DNA分子标记在实验动物遗传分析中的应用

DNA分子标记技术很多,基本都是建立在RFLP、PCR和重复顺序的基础上的。本文重点介绍了限制性片段长度多态性（RFLP）标记、随机扩增多态性DNA（RAPD）标记、微卫星DNA（STR）标记、DNA指纹（DFP）标记、扩增片段长度多态性（AFLP）标记等几种重要的DNA分子标记技术的定义、结构、分布、组成、保守性、优点及丰富的多态性等。并重点介绍了微卫星DNA（STR）标记在分子遗传监测、遗传多样性分析和遗传血缘关系及个体识别等领域的应用。     

微卫星DNA标记及其在鱼类遗传多样性研究中的应用

微卫星DNA作为第二代分子遗传标记是高等真核生物基因组中种类多、分布广、具有高度的多态性和杂合度的分子标记,由于其具有多态性检出率高、信息含量大、共显性标记、实验操作简单、结果稳定可靠等优点,已经成为种群遗传学研究中被广泛应用的分子遗传标记。微卫星DNA标记技术在鱼类的群体遗传结构的分析、物种遗传多样性的鉴定以及遗传基因连锁图谱的构建等方面已初步得到应用。该文就微卫星技术的原理方法,在鱼类遗传多样性研究中的应用概况以及应用范围和注意事项等方面进行综述。为微卫星技术在鱼类遗传多样性研究中应用提供了理论参考。     

海参微卫星DNA的筛选与克隆的初步分析

微卫星DNA(microsatelliteDNA)广泛存在于真核生物的基因组中。由于其具有突变频率快、多态性丰富、呈共显性遗传、通用性等特点,已成为近年来被广泛应用的分子遗传标记。对刺参基因组DNA的提取及其微卫星的筛选与克隆进行了初步分析。     

微卫星分子标记在濒危动物保护遗传学研究中的应用

微卫星DNA广泛分布于真核生物基因组中,具有多态性高、共显性遗传、选择中性、易于操作等特点,是一种极具应用价值的分子遗传标记,近年来在濒危动物保护遗传学研究中得到越来越多的应用。微卫星DNA高度多态性提供的高分辨率遗传信启,使其不仅适合个体水平的亲子鉴定与交配系统研究,而且也已成为种群遗传结构与多样性分析的有效分子标记。微卫星分析所需的DNA量极少,用非损伤性方法获取的极少量样品或陈旧样品就能用于有效分析,方便了濒危动物野外调查工作的开展,并且可以利用年代久远的馆藏历史标本揭示种群的重要历史进程。另外,某些微卫星DNA大小在近缘物种间可相互区分,这使得部分物种的DNA分子鉴别将更为简便。但微卫星分子标记的座位筛选和特异引物开发耗时费力,一定程度上限制了其广泛应用。针对不同的研究目的选择合适的分子标记方法将有助于更好的揭示问题本质。     

微卫星遗传标记及其在昆虫种群遗传学中的应用

微卫星是一类短串联重复的寡核苷酸序列,广泛地分散于各类真核生物基因组中,它具有多态性高、检测结果稳定可靠等特点,是目前较为理想的群体遗传研究的分子标记之一。该文阐述了微卫星DNA构成及特点,多态性形成机制、位点获得途径,列举了微卫星遗传标记在昆虫种群遗传学研究中的应用实例,并展望了该技术的应用前景。     

ISSR标记技术及其在遗传多样性研究中的应用

ISSR(Inter-Simple Sequence Repeat)技术是在PCR中直接使用微卫星序列进行DNA扩增的一种DNA分子标记。章主要介绍了ISSR标记的原理、方法、特点及其在遗传多样性研究中的应用。ISSR标记方法具有无需知道任何靶标序列的微卫星背景信息、遗传多态性高、检测快速等特点,在遗传多样性研究中具有广泛的应用前景。     

鸡的遗传多样性的研究方法及研究进展

遗传多样性是生物学研究中的一个重要领域,研究鸡的遗传多样性,不仅能加强生物多样性的保护。同时对起源进化、分类鉴定及遗传育种等都有重要的意义。本文对目前DNA水平鸡的遗传多样性的研究方法和研究进展进行了详细的阐述。重点介绍了DNA分子标记的特征;概括了在鸡遗传多样性分子标记的方法,包括微卫星分子标记(SSR)、扩增片段长度多态性(AFLP)、随机扩增多态性标记(RAPD)、限制性片段长度多态性标记(RFLP)和单核苷酸多态性标记(SNP)。本文综述了最近有关鸡DNA水平的遗传多样性的研究方法在系统学、遗传结构、生物地理等研究中的应用情况;提出在研究鸡遗传多样性时,可根据研究的目的,选择合适的方法。     

