Survival of bacterial DNA and culturable bacteria in archived soils from the Rothamsted Broadbalk experiment

Dried soil samples from many sources have been stored in archives world-wide over the years, but there has been little research on their value for studying microbial populations. Samples collected since 1843 from the Broadbalk field experiment on crop nutrition at Rothamsted have been used to document changes in the structure and composition of soils as agricultural practices evolve, also offering an invaluable record of environmental changes from the pre- to post-industrial era in the UK. To date, the microbial communities of these soils have not been studied, in part due to the well-documented drop in bacterial culturability in dried soils. However, modern molecular methods based on PCR amplification of DNA extracted directly from soil do not require bacterial cells to be viable or intact and may allow investigations into the legacy of bacteria that were present at the time of sample collection.

In a preliminary study, to establish if dried soils can provide a historical record of bacterial communities, samples from the Broadbalk soil archive dating back to 1868 were investigated and plots treated with either farmyard manure (FYM) or inorganic fertilizer (NPK) were compared. As anticipated, the processes of air-drying and milling greatly reduced bacterial viability whilst DNA yields declined less and may be preserved by desiccation. A higher proportion of culturable bacteria survived the archiving process in the FYM soil, possibly protected by the increased soil organic matter. The majority of surviving bacteria were firmicutes, whether collected in 2003 or in 1914, but a wide range of genera was detected in DNA extracted from the samples using PCR and DGGE of 16S rRNA genes. Analysis of DGGE band profiles indicated that the two plots maintained divergent populations. Sequence analysis of bands excised from DGGE gels, from a sample collected in 1914, revealed DNA from - and β-proteobacteria as well as firmicutes. PCR using primers specific for ammonia oxidizing bacteria showed similar band profiles across the two treatments in recently collected samples, however older samples from the NPK plot showed greater divergence. Primers specific for the genus Pseudomonas were designed and used in real-time quantitative PCR to indicate that archived soil collected in 1868 contained 10-fold less pseudomonad DNA than fresh soil, representing around 10⁵ genomes g⁻¹ soil. Prior to milling, dramatically less pseudomonad DNA was extracted from recently collected air-dried soil from the NPK compared to the FYM plot; otherwise, the two plots followed similar trends. Overall bacterial abundance, diversity and survival during the archiving process differed in the two soils, possibly due to differences in clay and soil organic matter content. Nevertheless, the results demonstrate that air-dried soils can protect microbial DNA for more than 150 years and offer an invaluable resource for future research.     

鱼糜制品中基因组DNA提取方法的比较

将分别采用普通酚-氯仿抽提法和试剂盒(柱吸附法)提取DNA的两种方法加以比较,以确定最适提取鱼糜制品DNA的方法.将鱼肉和鱼糜制品作为原料,用两种方法提取基因组DNA,利用分光光度计测定其A260与A280的吸光值,计算比率估计核酸的纯度,并利用琼脂糖凝胶电泳观察DNA片段在凝胶中的位置;并用线粒体16S rRNA基因PCR扩增产物鉴定所提取的基因组DNA.     

陆地棉SSR分子标记优化体系的建立

针对陆地棉富含酚类、多糖等次生产物的特点,建立了一种简便、快速的提取高纯度DNA的方法.同时建立了适合陆地棉SSR标记的优化体系和试验方案,并对所构建的QTL定位群体进行了SSRPCR检测,得到了清晰的多态性SSR标记,为SSR分子标记技术在棉花育种中的应用打下了基础.     

多子领春木RAPD反应体系的研究

利用RAPD(随机扩增多态性DNA)技术对濒危植物多子领春木进行了RAPD反应体系的研究.以多子领春木叶片为材料,采用改良的CTAB法提取其基因组DNA,进行RAPD反应条件的优化.通过单因子试验,分别研究了模板DNA用量、Mg2 浓度、dNTPs浓度、Taq酶的用量和引物浓度对RAPD反应的影响,建立了适于多子领春木RAPD分析的有效稳定的反应体系,即在25μL总反应体系中,模板DNA的最适用量为10 ng、Mg2 的适宜浓度为2.0 mmol·L-1、dNTPs的适宜浓度2.2 mmol·L-1、Taq酶的用量为1U、随机引物的浓度以2.5 μmol·L-1为佳.     

