西施舌5个地理群体ITS1序列变异及系统发生分析

利用核糖体rRNA基因第一内转录间隔区(ITS1)序列,对我国山东日照(RZ)、江苏连云港(LYG)和启东(QD)、福建长乐(CL)、广西北海(BH)沿海西施舌5个野生群体的遗传结构进行了分析。经PCR扩增、克隆、非克隆测序,获得长度为816—843bp的ITS1核苷酸序列,其平均G+C含量(60.8%)显著高于A+T含量。在西施舌5个群体93个个体118个序列中共检测到153个变异位点,多态位点比例为18.3%,其中,简约信息位点为63个,共有63种基因型。群体间遗传距离在0.74%—3.38%之间,群体内遗传距离为0.72%—1.06%之间。CL群体与其他4个群体间的遗传距离明显高(3.17%—3.38%)。AMOVA分析结果显示长乐群体有明显的遗传分化(FST=0.670—0.702,P(0.001)。以中国蛤蜊为外群构建的NJ树和ML树显示CL群体聚为一支,非长乐群体互相重叠聚为另一支,研究结果显示,长乐群体已经形成了具有明显遗传结构的地理种群。     

西施舌的核型分析

以西施舌囊胚期胚胎为材料,5—10μg／ml秋水仙素海水处理2h,50％海水蒸馏水低渗0．5h,热片法制片,对西施舌的核型进行分析。结果表明,西施舌的二倍体数目2n＝38,核型为14m 16sm 8st,NF＝68。染色体实际长度CL为1．10—3．47μm,染色体总长度TCL为80．72μm。整个核型的染色体长度依次减小,相邻各对之间差异不明显。其中l、3、7、10、11、16和19号为中着丝粒染色体;5、6、8、9、13、14、17和18号为亚中着丝粒染色体;2、4、12和15号为亚端着丝粒染色体。     

西施舌的繁殖生物学

对福建省长乐沿海的西施舌繁殖生物学研究结果表明,年满一龄的西施舌开始性发育,生物学最小型为壳长46．5mm,壳高37mm,体重18．3g。性比率与个体大小有关,存在雄性先熟和雌雄同体的现象。体长91～132mm,个体绝对平均排卵量为429～317万粒／个,个体相对平均排卵量为2．648～1．210万粒/g。性腺发育丰满度指数(尺)在2～6月间,随着水温上升而递增。成熟期为4月中旬～6月中旬,水温16．5～26．3℃;生殖期为5月上旬～7月下旬,水温21．8～28．6℃。西施舌的禁捕期应确定为4～7月份。     

西施舌早期胚胎发育温度效应的研究

１９８２年６月￣１９９５年８月,作者在工乐漳港海蚌汤,对西施舌的受精卵发育成直线铰合幼虫的有效温度,进行了研究。观察结果：西施舌的早期胚胎发育有效温度为１７￣２８℃,其最适温度范围与亲体所处的环境温度有着密切关系,它随着亲体环境温度的升降而相应地在适温范围内变动。     

西施舌栖息环境及人工养殖的研究

本文报道近年来作者在福建省长乐进行的西施舌栖息环境及人工养殖的研究结果。西施舌栖息于低潮线附近至干潮线以下１０ｍ左右的水深,底质以砂为主,适应水温８－３０℃,最适水温１７－２８℃,适应盐度１７－３５‰,最适盐度２０－２８‰,主要摄食浮游植物中的硅藻及水中悬浮物。西施舌干露后体腔液失水率超过８％时,出现死亡。养殖西施舌的场所,应选择在细砂底质,地下渗透水盐度在１７‰以上滩涂垦区为适。     

