糖合成1,2,4-丁三醇的能力,确定限速步骤,并通过增加关键基因xylD与kdcA的拷贝数提高1,2,4-丁三醇产量。【结果】在30℃、200 r/min、接种量1%、以30 g/L木糖为底物的情况下,重组菌的1,2,4-丁三醇的产量达到了1.2 g/L,在5 L发酵罐中的产量达到了3.7 g/L。【结论】在热带假丝酵母中实现以木糖为底物的1,2,4-丁三醇代谢途径,并通过在基因组上增加关键基因xylD与kdcA的拷贝数,获得了一株高产1,2,4-丁三醇的重组酵母菌株,这为后续在热带假丝酵母中进一步提高1,2,4-丁三醇产量奠定了基础。     

A genetic linkage map for the apicomplexan protozoan parasite Eimeria maxima and comparison with Eimeria tenella

Eimeria maxima is one of the seven Eimeria spp. that infect the chicken and cause the disease coccidiosis. The well characterised immunogenicity and genetic diversity associated with E. maxima promote its use in genetics-led studies on avian coccidiosis. The development of a genetic map for E. maxima, presented here based upon 647 amplified fragment length polymorphism markers typed from 22 clonal hybrid lines and assembled into 13 major linkage groups, is a major new resource for work with this parasite. Comparison with genetic maps produced for other coccidial parasites indicates relatively high levels of genetic recombination. Conversion of ∼14% of the markers representing the major linkage groups to sequence characterised amplified region markers can provide a scaffold for the assembly of future genomic sequences as well as providing a foundation for more detailed genetic maps. Comparison with the Eimeria tenella genetic map produced 10 years ago has revealed a less biased marker distribution, with no more than nine markers mapped within any unresolved heritable unit. Nonetheless, preliminary bioinformatic characterisation of the three largest publicly available genomic E. maxima sequences suggest that the feature-poor/feature-rich structure which has previously been found to define the first sequenced E. tenella chromosome also defines the E. maxima genome. The significance of such a segmented genome and the apparent potential for variation in genetic recombination will be relevant to haplotype stability and the longevity of future anticoccidial strategies based upon multiple loci targeted by novel chemotherapeutic drugs or recombinant subunit vaccines.     

热带假丝酵母脂肪醛脱氢酶基因CtAld1和CtAld2的功能评价

【目的】热带假丝酵母是发酵法生产二元酸的重要工业菌株,具有较高的ω-氧化活性。脂肪醛脱氢酶在ω-氧化途径中起重要作用,催化脂肪醛生成脂肪酸,但其具体催化功能及对细胞生理影响还未被系统研究。本文通过删除脂肪醛脱氢酶基因CtAld1和CtAld2鉴定了其在ω-氧化途径中的功能。【方法】通过基因组信息挖掘获得热带假丝酵母脂肪醛脱氢酶基因CtAld1和CtAld2序列,在此基础上,通过同源重组敲除CtAld1和CtAld2基因。考察突变株的生长和胞内脂肪醛脱氢酶活性变化,并评价CtAld1和CtAld2基因敲除对细胞二元酸合成能力的影响。【结果】分别获得了热带假丝酵母突变株XZX-1(ΔCtAld1/ΔCtAld1)、XZX-2(ΔCtAld2/ΔCtAld2)和XZX-12(ΔCtAld1/ΔCtAld1,ΔCtAld2/ΔCtAld2)。在以十二烷为唯一碳源的培养基中,敲除CtAld2基因显著抑制细胞的生长,胞内脂肪醛脱氢酶活性降低为出发菌株的30%;敲除CtAld1基因尽管会使细胞损失一部分醛脱氢酶活性,但能够一定程度地提升细胞在十二烷中的生长性能。敲除CtAld1或CtAld2会降低菌株二元酸产量,组合敲除CtAld1和CtAld2严重削弱菌株十二碳二元酸的合成能力。【结论】CtAld2对热带假丝酵母细胞的生长和十二碳二元酸的合成具有重要作用,缺失CtAld1或CtAld2基因降低细胞的二元酸合成能力。CtAld1和CtAld2可作为热带假丝酵母ω-氧化途径代谢工程改造的潜在靶点。     