杂交水稻品种指纹鉴定研究进展

从蛋白质指纹和DNA指纹两个方面,综述了杂交水稻品种指纹鉴定研究的进展和不足。对SSR标记在品种指纹鉴定上的应用前景,单一特异标记的筛选和实验室标准检测技术的建立作了探讨和展望,以利于加快杂交水稻品种指纹鉴定的发展和应用。     

柏氏鲤与荷包红鲤抗寒品系杂交子二代的RAPD分析

采用随机扩增多态性DNA（RAPD）分子标记技术，对荷包红鲤抗寒品系与柏氏鲤进行杂交所获得的F2代个体进行亲子鉴定分析。从180个随机引物中筛选出5个多态性较丰富的引物，并对其产生的多态性DNA片段进行统计分析，结果显示：双亲的RAPD谱带全部在F2代中表现出来，而子代中产生的所有谱带在双亲中的表现率在AB02、AB05、AC20、AI10、AI13几个引物中分别为87．0％、100．0％、92．1％、82．8％、92．6％。这也表明BAPD技术在对鲤的亲子鉴定中具有很强的可行性。     

刺参微卫星DNA分子标记的分离及筛选

采用磁珠富集法分离刺参Apostichopus japonicus的微卫星分子标记,共获得阳性克隆123个,其中106个含微卫星DNA序列。分析结果表明:完美型有48个,占45.28%,非完美型有39个,占36.79%,复合型有19个,占17.92%;重复碱基数以双核苷酸重复最常见,占84.91%,双核苷酸中又以CA/GT重复所占比例最大;在重复次数方面,以重复数为3~10次、11~18次和≥35次最为常见,各占39.62%、26.42%和16.98%,重复数达100次以上的有5个,最多的达到148次,有28个微卫星序列微卫星含量丰富。筛选的22对引物中,可产生出扩增产物的有17对,其中16对引物的产物带在预测区域内出现;有7对引物在中国、俄罗斯刺参模板中表现出多态性。文中还对刺参微卫星的特点进行分析,对刺参微卫星引物的PCR筛选结果进行了探讨。     

饥饿及再投喂对日本囊对虾蛋白质代谢的影响

研究了体重为(13.08±2.40)g、体长为(10.37±0.73)cm的日本囊对虾Marsupenaeus ja-ponicus在饥饿和再投喂状态下蛋白质代谢的变化。对照组C连续饱食投喂10 d,试验组S10、S15、S25分别饥饿10、15、25 d后再恢复饱食投喂10 d。结果表明:在饥饿状态下,日本囊对虾肌肉中蛋白质的含量在饥饿前15 d迅速上升,饥饿第15~25 d趋于平稳,肝胰脏中蛋白质的含量则一直较稳定;恢复投喂后,肌肉中蛋白质的含量下降,而肝胰脏中蛋白质的含量上升。血浆中蛋白质的含量在饥饿前期(0~5 d)保持平稳,但在之后的第5~15 d则迅速下降,最后又恢复至最初水平;恢复投喂后,除S25组外其余各组血浆中蛋白质的含量都呈上升趋势,且试验组显著高于对照组(P<0.05)。肌肉中RNA/DNA的值在饥饿初期(0~5 d)略有下降,之后保持稳定,肝胰脏中RNA/DNA的值则在短期下降后有所上升;恢复投喂后,肌肉中RNA/DNA的值除S25组外其余各组均呈上升趋势,而肝胰脏中RNA/DNA的值除C组外其余各组均显著上升(P<0.05)。     

直接扩增甜瓜小卫星DNA指纹图谱

以小卫星DNA YNZ22核心序列为引物,甜瓜DNA为模板,在甜瓜上研究了直接扩增小卫星DNA(DAMD)的优化反应体系,结果表明:在20μL的反应体系中,5种主要成分Taq DNA聚合酶、Mg2 、引物、模板DNA和dNTPs的最适浓度分别为1U,2.5 mmol/L,0.5 mmol/L,30 ng,5 mmol/L。在优化的DAMD-PCR反应体系下,利用该引物在28个甜瓜品种上构建了直接扩增小卫星DNA(DAMD)的指纹图谱,分析结果表明,该引物在28个甜瓜品种上共扩增出13位点,其中9位点具有多态性,多态性条带比率为69%,可一次性从28个甜瓜品种中鉴别出其中的21个,鉴别率高达75%。     

贵州荞麦重要种质的遗传多样性分析及分子身份证构建

为精准鉴定贵州荞麦种质资源,采用SSR分子标记对60份荞麦种质进行遗传多样性分析,构建DNA分子身份证数据库。结果显示,从100对SSR引物中筛选出16对稳定性好、多态性丰富的引物,在60份供试种质中共扩增出174个多态性条带;Shannon’s信息指数、Nei’s多样性指数、多态信息指数均值分别为0.337、0.206、0.693,引物的多态性较好,能有效揭示60份荞麦种质的遗传多样性;在Dice遗传相似系数为0.374时,所有供试材料可聚为A、B、C 三组;当Dice遗传相似系数为0.484时,将苦荞组（A组）更细分为A1、A2两个小组。采用毛细管电泳及8%的聚丙烯酰胺凝胶（PAGE）电泳对SSR标记扩增产物进行双验证,2种方法的聚类结果一致。结果表明,本研究开发的高效性SSR分子标记能够有效地鉴定贵州荞麦重要种质的遗传多样性且用于构建分子身份证。