基于ITS-1基因的菜粉蝶地理种群遗传分化研究

为了探讨我国不同地区菜粉蝶种群之间的遗传分化情况, 本研究运用克隆测序法, 对我国16个地区79只菜粉蝶Pieris rapae rDNA的ITS-1基因序列进行测定并分析; 同时, 以东方菜粉蝶Pieris canidia为外群, 重建了它们的系统发生树, 初步探讨了它们的生物地理学分化格局。序列分析结果显示: 所测菜粉蝶的ITS-1序列长度介于337~458 bp之间。经序列比对后, 在347个位点中共有281个保守位点, 63个变异位点, 16个简约信息位点和47个单突变位点; AMOVA软件分析其核苷酸多态性指数(nucleotide diversity) Pi为0.014, 每位点Theta (per site) Eta为0.041, 遗传分化指数(F_ST)为0.351 (P<0.05); DnaSP软件共检测出48个单倍型(haplotype)序列, 单倍型多样性(haplotype diversity)的频率为0.989。系统发生分析结果表明: 现存的菜粉蝶各地理种群与其地理分布没有直接的对应关系根据系统发生树结果推测, 该蝶种在我国的最早建群可能发生于辽宁、内蒙古及北京一带, 其后, 通过幼虫寄主植物的水陆运输以及成虫的迁飞向我国的不同地区扩散。     

天麻ITS-1测序及单核苷酸多态性变异位点分析

目的:选取天麻3种变型,测定ITS-1序列并进行对比分析,找出变异信息位点,对不同变异类型亲缘关系作出判定.方法:采用改良的CTAB法提取DNA,利用合成的特异引物对ITS区进行扩增,并对目的片段测序分析.结果:ITS-1共计209bp,序列片段上有27个单核苷酸变异位点,ITS-1单核苷酸多态性以碱基转换为主,绿天麻最高达80.8％,乌天麻最低也达70％;黄天麻居中达71.4％.在多态性位点数及多态性频率上,绿天麻均表现为最高分别为26、8.04;黄天麻表现居中为21、9.95;乌天麻表现最低为20、10.45.结论:乌天麻是占老类型,绿天麻是较为进化的类型,黄、绿天麻是由乌天麻进化而来.     

基于ITS2和16S rRNA的西施舌群体遗传差异分析

目前,我国西施舌群体分子遗传差异研究结果存在争议。分析我国南北沿海（9个群体）、与广西北海毗邻的越南（1个群体）西施舌核DNA的内转录间隔区2（ITS2）和线粒体DNA的16S rRNA基因（16S）片段核苷酸序列及其二级结构,为解决争议问题提供分子生物学资料。扩增获得西施舌ITS2片段和16S序列,其长度分别为389-402 bp和306 bp,加之下载序列共147条;序列分析显示,74个ITS2序列共有17种基因型,73个16S序列有15种单倍型,其中,长乐（CL）群体独享9种ITS2基因型和5种16S单倍型,非长乐群体（nCL）多数为群体间交叉共享1种或几种基因（单倍）型;基因（单倍）型核苷酸变异位点占5.7%（ITS2）和11.8%（16S）;基于ITS2和16S的CL群体和nCL群体间的遗传距离与群体内遗传距离之比分别为2.42和11.08,nCL群体间的平均遗传距离均为0.007;二级结构显示CL群体ITS2的9种基因型和16S的5种单倍型均区别于nCL群体,nCL的ITS2和16S二级结构分别相似;ITS2和16S基因的系统发育分析显示,CL西施舌形成支持率很高（98,96）的单系支,而nCL群体则交叉聚为另一支（98,96）。研究结果揭示,福建西施舌是腔蛤蜊属（Coelomactra）的一个新种。     

西施舌的耗氧率与排氨率研究

采用室内实验生态学方法研究了不同栖息水温和不同溶解氧水平下处于标准代谢状态的西施舌耗氧率与排氨率,并测定了窒息点.结果表明,在25 ℃时,水中DO≥3.11±0.15 mg·L^-1时,西施舌的耗氧率和排氨率分别为0.7±0.05 mg·g^-1·h^-1和2.56±0.05 μmol·g^-1·h^-1,处于相对稳定状态;当DO低于此值则代谢出现异常,耗氧率随DO下降而下降,直到窒息为止,其窒息点为1.22±0.06 mg·L^-1,而排氨率也呈直线下降,但排氨停止滞后于耗氧停止.耗氧率与栖息水温呈二次线型关系:OCR=-0.0027T²+0.1367T-0.9557,R²=0.972;水温为25.3 ℃时,西施舌的耗氧率达到最大,为0.77 mg·g^-1·h^-1.处于适温状态(15 ℃和20 ℃)的O/N值要高于低温(10 ℃)和高温(25 ℃和30 ℃)时的O/N值,西施舌在适宜条件下更多地依赖于脂肪供能维持标准代谢,而在环境不适时则更多地调用机体的蛋白质来维持生理代谢需要.     