A genetic map of Phycomyces blakesleeanus

Summary Complementation tests among Phycomyces auxotrophic strains revealed the existence of four genes with mutants requiring riboflavin, three genes with purine auxotrophs, two with nicotinic acid auxotrophs, and two with lysine auxotrophs. A total of 134 sexual crosses between strains carrying mutations affecting phototropism (madA-madE), carotenoid biosynthesis (carA), auxotrophy (ribA-ribD, purA-purC, lysA and lysB, nicA and nicB, and leuA) and resistance to 5-fluorouracil (furA and furB) were studied; mating type (sex) was also included as a marker. The results from random spore analysis, tetrad analysis, and gene-centromere distances shows that these markers are distributed into 11 linkage groups.     

Increased number of Arginine-based salt bridges contributes to the thermotolerance of thermotolerant acetic acid bacteria, Acetobacter tropicalis SKU1100

Thermotolerant acetic acid bacteria (AAB), Acetobacter tropicalis SKU1100, can grow above 40 °C. To investigate the basis of its thermotolerance, we compared the genome of A. tropicalis SKU1100 with that of mesophilic AAB strain Acetobacter pasteurianus IFO3283-01. The comparative genomic study showed that amino acid substitutions from large to small residue and Lys to Arg occur in many orthologous genes. Furthermore, comparative modeling study was carried out with the orthologous proteins between SKU1100 and IFO3283-01 strains, indicating that the number of Arg-based salt bridges increased in protein models. Since it has been reported that Arg-based salt bridges are important factor for thermo-stability of protein structure, our results strongly suggest that the increased number of Arg-based salt bridges may contributes to the thermotolerance of A. tropicalis SKU1100 (the thermo-stability of proteins in A. tropicalis SKU1100).     

Population structure and genetic diversity of Huperzia serrata (Huperziaceae) based on amplified fragment length polymorphism (AFLP) markers

The levels and pattern of the genetic variation within and among natural populations of Huperzia serrata were investigated using amplified fragment length polymorphism markers. Seven primer combinations used in the study amplified 615 discernible bands with 532 (86.5%) being polymorphic, indicating a considerable high level of genetic diversity at the species level. AMOVA analysis revealed a low level of genetic differentiation among the ten populations. The UPGMA cluster of all samples showed that individuals from the same population occasionally failed to cluster in one distinct group. A Mantel test showed no significant correlation between genetic distance and geographical distance (r = 0.278, P = 0.891), suggesting that the gene flow was not restricted geographically. A number of factors that might affect the genetic profiles of H. serrata included clonal growth, selective effect of niche and outcrossing, as well as the effective wind-dispersal of spores.     

甘蓝型油菜BnCYP78A8的克隆及序列分析

采用Genome walking方法,首次克隆到甘蓝型油菜BnCYP78A8的基因组序列,根据基因特异性引物克隆到其编码序列。基因组序列长1 679bp,有1个内含子和2个外显子。编码序列长1 605bp,编码534个氨基酸。序列比对分析表明,其氨基酸序列与拟南芥细胞色素P450单加氧酶基因(AtCYP78A8)的相似度高达88%。生物信息学分析显示,该蛋白含有1个细胞色素P450特有的亚铁血红素配合基结合位点和1个跨膜结构域。实时荧光定量PCR分析结果表明,BnCYP78A8在甘蓝型油菜的各个器官组织均有表达,根中表达量最高,表明该基因可能参与根的生长发育。     

The density distribution of gene loci over the genetic map ofEscherichia coli: Its structural,functional and evolutionary implications

Summary A quantitative analysis was carried out on the dispersion of gene loci over theE. coli genetic map. Therefore, the map was divided into regions characterized by an homogeneous gene density. This created a distribution pattern of gene loci that contained a symmetry axis located near to the origin of DNA replication. The pattern could be subdivided into a set of 22 functional domains containing gene loci whose products revealed a biochemical or functional relatedness. A correlation was found between the boundary positions of these domains and the distribution of F plasmid- and DNA insertion sites over theE. coli chromosome. The structural, functional and evolutionary implications of these findings are discussed.     

