长苞铁杉nrDNA ITS区假基因化拷贝的克隆及序列分析研究

NrDNA作为一个重要的工具,在许多分类等级上被广泛地应用于系统发育的分析。以长苞铁杉(Nothotsuga longibracteata)为实验材料,通过nrDNA ITS区特异拷贝的克隆及假基因化分析,在序列水平上探讨假基因化的特征,进而探讨引起多拷贝基因家族假基因化的分子机制。实验结果表明:假基因化拷贝的序列长度为1 745 bp,比其它拷贝短11 bp-12 bp;GC含量明显偏低,特别是5.8 S区和ITS2区的GC含量分别比其它克隆低6.15%-6.17%和5.75%-6.15%;5.8 S区和ITS2区序列变异大,遗传距离(d)分别为0.0718 0.0221和0.0691 0.0178;在系统发育的分析中出现异常的长枝现象。     

帘蛤科贝类rDNA内转录间隔区序列的研究

根据18SrDNA、5．8SrDNA和28SrDNA保守序列设计引物,应用聚合酶链式反应（PCR）扩增了文蛤（Meretrix meretrix L．）、青蛤（Cyclina sinensis G）、硬壳蛤（Mercenaria mercenaria L．）和江户布目蛤（Protothaca jedoensis L．）4种帘蛤科贝类的第一内转录间隔区（ITS1）和第二内转录间隔区（ITS2）序列,并进行了测序。结果表明,文蛤、青蛤、硬壳蛤和江户布目蛤的ITS1扩增产物大小分别为978bp、663bp、757bp和942bp,GC含量分别为61．55％、60．78％、62．48％和64．86％～64．97％,其中ITS1序列长度分别为900bp、585bp、679bp和864bp,是迄今已报道双壳贝类中变化范围最大的,GC含量分别为61．67％、61．03％、63．03％和65．51％～65．62％,江户布目蛤种内ITS1序列有个体差异;ITS2扩增产物大小分别为644bp、618～620bp、593bp和513～514bp,GC含量分别为61．18％、61．29％～61．81％、62．73％和61．48％61．60％,其中ITS2序列长度分别为412bp、386～388bp、361bp和281～282bp,GC含量分别为65．29％、65．21％～66．06％、67．87％和67．38％～67．62％,青蛤和江户布目蛤种内ITS2序列有个体差异。4种蛤ITS1和ITS2序列种间差异很大,有明显的长度多态性,ITS2种间序列相似度73．0％～89．1％,与ITS1的种间序列相似度48．7％～81．5％相比略高。此外,在4种蛤ITS1和ITS2序列中各发现2个与rRNA加工有关的保守区。通过对ITS1和ITS2序列的组装获得了4种蛤5．8SrRNA基因完整序列,序列长度都是157bp,GC含量57．96％～58．60％,4种蛤5．8SrRNA基因相对保守,种间序列差异度0-6．0％,共有10个变异位点,其中转换4处,颠换6处,硬壳蛤和江户布目蛤5．8SrRNA基因序列完全相同。以ITS2序列（包含5．8SrRNA和28SrRNA基因部分序列）为标记,调用北极蛤科的Arctica islandica相应序列数据作外群,构建了帘蛤科贝类的系统发育树,其拓扑结构显示江户布目蛤与硬壳蛤亲缘关系最近,青蛤与其他3物种的亲缘关系最远。     

长苞铁杉的核型分析及其分类学意义

本文首次分析了特产我国的长苞铁杉亚属(Subgen. Paleotsuga)的代表种长苞铁杉Tsuga longibracteata的核型,它全由中部和近中部着丝粒染色体组成,核型公式为K(2n)=24=22m 2sm,属“lA”类型。第12号染色体具“长着丝点区域”。染色体相对长度组成为2n=12M_2 10M_1 2S。作者发现铁杉亚属(Subgen. Tsuga)植物的核型全为“2A”类型。胞核学资料支持对长苞铁杉亚属和铁杉亚属的划分并表明前者(长苞铁杉)较为原始。长苞铁杉应隶于长苞铁杉亚属、长苞铁杉组。     