鲫鱼微卫星DNA的分离及多态性分析

目的：用近缘物种鲤微卫星引物来分离鲫鱼微卫星标记并对其多态性进行分析。方法：以鲫鱼基因组DNA为模板,采用6对鲤微卫星引物进行PCR扩增,PCR产物经8%的非变性聚丙烯酰胺凝胶电泳和银染色检测。结果：筛选出2个以AC和TA为重复单元的鲫鱼新的微卫星标记。多态性分析表明,这2个微卫星标记的遗传杂合度分别为0.611和0.644,多态信息含量为0.536和0.572,属于高度多态性标记。结论：该研究筛选的2个微卫星标记可应用于鲫鱼遗传多样性、遗传连锁图谱构建及分子标记辅助育种等方面的研究。     

微卫星分子标记及其在动物遗传育种中的研究进展

微卫星分子标记在基因组中具有含量丰富、多态性、共显性和易于检测等优点,是动物遗传育种应用中一种重要的分子标记技术。随着科学技术的飞速发展,微卫星标记的研究方法也日新月异:生物信息学技术的兴起使得微卫星位点的获得越来越方便;高通量测序技术的成熟使得开发新微卫星标记更加简单高效;自动化的序列分析仪器使得微卫星DNA的检测越来越快速准确;同时微卫星标记的应用范围也越来越广泛,其功能的开发正逐渐从刚开始的个别位点研究转变到全基因组微卫星分析。本研究简单介绍了微卫星分子标记的特点,重点综述了目前微卫星分子标记的获得、开发和筛选方法,及其在动物遗传育种中的应用,将为微卫星分子标记的研究提供理论支持。     

微卫星DNA标记技术及其在昆虫学上的应用

微卫星DNA标记作为一种多态性和稳定性高、重复性好、呈共显性的分子遗传标记技术,目前已被广泛应用于昆虫学的研究中。本文介绍了微卫星DNA标记的基本原理和特点,并综述了近年来该技术在昆虫种群遗传结构及分化、生物学特性与习性、遗传图谱的构建、基因定位以及系统发生等领域中的应用。     

Holotoxin content in the tissues of the holothurian Stichopus japonicus at different seasons of the year and their effect on oocyte maturation

Seasonal variations and distribution of holotoxins in tissues of the sea cucumber Stichopus japonicus have been studied. It was shown that holotoxin content in the ovaries is higher than that in other organs during the breeding season. In the course of a year, the amount of glycosides in other holothurian organs changed a little. It has been found out that high concentrations of glycosides inhibit oocyte maturation in the holothurian Stichopus japonicus.     

海参i型溶菌酶基因及其编码产物的结构特点

通过RT-PCR 和 RACE PCR技术,从海参(Stichopus japonicus)体壁中克隆得到一种溶菌酶基因(GenBank：EF036468).生物信息软件分析表明,其中全长cDNA为 713 bp,5′非编码区（UTR）246 bp,3′UTR 29 bp,开放阅读框438 bp,编码145个氨基酸,包括溶菌酶成熟肽124个氨基酸和信号肽21个氨基酸.对海参溶菌酶与多种无脊椎动物的c、g和i型溶菌酶进行分析比较,发现它与i型溶菌酶有较高的同源性,并具有i型溶菌酶高度保守的2个活性位点,即Glu34和Ser50.活性位点附近具有i型溶菌酶的一段特有的氨基酸保守序列MDVGSLSCG(P/Y)(Y/F)QIK,所以推断克隆的海参溶菌酶为i型.另外,通过搜索蛋白保守结构域数据库,发现海参溶菌酶与医用水蛭失稳酶相似性最高,并且这2个酶的三级结构模型也极其相似.因此推测,海参i型溶菌酶具有双功能特性,既能作用于细菌细胞壁的糖苷键使细胞裂解,又具有失稳酶的一些生化功能,能够水解纤维蛋白,这些特点在海参自溶过程中发挥重要的作用.     

Genetic differentiation among populations and color variants of sea cucumbers (Stichopus japonicus) from Korea and China

The Far Eastern sea cucumber, Stichopus japonicus, is a favored food in Eastern Asia, including Korea, Japan, and China. Aquaculture production of this species has increased because of recent declines in natural stocks and government-operated stock release programs are ongoing. Therefore, the analyses of genetic structure in wild and hatchery populations are necessary to maintain the genetic diversity of this valuable marine resource. In addition, given that sea cucumber color affects market price, with the rare, possibly reproductively isolated, red type being the most valuable, an understanding of the genetic structure and diversity in color variation of green and red types is necessary. We analyzed the genetic structure of wild and hatchery-produced green type S. japonicus from Korea and China, and wild red type from Korea using 9 microsatellite makers. The number of alleles per locus ranged from 11 to 29 across all populations. The mean allele numbers of the green types from Korea (10.6) and China (10.1) were similar, but differed slightly from that of the red type (9.1). Pairwise multilocus F(ST) and genetic distance estimations showed no significant differences between the green types from Korea and China, whereas the differences between the green and red types were significant. This was clearly illustrated by a UPGMA dendrogram, in which the two close subclusters of green types were completely separated from the red type. In addition, the allele frequencies of the green and red types were significantly different. Assignment tests correctly assigned 100% (quality index 99.97%) of individuals to their original color types and demonstrated the feasibility of microsatellite analysis for discrimination between color types.     