Comparative sequence analysis of the SALT OVERLY SENSITIVE1 orthologous region in Thellungiella halophila and Arabidopsis thaliana

To provide a framework for studies to understand the contribution of SALT OVERLY SENSITIVE1 (SOS1) to salt tolerance in Thellungiella halophila, we sequenced and annotated a 193-kb T. halophila BAC containing a putative SOS1 locus (ThSOS1) and compared the sequence to the orthologous 146-kb region of the genome of its salt-sensitive relative, Arabidopsis thaliana. Overall, the two sequences were colinear, but three major expansion/contraction regions in T. halophila were found to contain five Long Terminal Repeat retrotransposons, MuDR DNA transposons and intergenic sequences that contribute to the 47.8-kb size variation in this region of the genome. Twenty-seven genes were annotated in the T. halophila BAC including the putative ThSOS1 locus. ThSOS1 shares gene structure and sequence with A. thaliana SOS1 including 11 predicted transmembrane domains and a cyclic nucleotide-binding domain; however, different patterns of Simple Sequence Repeats were found within a 540-bp region upstream of SOS1 in the two species.     

An integrated physical and genetic map of the nematode<Emphasis Type="Italic"> Pristionchus pacificus</Emphasis>

The free-living nematode Pristionchus pacificus is one of several species that have recently been developed as a satellite system for comparative functional studies in evolutionary developmental biology. Comparisons of developmental processes between P. pacificus and the well established model organism Caenorhabditis elegans at the cellular and genetic levels provide detailed insight into the molecular changes that shape evolutionary transitions. To facilitate genetic analysis and cloning of mutations in P. pacificus, we previously generated a BAC-based genetic linkage map for this organism. Here, we describe the construction of a physical map of the P. pacificus genome based on AFLP fingerprint analysis of 7747 BAC clones. Most of the SSCP markers used to generate the genetic linkage map were derived from BAC ends, so that the physical genome map and the genetic map can be integrated. The contigs that make up the physical map are evenly distributed over the genetic linkage map and no clustering is observed, indicating that the physical map provides a valid representation of the P. pacificus genome. The integrated genome map thus provides a framework for positional cloning and the study of genome evolution in nematodes.Communicated by G. Jürgens     

SSRs transferability and genetic diversity of Tunisian Festuca arundinacea and Lolium perenne

SSR primers specific to Lolium perenne generated a total of 96 alleles and 124 genotypes within Festuca arundinacea and Lolium perenne accessions. Their highly transferability (100 %) across genera was evidenced. Six alleles specific to loci H01F02, H02C11 and K01A03 and only 5/96 common alleles between both species (60, 140, 144, 190 and 192) expressed the differentiation between species. Besides, based on the Wrights fixation indices, the genetic variation within each species was attributable to differences within populations with a significant deficiency of heterozygous. The unweighted pair group method with arithmetic averaging dendrogram based on the Nei’s distances and the principal coordinate analysis based on Jaccard coefficient similarity distinguished each genus independently of the geographical origin. However, typically continuous genetic diversity and a low level of gene flow (N_m: 0.29–2.47) expressed the relatively closely relationships of both genera and suggest a possible hybridization in nature.     