利用核核糖体DNA内转录间隔区(ITS)探讨广义羽藓科系统发育

利用核核糖体DNA ITS序列,探讨了苔藓植物广义羽藓科的系统发育,摸索出适于扩增ITS片段的最适反应条件。实验共得到广义羽藓科6个种的ITS序列,它们分别是：Abieti-nela abietina(AJ417494),Anomodon minar(AJ344145),Chaopodium aciculum(AJ315968),Tuidium pristocalyx(AJ416443),Thuidium assimile(AJ416442),Herpetineuron toccoae(AJ315967),其中后5个种是国际上首次得到的。本文利用ITS序列构建羽藓科7属、11种植物的系统发育树,据Bootstrap严格一致树表明：广义的羽藓科为并系发育,可分为两个主要的分支,牛舌藓属Anomodon,羊角藓属Herpetineuron和多枝藓属Happohymenium等为一支,而山羽藓属Abietinella,羽藓属Thuuidium,沼羽藓属Helodium和麻羽藓属Claopodium等为另一主要分支,从分子水平上支持了据形态特征把原牛舌藓亚科的牛舌藓属,羊角藓属,多枝藓属提升为牛舌藓科的结论。     

以ITS1-5.8S rDNA-ITS2序列为标记的肺孢子菌分子系统发育研究

为了阐明肺孢子菌的系统发育及种内分类状况, 本实验对源于大鼠、长爪沙鼠和人的肺孢子菌5.8S核糖体RNA(rRNA)基因和rRNA内转录间隔区(the internal transcribed spacer, ITS)序列进行了扩增、克隆和序列测定. 分析了6种肺孢子菌的属内进化距离并与外囊菌属和酵母菌属比较. 研究结果表明, 肺孢子菌属含有包括长爪沙鼠源肺孢子菌在内的多个菌种. 构建的系统发育树也表明, 人和猴来源的肺孢子菌聚成1组, 4种鼠源肺孢子菌聚成另1组, 而4种鼠源肺孢子菌中, 大鼠源肺孢子菌Pneumocystis wakefieldiae与小鼠源肺孢子菌P. murina及另一大鼠源肺孢子菌P. carinii亲缘关系最近, 与长爪沙鼠源肺孢子菌关系最远.     

长苞铁杉天然林群落种内及种间竞争关系研究

通过各种竞争指数的比较筛选,得到较能客观反映长苞铁杉种内、种间竞争关系的竞争指数,定量地分析了长苞铁杉种内和种间竞争强度。结果表明:长苞铁杉种内竞争随胸径的增大而逐渐减少;种间与种内竞争关系顺序为:长苞铁杉-长苞铁杉>猴头杜鹃-长苞铁杉>青冈栎-长苞铁杉>其它树种-长苞铁杉。竞争强度与对象木的胸高直径服从双曲线回归关系,利用模型预测了长苞铁杉种内、种间的竞争强度。     

濒危植物长苞铁杉的年龄结构与空间格局的研究

长苞铁杉(Nothotsuga longibracteata)是我国特有的古老孑遗的单种属植物,对研究松科植物的系统发育及古生态、古气候具有重要价值.对福建天宝岩自然保护区长苞铁杉天然林的群落组成、年龄结构和各年龄级的空间分布格局进行了研究.通过设置60 m x72 m的样方,对样方内所有215株长苞铁杉个体定位并划分为16个年龄段,统计伴生种的种类及数量,用Ripley's K(d)函数对各年龄段个体进行空间格局分析.结果表明,伴生种主要为常绿和落叶阔叶树种,不利于长苞铁杉幼苗的更新;该种群经历了3个阶段性的更新期,可能与群落中林窗的形成或周期性的干扰有关;在35 m的半径范围内,长苞铁杉60年以下年龄级个体呈显著的聚集分布,60年以上年龄级个体则变为随机分布,主要是由于长苞铁杉的种子散布和生长特性、种群内和种群间的竞争、种群的自我稀疏过程造成的.     