海刺参(Stichopus japonicus)电压依赖性钙离子通道 β亚基基因及其编码产物的结构特点学分析

电压依赖性钙离子通道(voltage dependent calcium channels, VDCC)是细胞膜上 控制Ca^2+进入胞内的特殊蛋白质,VDCC β亚基具有调节其通道活性的作用．使用 RT- PCR 和 RACE-PCR技术,从海刺参(Stichopus japonicus)肠中克隆得到电压依赖性钙离子通道β亚基(SjCaβ)基因,所得序列提交GenBank,登录号EU853658．该基因全长1 867 bp,包含1个1 614 bp的开放阅读框(ORF),编码537个氨基酸．将SjCaβ与多种无脊椎动物钙离子通道β亚基进行分析比较,发现它们有较高的同源性,并具有钙离子通道β亚基基因内保守的2个功能区域,即SH3功能区（src-homology 3 domain）和GK功能区(guanylate kinase domain)．在功能区域附近还具有电压依赖性钙离子通道β亚基特有的BID区(β interaction domain),氨基酸保守序列为PYDVVP-RP--- VGPSLKGYEVTDMMQKALFDF,进一步确定了得到的cDNA序列为海刺参电压依赖性钙离子通道β亚基．对SjCaβ的二级结构进行了预测,构建了系统进化发育树,并运用同源建模法构建了SjCaβ的三维结构模型．研究结果对深入研究海刺参离子通道的结构以及在信号传导过程中的作用有着重要意义,并为研究海刺参自溶等问题提供一定的理论基础．     

微生物方法降解刺参养殖池塘中氨态氮的研究进展

随着刺参养殖业的迅速发展,刺参池塘养殖暴露出诸多问题,如水体氨态氮含量高、病害严重、单位产量下降等.传统药物弊端较多,微生物方法成为降解水体中氨态氮含量和预防病害发生的新的有效途径.综合阐述了刺参池塘养殖过程中氨态氮对刺参的危害和微生物降解氨态氮的利弊,并介绍了水体中氨态氮高效降解菌株的筛选以及现有微生态制剂存在的问题,对微生物方法降解刺参养殖池塘氨态氮的前景进行了展望.     

Expression,purification, and characterization of arginine kinase from the sea cucumber Stichopus japonicus

The arginine kinase gene of sea cucumber Stichopus japonicus was cloned and inserted into the prokaryotic expression plasmid pET-21b. The protein was expressed in a soluble and functional form in Escherichia coli and purified by Blue Sepharose CL-6B, DEAE-32, and Sephadex G-100 chromotography with a final yield of 83 mgL(-1) of LB medium. The specific activity, electrophoretic mobility, and isoelectric focusing were all identical with those of arginine kinase that was purified from sea cucumber muscle. The fluorescence emission spectrum of arginine kinase had a maximum fluorescence at a wavelength of 330 nm upon excitation at 295 nm. These results are the first report of this purified protein.     

Thermal inactivation and unfolding of a dimeric arginine kinase

Thermal inactivation and unfolding of the dimeric arginine kinase (AK) from sea cucumber Stichopus japonicus was investigated. The activation energy was calculated to be 388 kJ/mol. Based on the analysis of the denaturation course at 58 degrees C, a model is suggested for the thermal unfolding of this dimeric AK. In addition, the effect of free Mg(2+) and the potential biological significance on the thermal unfolding of dimeric AK is discussed.     

Development and characterization of 70 novel microsatellite markers for the sea cucumber (Apostichopus japonicus)

The sea cucumber (Apostichopus japonicus) is an important item in Asian cuisine. It is currently produced through aquaculture, especially in China, after being overexploited in the wild in the 1990s. We isolated 70 novel polymorphic microsatellite loci using an enrichment-colony hybridization protocol. All loci were characterized in 48 individuals from a natural population in Rongcheng (Shandong, China) using genomic DNA isolated from muscle tissue. The number of alleles ranged from 2 to 17 (mean 7.0), and the observed and expected heterozygosities varied from 0.0010 to 1.0000 and from 0.2125 to 0.9477, respectively. Thirty-one of the 70 loci exhibited departure from Hardy-Weinberg equilibrium. These microsatellite markers should be useful resources for population genetic studies and for molecular marker-assisted breeding of A. japonicus.     

INDUCTION OF OOCYTE MATURATION BY DISULFIDE-REDUCING AGENT IN THE SEA CUCUMBER, STICHOPUS JAPONICUS

1-Methyladenine (1-MeAde) is known to be a natural inducer of starfish oocyte maturation. Disulfide-reducing agents such as dithiothreitol (DTT) and 2, 3-dimercapto-1-propanol (BAL) are known to mimic the action of 1-MeAde in inducing starfish oocyte maturation. Although 1-MeAde failed to induce oocyte maturation in sea cucumbers, breakdown of germinal vesicles and subsequent meiotic behaviour of chromosomes were induced by the treatment with DTT in the pronase-treated oocytes of the sea cucumber, Stichopus japonicus. These findings suggest that reduction of disulfide bonds plays an important role in triggering oocyte maturation in some marine forms such as echinoderms.