根据rRNA基因ITS-2序列研究舌蝇属的种系发生

舌蝇又称采采蝇 ,是传播非洲锥虫病 (又称非洲睡眠病 )的重要媒介 .采用PCR方法得到的不同种采采蝇的核糖体DNA内部转录间隔序列 2 (ITS 2 )来分析采采蝇的种系发生 .用简约法 (parsimony)产生的进化树证实了所有采采蝇的单源种类发生并且表明Austenina亚属最早分化 ,然后依次是Glossina和Nemorhina亚属 .经典的形态和生化分类法对Glossinaausteni的进化地位一直有争议 .通过ITS 2位点序列的分析 ,认为G .austeni可以成为独立的亚属和Glossina亚属成为姊妹亚属关系 .     

Mitogenome evidence for the existence of cryptic species in Coelomactra antiquata

Cryptic species which improve our understanding of species diversity and evolutionary histories within marine animals have been increasingly revealed in the marine realm. Coelomactra antiquate is an important commercial bivalve species, but has been suffering from severe population decline due to over-exploitation and deterioration of environmental conditions. To test the hypothesis that cryptic species might exist in C. antiquate presented in previous study, four complete mitogenomes of C. antiquate from northern and southern China were determined here. Comprehensive comparative analysis of the mitochondrial genomes of C. antiquate between northern and southern population reveals significant differences in genome composition, protein coding genes, tRNA genes, non-coding regions, genetic divergence and phylogenetic analysis. The results provide strong mitogenome evidence for the existence of cryptic species in C. antiquate. Besides, our results also support that comprehensive comparative analysis of mtDNA represents an accessible and powerful tool to identify cryptic species.     

Growth and Compositional Changes in Kiwifruit Berries from Three Californian Locations

Kiwifruit [Actinidia deliciosa (A. Chev.) C. F. Liang et A.R. Ferguson var. deliciosa cv. Hayward] berries were sampledfrom three Californian locations, two sites in the central valley(near Davis and Fresno), the other on the coast (near Watsonville).Samples were analyzed for nitrogen, lipid, starch, soluble sugars,organic acids and ash, at regular intervals from flowering untilharvest. The results of the analyses of the fruit from the twocentral valley sites were similar, but markedly different fromthose from the cooler coastal site Sugar/acid ratios were consistentlyhigher in fruit from the coast than the central valley. Thismay prove a useful refinement to the current maturity index. Actinidia deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson var. deliciosa cv. Haywood, kiwifruit, fruit growth, fruit composition, seasonal changes     

Complete mitochondrial genome of Coelomactra antiquata (Mollusca: Bivalvia): The first representative from the family Mactridae with novel gene order and unusual tandem repeats

The complete mitochondrial genome plays an important role in the accurate inference of phylogenetic relationships among metazoans. Mactridae, also known as trough shells or duck clams, is an important family of marine bivalve clams in the order Veneroida. Here we present the complete mitochondrial genome sequence of the Xishishe Coelomactra antiquata (Mollusca: Bivalvia), which is the first representative from the family Mactridae. The mitochondrial genome of C. antiquata is of 17,384 bp in length, and encodes 35 genes, including 12 protein-coding, 21 transfer RNA, and 2 ribosomal RNA genes. Compared with the typical gene content of animal mitochondrial genomes, atp8 and tRNAS₂ are missing. Gene order of the mitochondrial genome of C. antiquata is unique compared with others from Veneroida. In the mitochondrial genome of the C. antiquata, a total of 2189 bp of non-coding nucleotides are scattered among 26 non-coding regions. The largest non-coding region contains one section of tandem repeats (99 bp × 11), which is the second largest tandem repeats found in the mitochondrial genomes from Veneroida. The phylogenetic trees based on mitochondrial genomes support the monophyly of Veneridae and Lucinidae, and the relationship at the family level: ((Veneridae + Mactridae) + (Cardiidae + Solecurtidae)) + Lucinidae. The phylogenetic result is consistent with the morphological classification. Meanwhile, bootstrap values are very high (BP = 94–100), suggesting that the evolutionary relationship based on mitochondrial genomes is very reliable.     