核糖体DNA的内转录间隔区序列标记在真菌分类鉴定中的应用

传统的真菌分类主要根据真菌菌株的形态特征、生长特性与生理生化指标进行,而分子生物学技术的发展提升了真菌分类鉴定研究的手段。真菌核糖体DNA内转录间隔区(ITS)在进化上比编码区快,种内的不同菌株之间高度保守,但在种间变化极大,故可为真菌学的研究提供丰富的遗传信息。简要综述了ITS序列分析技术在真菌分类鉴定中的应用现状、相关问题及前景。     

长苞铁杉林乔木层优势种群种间关联及尺度效应研究

通过设定不同尺度的样方面积,根据2×2联列表,运用方差分析、联结系数AC、2统计量度量、 Ochiai指数系列技术,测定天宝岩国家级自然保护区长苞铁杉群落乔木层10个优势种群间的总体联结关系、 种对间联结显著性和关联系数。结果表明:长苞铁杉群落乔木层10个优势种群的总体种间关联性均呈正关 联,反映该群落处于稳定的顶极阶段;10个优势种群的种间联结关系中,有26个种对在不同尺度样方设计中 均未达到一般显著水平,种对间的相互独立性较强;样方设计为100 m2时,各项指标上规律性比较明显,它的 研究结果与实际情况相吻合。群落虽处于比较稳定的顶极阶段,但应适当调整群落结构,为长苞铁杉的生长 更新提供良好的生态环境。     

奥利亚罗非鱼与尼罗罗非鱼rDNA内转录间隔区序列特征

核糖体DNA内转录间隔区(internal transcribed spacers,ITS)是经常被用作种和种群水平系统研究的分子序列.本文分离了奥利亚罗非鱼(Oreochromis aureus)、尼罗罗非鱼(O.niloticus)内转录间隔区,包括部分185序列,ITS1、5.8S、ITS2全序列及部分28S序列.4尾奥利亚罗非鱼的10个克隆序列分析表明,其存在长度不同的a、b两种类型ITS1.a型长为536 bp,GC含量为69.96%;b型长为520 bp,GC含量为69.04%～69.42%.4尾尼罗罗非鱼的10个克隆序列分析表明,其只存在a型ITS1,长为536～540 bp,GC含量为69.42%～70.19%.与b型ITS1相比,a型ITS1在16～31 nt有16 bp片段(GGCCCGCCTCGGCGC)的插入.奥利亚罗非鱼和尼罗罗非鱼共20条ITS序列中,5.8S长度均为157 bp,GC含量为56.69%～57.96%;ITS2为408 bp,GC含量为72.79%～74.26%.奥利亚罗非鱼和尼罗罗非鱼ITS区序列相似性高达98.2%,表明这两种罗非鱼亲缘关系很近.此外,本文对14尾奥利亚罗非鱼、15尾尼罗罗非鱼以及15尾奥尼罗非鱼[O.aureus(♂)×O.niloticus(♀)]ITS1的扩增结果显示,奥利亚罗非鱼均有a、b两种类型ITS1;15尾尼罗罗非鱼中1尾为a、b两类型ITS1,14尾为a型ITS1;15尾奥尼罗非鱼中则有6尾具有a、b两类型ITS1,9尾为单一的a型ITS1.分析表明,奥利亚罗非鱼在ITS1这个位点一致性高,但尼罗罗非鱼中有1尾混杂了奥利亚罗非鱼的基因,同时也说明分子生物学手段应用于种质鉴定比形态学手段更为精确.     

Genotyping of a Miso and Soy Sauce Fermentation Yeast,Zygosaccharomyces rouxii,Based on Sequence Analysis of the Partial 26S Ribosomal RNA Gene and Two Internal Transcribed Spacers

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I–VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type α and type β) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species.     