氟康唑对热带念珠菌活性氧和线粒体膜电位的影响

为了探讨氟康唑作用机制,观察它对热带念珠菌作用后存活率、活性氧(Reactive oxygen species,ROS)、线粒体膜电位(Mitochondrial membrane potential,△Ψm)和细胞周期的变化。参照NCCLS M27-A方案的微量稀释法测定氟康唑对热带念珠菌的最低抑菌浓度(MIC);热带念珠菌与不同浓度氟康唑共同培养后用流式细胞术(Flow cytometry,FCM)分析热带念珠菌存活率、ROS、线粒体膜电位△Ψm和细胞周期的变化。结果表明,氟康唑作用后,热带念珠菌氟康唑耐药株的存活率、ROS、线粒体膜电位△Ψm和细胞周期各期比例均没有明显变化;而热带念珠菌氟康唑敏感株的存活率和线粒体膜电位△Ψm明显下降,ROS明显升高,而且大部分热带念珠菌阻滞于G2/M期,并出现明显凋亡峰,呈一定的时间剂量依赖关系。自由基清除剂谷胱甘肽抑制热带念珠菌ROS的产生,阻止细胞周期G2/M期阻滞和降低凋亡。由此可见,氟康唑可能通过刺激热带念珠菌产生过多ROS,并使线粒体膜电位△Ψm下降,从而诱导热带念珠菌凋亡。     

Mutation as an origin of genetic variability in Helicobacter pylori

The availability of two complete Helicobacter pylori genome sequences and recent studies of its population genetics have provided a detailed picture of genetic diversity in this important human gastric pathogen. It is believed that, in addition to genetic recombination, de novo mutation could have a role in generating the high level of genetic variation in H. pylori.     

重楼属植物遗传多样性的SCOT标记

为评估重楼属植物的基因资源开发前景,初步探讨了SCOT标记技术在重楼属植物遗传多样性研究上的应用。采用目标起始密码子多态性(Start codon targeted polymorphism,SCOT)技术,对重楼属9个种40份材料进行基因组DNA多态性分析。结果表明:四川地区重楼属植物具有丰富的遗传多样性,40份供试材料可聚为4类:狭叶重楼单独聚为第一类;球药隔重楼、卵叶重楼、金线重楼及滇重楼聚为第二类;黑籽重楼与七叶一枝花聚为第三类;五指莲与毛重楼聚为第四类。说明SCOT标记能够对重楼属植物进行准确的分子鉴定,为重楼属植物的种类鉴定和种间的分类地位提供分子生物学依据,同时也为深入探究重楼皂苷合成关键酶基因定位研究提供重要参考。     

Development and Characterization of a Genetic Linkage Map of Cryptococcus neoformans var. neoformans Using Amplified Fragment Length Polymorphisms and Other Markers

A segregating population of single basidiospore isolates from a sexual cross was used to generate the first moderately dense genetic linkage map of Cryptococcus neoformans var. neoformans (Serotype D). Polymorphic DNA markers were developed using amplified fragment length polymorphisms, random amplified polymorphic DNA, and gene-encoding sequences. These markers were used to analyze 100 meiotic progeny. All markers were tested for distorted segregation with a goodness of fit test. Of the total of 181 markers, 148 showed balanced (1:1) segregation ratios. Segregation distortion was observed for 33 markers. Based on all the markers, a linkage map was generated that consists of 14 major linkage groups with 127 markers, several small linkage groups, and 2 linkage groups that consist only of highly skewed markers. The genetic distance of the linkage map is 1356.3 cM. The estimated total haploid genome size for C. neoformans var. neoformans was calculated using Hulberts method and yielded a map size of 1917 cM. The number of major linkage groups correlates well with the proposed number of 13 chromosomes for C. neoformans var. neoformans. Several genes, including CAP64, CnLAC, and the mating-type locus, were mapped, and their associations were consistent with published data. To date, 6 linkage groups have been assigned to their corresponding chromosomes. This linkage map should provide a framework for the ongoing genome sequencing project and will be a useful tool for studying the genetics and pathogenicity of this important medical yeast.     

Integrin α5 during early development of Xenopus laevis

The full length sequence of the Xenopus integrin α₅ subunit is reported. Analysis of cloned cDNA fragments reveals that alternative polyadenylation of α₅ mRNA occurs in the embryo. Furthermore, a variant form of the α₅ mRNA is expressed which encodes an integrin α₅ subunit with a truncated cytoplasmic domain. Integrin α₅ mRNA and protein are expressed in oocytes, eggs and throughout development. Spatial expression of α₅ mRNAs is first detected by whole mount in situ hybridization in presumptive neural crest cells and in the somitic mesoderm from the midgastrula stage onwards. In contrast, the α₅ protein is present on newly formed plasma membranes beginning at first cleavage. During neurulation, the integrin α₅ subunit disappears from the outer layer of the ectoderm, the notochord and the neural tube and accumulates in the sensorial layer of the ectoderm, the somites and the neural crest cells. These results provide evidence for the position specific regulation of α subunit expression in early vertebrate embryos.     

