Xα21基因导入水稻及转基因植株的鉴定

以水稻愈伤组织为受体、质粒pCXK1301为Xα21基因供体,用农杆菌EHA105和PDS－1000／He基因枪转化受体,经Hyg浓度梯度筛选,获得一些转基因水稻植株,GUS检测、PCR扩增和Southern杂交分析表明,报告基因（GUS）、选择标记基因（Hyg^r）和抗白叶枯病基因Xα21都已整合入转化水稻基因组中,Southern杂分析显示了Xα21基因在转化水稻基因组中整合的拷贝数。田间接菌鉴定表现抗白叶枯病。     

甲基营养型酵母表达载体系统研究进展

酵母作为生产真核异源蛋白的宿主菌 ,具备了易于分子遗传操作 ,原核微生物的生长性质及真核蛋白的翻译后加工修饰等特点 .其中Ｓａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ (酿酒酵母 )是第一个作为异源蛋白基因表达系统并加以应用的酵母 .人们认为Ｓ .ｃｅｒｅｖｉｓｉａｅ并非表达外源基因的最理想的酵母宿主菌 .在一群优于Ｓ .ｃｅｒｅｖｉｓｉａｅ的表达菌中 ,甲基营养型酵母Ｐｉｃｈｉａｐａｓｔｏｒｉｓ研究得最多 ,最具应用潜力 .在 2 0世纪 70年代期间 ,Ｐ .ｐａｓｔｏｒｉｓ是单细胞蛋白 (Ｓｉｎｇｌｅ ｃｅｌｌｐｒｏｔｅｉｎ…     

Genetic Bottlenecks Driven by Population Disconnection

Abstract: Connectivity among populations plays a crucial role in maintaining genetic variation at a local scale, especially in small populations affected strongly by genetic drift. The negative consequences of population disconnection on allelic richness and gene diversity (heterozygosity) are well recognized and empirically established. It is not well recognized, however, that a sudden drop in local effective population size induced by such disconnection produces a temporary disequilibrium in allelic frequency distributions that is akin to the genetic signature of a demographic bottleneck. To document this effect, we used individual‐based simulations and empirical data on allelic richness and gene diversity in six pairs of isolated versus well‐connected (core) populations of European tree frogs. In our simulations, population disconnection depressed allelic richness more than heterozygosity and thus resulted in a temporary excess in gene diversity relative to mutation drift equilibrium (i.e., signature of a genetic bottleneck). We observed a similar excess in gene diversity in isolated populations of tree frogs. Our results show that population disconnection can create a genetic bottleneck in the absence of demographic collapse.     

连续施用发酵猪粪对土壤中四环素抗性基因数量的影响

为了研究连续施用发酵猪粪之后,土壤中抗性基因的数量以及发酵猪粪的施用量对土壤抗性基因的影响,采用实时荧光定量PCR方法,对稻麦轮作模式下连续6年施用4.5t/hm2和9.0t/hm2发酵猪粪的土壤进行了四环素抗性基因(TRGs)的检测和定量分析.在施用发酵猪粪的土壤中能够检测到9种TRGs.施用发酵猪粪显著增加了土壤中tet G、tet L、tet B(P)、tet O、tet W的绝对数量,其中tet B(P)、tet W、tet O的绝对数量受到了施肥用量的影响;而tet Z、tet C和tet S的绝对数量则没有受到施肥的影响.上述8种TRGs在同一施肥处理的0~5cm、5~10cm以及10~20cm土壤中的相对丰度分布均无显著差异.发酵猪粪的施用也显著增加了tet G、tet L、tet B(P)和tet O的相对丰度,但是仅有tet O的相对丰度受到了施肥用量的显著影响.本研究表明,在稻麦轮作模式下,农田土壤中的TRGs数量会受到发酵猪粪中残留TRGs的影响,连续施用该粪肥会显著增加土壤中tet G、tet L、tet B(P)和tet O的绝对数量和相对丰度.因此,需要优化畜禽粪堆肥发酵工艺,以减少抗性基因在粪肥中的残留.     

Interspecific and intraspecific identification of Dendrobium based on the psbAtrnH intergenic region sequences and the 5S rRNA gene spacer sequences北大核心CSCD

This research aimed to investigate the interspecific and intraspecific identification of Dendrobium by using the multi-locus method so as to provide a molecular basis for Dendrobium identification through the combination of chloroplast psbA-trnH intergenic region sequences and ribosome 5S rRNA gene spacer sequences. PCR direct sequencing was applied to detect the chloroplast psbA-trnH intergenic region sequences as well as the ribosome 5S rRNA gene spacer sequences of 12 Dendrobium species, while the psbA-trnH intergenic region sequences of Dendrobium denneanum dq-2 variety and dq- 5line were cloned and sequenced for single nucleotide polymorphism (SNP) analyzing. The sequences were analyzed by the software Sequencher4.14, Bioedit7.0, MEGA5.2 and Dansp5.0; the interspecific and intraspecific Kimara-2-Parameter(K2P) distances were also calculated. The phylogenetic tree (using Neighbor joining method) was constructed with Bulbophyllum odoratissimum and Bletilla striata as outgroup. The results showed an average length of chloroplast psbA-trnH gene sequences in Dendrobium as 742.3 bp, with 72 variable sites, including 33 information sites; the average length of the ribosome 5S rRNA gene spacer sequences in Dendrobium was 336.4 bp, with 213 variable sites including 139 information sites. Using psbAtrnH intergenic region sequences in combination with ribosome 5S rRNA gene spacer sequences can not only identify D. denneanum, D. hancockil, D. thysiflorum, D. devonianum, D. moniliforme, D. chrysotoxum, D. officinale, D. heterocarpum and D. nobile, but also differentiate D. officinale from different geographical populations, and distinguish the dq-2 variety and dq 5line with SNP in the multi locus of D. denneanum.     