Study on Sequences of Ribosomal DNA Internal Transcribed Spacers of Clams Belonging to the Veneridae Family (Mollusca: Bivalvia)

The first and second internal transcribed spacer (ITS1 and ITS2) regions of the ribosomal DNA from four species, Meretrix meretrix L., Cyclina sinensis G., Mercenaria mercenaria L., and Protothaca jedoensis L., belonging to the family Veneridae were amplified by PCR and sequenced. The size of the ITS1 PCR amplification product ranged from 663 bp to 978 bp, with GC contents ranging from 60.78% to 64.97%. The size of the ITS1 sequence ranged from 585 bp to 900 bp, which is the largest range reported thus far in bivalve species, with GC contents ranging from 61.03% to 65.62%. The size of the ITS2 PCR amplification product ranged from 513 bp to 644 bp, with GC contents ranging from 61.29% to 62.73%. The size of the ITS2 sequence ranged from 281 bp to 412 bp, with GC contents ranging from 65.21% to 67.87%. Extensive sequence variation and obvious length polymorphisms were noted for both regions in these species, and sequence similarity of ITS2 was higher than that of ITS1 across species. The complete sequences of 5.8S ribosomal RNA gene were obtained by assembling ITS1 and ITS2 sequences, and the sequence length in all species was 157 bp. The phylogenetic tree of Veneridae clams was reconstructed using ITS2-containing partial sequences of both 5.8S and 28S ribosomal DNA as markers and the corresponding sequence information in Arctica islandica as the outgroup. Tree topologies indicated that P. jedoensis shared a close relationship with M. mercenaria and C. sinensis, a distant relationship with other species.     

Isolation and characterization of polymorphic microsatellite markers in the endangered Nothotsuga longibracteata (Pinaceae)

Twelve polymorphic microsatellite loci were isolated and characterized from an AC‐enriched genomic library of Nothotsuga longibracteata. The average allele number of these microsatellites was 8.3 per locus, ranging from two to 13. The ranges of observed and expected heterozygosities were 0.03–0.97 and 0.09–0.88, respectively. These polymorphic markers provide useful tools for the study of evolutionary history and conservation genetics of N. longibracteata.     

Phylogenetic relationships ofPythium species based on ITS and 5.8S sequences of the ribosomal DNA

The sequences of ITS regions in 30 species and two groups of the genusPythium were resolved. In the phylogenetic trees, the species were generally divided into two clusters, referred to here as the F and S groups. The species in the two groups correspond in terms of their sporangial morphology, with the F group being filamentous/lobulate and the S group being spherical. Genetic divergence within the F group was lower than that within the S group. Other morphological characteristics such as oogonial structure and sexual nature appeared to be unrelated to the groupings in these trees. An alignment analysis revealed common sequences to all the species and arrangements specific to each F or S group. It was found that the ITS region was a good target in designing species-specific primers for the identification and detection ofPythium species. In the tree based on 5.8S rDNA sequences, oomycetes are distantly related to other fungi but separated from algae in Chromista.     

High evolutionary divergence of the 5.8S ribosomal DNA in Mimulus glaucescens (Scrophulariaceae)

Ribosomal DNA sequences for the ITS 1, 5.8S, ITS 2 and adjoining regions of the 18S and 25S were obtained from Mimulus glaucescens (Scrophulariaceae) via cloned PCR products. The spacer sequences were completely unrelated to other plant taxa, although spacer lengths were approximately the same. Interestingly, the Mimulus 5.8S sequence was much more divergent than other higher-plant rDNA sequences. Consideration of the secondary structure of the 5.8S rRNA shows that most of the changes in Mimulus are compensatory and preserve the basic secondary structure of the mature RNA molecule.     

Molecular phylogeny of Pneumocystis based on 5.8S rRNA gene and the internal transcribed spacers of rRNA gene sequences

To clarify the phylogenetic relationships and species status of Pneumocystis, the 5.8S rRNA gene and the internal transcribed spacers (ITS, 1 and 2) of Pneumocystis rRNA derived from rat, gerbil and human were amplified, cloned and sequenced. The genetic distance matrix of six Pneumocystis species compared with other fungi like Taphrina and Saccharomyces indicated that the Pneumocystis genus contained multiple species including Pneumocystis from gerbil. The phylogenetic tree also showed that Pneumocystis from human and monkey formed one group and four rodent Pneumocystis formed another group. Among the four members, Pneumocystis wakefieldiae was most closely related to Pneumocystis murina and Pneumocystis carinii, and was least related to gerbil Pneumocystis.