三种产地火麻仁中CannabisinA的含量测定

为建立同时测定广西,甘肃,云南三个产地火麻仁中Cannabisin A的含量测定方法,本文采用高效液相色谱法对三种产地火麻仁中Cannabisin A 进行含量测定.用Waters C18(250 mm×4.6 mm,5 μm)色谱柱,流动相:乙腈-0.1%磷酸溶液(22∶ 78);流速1.0 mL/min;检测波长:257 nm,柱温25 ℃.结果表明:Cannabisin A在0.1～0.50 μg范围内线性关系良好,r=0.9996,加样回收率100.31%,RSD=0.63%(n＝6).三种产地的火麻仁中Cannabisin A含量分别为0.343%,0.250%,0.455%.不同产地火麻仁中Cannabisin A含量有一定的差异,该法操作简便,准确,重复性好,可用于火麻仁中Cannabisin A的含量测定.     

Molecular phylogeny of Trichomonadidae family inferred from ITS-1, 5.8S rRNA and ITS-2 sequences

The Trichomonads have been the subject of several molecular studies that reported some discrepancies both at the lower and higher taxonomic levels. The purpose of this study was to make an extensive phylogenetic analysis of the Trichomonadidae using ITS-1/5.8S/ITS-2 sequences, to better understand its phylogeny and the usefulness of this marker. ITS-1/5.8S/ITS-2 sequences of 36 strains from 14 species belonging to Trichomonadidae and Monocercomonadidae were analysed, in which 20 were newly determined. Maximum likelihood, maximum parsimony, neighbour joining, and Bayesian phylogenetic methods were employed in order to reconstruct and compare the evolutionary history of this group. Tetratrichomonas gallinarum and four strains of Tetratrichomonas sp. isolated from bull genital organs were found closely related, confirming the classification of the latter, probably as a new species. The monophyly of Tritrichomonadinae and Trichomonadinae subfamilies were corroborated, with the exclusion of Trichomitus batrachorum from the latter since it grouped consistently with Hypotrichomonas acosta. Tritrichomonas foetus, Tritrichomonas suis and potentially also Tritrichomonas mobilensis seemed to correspond to the same species. Monocercomonas sp. and Ditrichomonas honigbergii emerged as independent lineages, with their phylogenetic positions undetermined. Neither Trichomonadidae nor Monocercomonadidae were supported as monophyletic groups. The ITS-1/5.8S/ITS-2 seems to be a reliable locus for phylogenetic studies in the Trichomonadida, mainly at lower taxonomic levels, and at least up to the family level.     

新疆3种藜科盐生植物NHX基因的克隆与序列分析比较

从新疆野生植物盐角草(Salicornia europaea)、盐爪爪(Kalidium foliatum)和盐穗木(Halostachys caspica)中分别克隆了约1.7kb的NHX基因cDNA片段,此片段均包含了NHX完整基因,三者之间有较高的同源性,盐角草NHX基因与盐爪爪NHX基因同源性达92.81%,盐角草与盐穗木同源性达92.19%,盐爪爪与盐穗木同源性达97.66%.它们与其它几种藜科盐生植物如滨藜、碱蓬、灰绿藜的NHX基因同源性也很高,达到80%以上,与拟南芥同源性也达到86%.此基因在植物尤其是藜科盐生植物中高度保守,其编码的功能性蛋白可能在影响植物耐盐中起作用.     

Sequence Analysis of cytb Gene in Echinococcus granulosus from Western China

Echinococcus granulosus is the causative agent of cystic echinococcosis with medical and veterinary importance in China. Our main objective was to discuss the genotypes and genetic diversity of E. granulosus present in domestic animals and humans in western China. A total of 45 hydatid cyst samples were collected from sheep, humans, and a yak and subjected to an analysis of the sequences of mitochondrial cytochrome b (cytb) gene. The amplified PCR product for all samples was a 1,068 bp band. The phylogenetic analysis showed that all 45 samples were identified as E. granulosus (genotype G1). Ten haplotypes were detected among the samples, with the main haplotype being H1. The haplotype diversity was 0.626, while the nucleotide diversity was 0.001. These results suggested that genetic diversity was low among our samples collected from the west of China based on cytb gene analysis. These findings may provide more information on molecular characteristics of E. granulosus from this Chinese region.     