基于esp基因和JCV标记的MST方法在五大流域典型水源地中的应用

非点源污染目前已经成为影响水体环境的重要污染。微生物源示踪技术(microbial source tracking,MST)是解决非点源污染的一项新技术,它可以确定污染的宿主来源。肠球菌esp基因和多瘤病毒JCV可以作为检测水体中人源粪便污染的分子标记,其灵敏度和特异性都很高。为分析五大流域水源地是否受到人源粪便的污染,对辽河、海河、淮河、长江和黄河五大流域典型水源地水样进行采集和检测,选取人源粪便特异病原微生物肠球菌的esp基因和多瘤病毒JCV建立了相应的MST分子检测方法。结果表明,五大流域的典型水源地采样点均有可能受到了人源粪便的污染,可为当地相关部门提供技术支持和数据参考。     

湖南石门雄黄矿区矿样中的砷氧化细菌的分离及鉴定

绿色的生物学方法处理含砷废水,对于缓解工业发展所带来砷污染问题,提高居民健康生活质量具有重要意义。以湖南石门雄黄矿区的尾矿池积水（SY）、地下400 m处的矿坑水（NY）和尾矿池底泥表层（XY）3个样本为研究对象,利用细菌富集和多次传代的方法,从这3个环境样本中获得可氧化三价砷的3个菌群。在砷质量浓度为l~5 g·L-1的培养基中分别检测上述3个菌群的耐受能力,发现在砷质量浓度高达5 g·L-1的条件下,上述菌群均能在2~3 d内达到109 cells·mL-1。利用上述菌群处理l g·L-1亚砷酸钠溶液,SY、XY和NY菌群可分别在25、20和35 h内将三价砷完全氧化,并且使溶液中的总砷质量浓度降低66.7%。进一步对3个菌群进行多次平板分离,利用五价砷和硝酸银的颜色反应,获得11株可氧化三价砷的目的菌株。对这些菌株16S rDNA进行NCBI的BLASTN序列比对发现,菌株SMY24、SMY33、SMY22、SMY32、SMY21和SMY31均属于假单胞菌属（Pseudomonas）,菌株SMY104属于不动杆菌属（Acinetobacter）,菌株SMY17、SMY25、SMY9和SMY1在所有的已知序列中,最大的相似率只有91%,最小的相似率为76%,且都是未曾报道纯培养的细菌。对这些菌株三价砷氧化能力进行测定发现,其中菌株SMY104和SMY21在20 h内能将三价砷几乎完全氧化。通过对这些菌株三价砷氧化酶基因进行初步检测,发现了多个菌株具有三价砷氧化酶基因（AoxB）。     

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

Amphibian metamorphosis provides a wonderful model to study the thyroid hormone (TH) signaling disrupting activity of environmental chemicals, with Xenopus laevis as the most commonly used species. This study aimed to establish a rapid and sensitive screening assay based on TH-response gene expression analysis using Pelophylax nigromaculatus, a native frog species distributed widely in East Asia, especially in China. To achieve this, five candidate TH-response genes that were sensitive to T3 induction were chosen as molecular markers, and T3 induction was determined as 0.2 nmol/L T3 exposure for 48 hr. The developed assay can detect the agonistic activity of T3 with a lowest observed effective concentration of 0.001 nmol/L and EC50 at around 0.118–1.229 nmol/L, exhibiting comparable or higher sensitivity than previously reported assays. We further validated the efficiency of the developed assay by detecting the TH signaling disrupting activity of tetrabromobisphenol A (TBBPA), a known TH signaling disruptor. In accordance with previous reports, we found a weak TH agonistic activity for TBBPA in the absence of T3, whereas a TH antagonistic activity was found for TBBPA at higher concentrations in the presence of T3, showing that the P. nigromaculatus assay is effective for detecting TH signaling disrupting activity. Importantly, we observed non-monotonic dose-dependent disrupting activity of TBBPA in the presence of T3, which is difficult to detect with in vitro reporter gene assays. Overall, the developed P. nigromaculatus assay can be used to screen TH signaling disrupting activity of environmental chemicals with high sensitivity.     

Cloning and expression analysis of geosmin synthase gene from Aphanizomenon gracile北大核心CSCD

Geosmin is a secondary metabolite responsible for earthy odors. The occurrence of geosmin has great impact on the quality of water environment. The gene essential for geosmin biosynthesis have been identified in several species. But little is known about the mechanism of geosmin synthesis in Aphanizomenon gracile. This study attempted to clone the gene involved in geosmin biosynthesis of Aphanizomenon gracile a nd a nalyze t he geosmin production u nder d ifferent e nvironments. T he high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) was used to amplify the full-length of geosmin synthase gene from Aphanizomenon gracile (WH-1). Real time PCR (RT-PCR) was applied to quantify the geosmin production in different light and temperature. As a result, geo, a geosmin synthase gene from Aphanizomenon gracile (WH-1) was cloned by hiTAIL-PCR. The full-length open reading frame (ORF) of geo was 2 262 bp, coding for a protein of 753 amino acids. Meanwhile, WH-1 was treated with different environment conditions and mRNA expression levels of geo were quantified by RT-PCR. It was found that low temperature (15 °C), high light intensity (35 μmol m-2 s-1) and continuous light illumination were beneficial to the expression of geo. The successful amplification of geosmin synthase gene verified that hiTAIL-PCR is an effective and simple procedure of low cost. The result provides fundamental knowledge on the monitoring and prevention for odorants.