An SSR-based linkage map of Capsicum annuum

There are five cultivated species of pepper, of which Capsicum annuum is the most widely cultivated as a vegetable or spice and the main experimental material of most pepper breeding programs. However, the number of simple sequence-repeat (SSR) markers known for C. annuum is limited. To develop SSR markers for Capsicum species, we constructed four SSR-enriched libraries from the genomic DNA of C.␣annuum, sequenced 1873 clones, and isolated 626 unique SSR clones. A higher percentage of these SSR markers were taken from dinucleotide motif libraries than from trinucleotide motif libraries. Primer pairs for the 626 SSR clones were synthesized and tested for polymorphisms; 594 amplified products were detected with the expected size. However, only 153 products were polymorphic between the parents of our mapping population. Using 106 highly reproducible pairs from the primer pairs, we constructed a linkage map of C. annuum in an intraspecific doubled haploid population (n=117) that contains nine previously reported SSRs as well as AFLP, CAPS, and RAPD markers and the trait of fruit pungency. The map contains 374 markers, including 106 new SSR markers distributed across all 13 linkage groups, and covers 1042 cM. The polymorphism information content (PIC) of these new SSR markers was calculated using 14 lines of Capsicum species. The average number of alleles per locus was 2.9 and the average PIC value was 0.46, even within C. annuum. The SSR markers developed in this study will be useful for mapping and marker-assisted selection in pepper breeding, and the linkage map provides a reference genetic map for Capsicum species.     

Localization of his genes on the Rhizobium trifolii RS800 linkage map

Summary We have studied N-methyl-N-nitro-N-nitrosoguanidine (NTG)- and Tn5-induced histidine auxotrophic mutants in Rhizobium trifolii. HisGE, hisD and hisH mutants have been characterized. Using the Kemper's equation we have located them on the R. trifolii linkage map. The hisGE and hisD genes are clustered in the same region and are closely linked to the spectinomycin marker. The hisH gene is located in another region equidistant from the streptomycin and rifampicin markers. The two regions carrying his genes are separated by a distance approximately one-third of the length of the chromosome.     

An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei

Burkholderia pseudomallei and B. mallei are Gram-negative bacterial pathogens that cause melioidosis in humans and glanders in horses, respectively. Both bacteria are classified as category B select agents in the United States. Due to strict select-agent regulations, the number of antibiotic selection markers approved for use in these bacteria is greatly limited. Approved markers for B. pseudomallei include genes encoding resistance to kanamycin (Km), gentamicin (Gm), and zeocin (Zeo); however, wild type B. pseudomallei is intrinsically resistant to these antibiotics. Selection markers for B. mallei are limited to Km and Zeo resistance genes. Additionally, there are few well developed counter-selection markers for use in Burkholderia. The use of SacB as a counter-selection method has been of limited success due to the presence of endogenous sacBC genes in the genomes of B. pseudomallei and B. mallei. These impediments have greatly hampered the genetic manipulation of B. pseudomallei and B. mallei and currently few reliable tools for the genetic manipulation of Burkholderia exist. To expand the repertoire of genetic tools for use in Burkholderia, we developed the suicide plasmid pMo130, which allows for the compliant genetic manipulation of the select agents B. pseudomallei and B. mallei using allelic exchange. pMo130 harbors an aphA gene which allows for Km selection, the reporter gene xylE, which allows for reliable visual detection of Burkholderia transformants, and carries a modified sacB gene that allows for the resolution of co-integrants. We employed this system to generate multiple unmarked and in-frame mutants in B. pseudomallei, and one mutant in B. mallei. This vector significantly expands the number of available tools that are select-agent compliant for the genetic manipulation of B. pseudomallei and B. mallei.