ITS-1 versus ITS-2 pyrosequencing: a comparison of fungal populations in truffle grounds

In a recent study pyrosequencing of the ribosomal internal transcribed spacer-1 (ITS-1) has validated the effectiveness of such technology in the survey of soil fungal diversity. Here we compare the two ITS regions, ITS-1 and ITS-2, of the fungal populations occurring in Tuber melanosporum/Quercus pubescens truffle grounds and sampled in two areas, one devoid of vegetation ("burned", brulé in French) where T. melanosporum fruiting bodies are usually collected, and outside the brulé. TS1F/ITS2 and ITS3/ITS4 were used respectively for the amplification of the ITS-1 and ITS-2 regions. Two amplicon libraries were built, one for inside and the other for outside. A set of 15.788 reads was obtained. After the removal of low quality sequences, 3568 and 3156 sequences were obtained from inside the brulé with the ITS-1 and ITS-2 primers respectively. The sequences obtained from outside the brulé were 4490 with the ITS-1 primers and 2432 with the ITS-2 primers. Most of the sequences obtained for both ITS fragments could be attributed to fungal organisms. The pair of primers, ITS1-F/ITS2, was more selective, producing fewer non-fungal sequences (1% inside, 3% outside), in addition to a higher number of sequences, than the pair ITS3/ITS4 (6% inside, 11% outside). Although differences are present in the taxa percentages between ITS-1 and ITS-2, both reveal that Ascomycota were the dominant fungal phylum and that their number decreased moving from inside the brulé to outside, while the number of Basidiomycota increased. Taken together, both the short ITS-1 and ITS-2 reads obtained by the high throughput 454 sequencing provide adequate information for taxon assignment and are suitable to correlate the dynamics of the fungal populations to specific environments.     

三株牛源亚洲1型口蹄疫病毒VP1基因的克隆与序列分析

以3株国内分离的亚洲1型口蹄疫病毒(分别命名为F1、F2、F3)为研究目标,根据GenBank中注册的FMDV VP1基因的序列设计1对引物,采用RT-PCR方法成功地扩增出含有VP1全基因的片段,并测定了3个毒株VP1基因的序列.结果表明,3株亚洲1型FMDV毒株VP1基因长度均为633 bp,编码211个氨基酸.3株毒株彼此之间的核苷酸序列同源性在82.8% ～99.1%之间,推导氨基酸序列同源性在89.1% ～99.1%之间.从系统发生树看,F1株与我国香港2005年牛毒株序列同源性99.5%,属同一遗传谱系,F2株、F3株与2005年引起河北省万全县、北京市延庆县、甘肃静宁县疫情的毒株分属同一个基因群.     

Sequence Diversity in MIC6 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations

Toxoplasma gondii is an opportunistic protozoan parasite that can infect almost all warm-blooded animals including humans with a worldwide distribution. Micronemes play an important role in invasion process of T. gondii, associated with the attachment, motility, and host cell recognition. In this research, sequence diversity in microneme protein 6 (MIC6) gene among 16 T. gondii isolates from different hosts and geographical regions and 1 reference strain was examined. The results showed that the sequence of all the examined T. gondii strains was 1,050 bp in length, and their A + T content was between 45.7% and 46.1%. Sequence analysis presented 33 nucleotide mutation positions (0-1.1%), resulting in 23 amino acid substitutions (0-2.3%) aligned with T. gondii RH strain. Moreover, T. gondii strains representing the 3 classical genotypes (Type I, II, and III) were separated into different clusters based on the locus of MIC6 using phylogenetic analyses by Bayesian inference (BI), maximum parsimony (MP), and maximum likelihood (ML), but T. gondii strains belonging to ToxoDB #9 were separated into different clusters. Our results suggested that MIC6 gene is not a suitable marker for T. gondii population genetic studies.