基于Special-Base模型检测转基因作物nos/plamid构建特异性

以转基因作物中常见的nos/plamid构建特异性序列为研究对象,通过同源性比较和焦磷酸测序dispensation order设计,建立Special-Base焦磷酸测序检测模型;利用转基因大豆Roundup Ready Soybean和转基因玉米Bt11、GA21、NK603等4种转基因作物为试验材料,针对nos/plamid构建特异性序列设计引物,经复合PCR扩增及焦磷酸测序,验证Special-Base焦磷酸测序检测模型有效性。结果表明:本研究建立的Special-Base焦磷酸测序模型能检测出样品中单一或多种转基因作物成分,提高了转基因作物检测的精确性、检测通量和自动化程度。     

稻作条件下不同施肥模式对盐碱化土壤细菌和古菌群落结构的影响

为探讨稻作条件下不同施肥模式对盐碱化土壤细菌和古菌群落结构的影响,实验选取脱硫废弃物(T1)、脱硫废弃物和有机肥混合不同梯度(T2、T3和T4)4个施肥处理盐碱土壤,不施肥土壤(CK)作为对照,利用Illumina Hiseq高通量测序技术解析不同处理土壤特性和细菌、古菌群落结构关系的特征,并对细菌和古菌群落结构与环境因子进行相关性分析。试验结果表明:与CK相比,4种处理均降低了古菌多样性,T2、T3和T4处理增加了细菌的多样性;变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)是所有土样中的优势菌群,占细菌总量的25.7%~31.2%和22.9%~28.1%。施用脱硫废弃物和有机肥料的处理Proteobacteria相对丰度提高了14.0%~36.4%,T1处理降低了9.2%。T1、T2和T3处理的Euryarchaeota相对丰度提高了41.6%~115.2%。不同施肥处理土壤细菌和古菌群落结构存在显著差异。施用脱硫废弃物和有机肥改变了土壤化学性质,其中T4提高了有机碳(6.74%)、碱解氮(37.20%)、速效磷(47.83%)和铵态氮(96.26%)。RDA分析结果显示,硝态氮对土壤细菌和古菌群落结构的影响最大,其次分别是全磷、pH、速效钾、电导率。研究解析了稻作条件下不同施肥模式对盐碱化土壤细菌和古菌群落结构的特征,确定了影响土壤细菌和古菌区系的主效环境因子,脱硫废弃物与有机肥最佳配比模式为T4处理组,该处理不仅提高了土壤养分含量,同时也增加了土壤细菌的多样性,有利于维持良好的土壤生态环境。     

复合垂直流人工湿地基质理化性质对nirS和nirK型反硝化菌多样性的影响

采用Illumina高通测序技术,探讨喀斯特地区复合垂直流人工湿地基质理化性质对nirS和nirK型反硝化菌多样性的影响。结果表明,复合垂直流人工湿地中4个池子（即一级垂直流下行池、一级垂直流上行池、二级垂直流下行池、二级垂直流上行池）的基质理化性质存在显著差异。其中,pH在中4个池子中均呈中性,DO呈先下降后升高趋势,下行池的基质电导率EC均比上行池要高;TN、TP含量随着人工湿地梯级呈显著下降趋势,未出现厌氧现象;NO3--N和NH4+-N含量在二级垂直流下行池中大量积累并达到峰值。在AlpHa和Beta多样性分析中,nirS、nirK型两类反硝化细菌丰度随着人工湿地梯级呈先降低后升高的趋势,多样性随人工湿地级数逐渐降低,且下行池丰度多样性显著高于上行池。基质中nirS型反硝化细菌鉴定为5门9属,nirK型为6门14属,变形菌门是nirS、nirK型反硝化菌群的共同优势菌门,相对丰度为55.36%~65.72%、30.06%~45.66%。其中,优势菌属为红杆菌属（Rhodobacter）、脱氯单胞菌属（Dechloromonas）、红假单胞菌属（Rhodopseudomonas）、慢生根瘤菌属（Bradyrhizobium）和氏菌属（Bosea）。红杆菌属和脱氯单胞菌属相对丰度在空间上随着人工湿地级数升高总体呈上升趋势,红假单胞菌属、慢生根瘤菌属和氏菌属在空间上随人工湿地级数升高总体呈下降趋势,此2类优势菌属在空间上存在显著差异（P<0.01）。韦恩图表明nirS型反硝化菌群丰度明显高于nirK型,且各级池中反硝化菌群相似度较高。冗余分析表明基质中nirS和nirK反硝化菌群的构建主要受pH、NH4+-N、DO、TP、TN影响,且nirS型对pH,以及nirK型对NH4+-N的响应更为强烈。综上,本文研究了人工湿地nirS、nirK型反硝化菌多样性特征及其与环境因子的关系,为复合垂直流人工湿地高效脱氮提供理论依据。     

广西环江喀斯特常绿落叶阔叶林土壤微生物类群的空间格局

运用地理统计学和Illumina 16S rRNA基因测序等手段,研究广西环江木论国家自然保护区25 hm~2喀斯特常绿落叶阔叶林土壤主要门级微生物类群的空间分布。结果表明:广西环江木论国家自然保护区喀斯特森林土壤中优势菌门为变形菌门(Protebacteria,34.5%)、放线菌门(Actinobacteria,30.7%)和酸杆菌门(Acidobacteria,12.2%);土壤细菌类群显示空间相关性的自相关范围为44.4～841.4m,其中大部分细菌类群的变程在研究区域范围(500 m)内;在研究区内,变形菌门的相对丰度从北到南逐渐升高,而放线菌门和厚壁菌门(Firmicutes)的相对丰度则从北到南逐渐降低,绿弯菌门(Chloroflexi)、酸杆菌门、奇古菌门(Thaumarchaeota)、拟杆菌门(Bacteroidetes)、芽单胞菌门(Gemmatimonadetes)、疣微菌门(Verrucomicrobia)的相对丰度呈斑片状分布,硝化螺旋菌门(Nitrospirae)的相对丰度呈单峰型分布,WS3(Latescibacteria)的相对丰度则呈间歇性的低值条带和高值条带状分布。可见,喀斯特土壤主要微生物类群相对丰度呈现不同的空间分布格局。     

大豆胞囊线虫侵染诱导五寨黑豆早期的转录组分析

利用Illumina Hi SeqTM2000对大豆胞囊线虫侵染前后的抗病大豆品种五寨黑豆的转录组进行检测。将测序的两个文库进行拼接,得到长度大于200bp的reads共20 980 831条,总长度约为4.23Gb,筛选到1 045个差异表达基因。Gene ontology功能显著性富集发现差异表达基因在生物过程注释基因最多,共有1 007个差异表达基因。在COG数据库中对基因产物进行直系同源分类,结果表明众多基因参与到碳水化合物及氨基酸的运输和代谢、次生代谢物质合成及信号传导机制过程,并受胞囊线虫侵染的诱导表达。KEGG代谢通路分析显示差异表达基因在苯丙烷类及氧化磷酸化代谢通路显著富集,说明这两个代谢通路在五寨黑豆抗胞囊线虫病中起重要作用。     

Large‐scale identification of microsatellites for a major invasive weed,Ageratina adenophora,using the Illumina sequencing technology

Microsatellites were identified and characterised for a worldwide invasive weed, Ageratina adenophora, based on the Illumina sequencing technology. Using the assembled genomic sequences obtained from the Illumina GAII sequencing platform, a total of 3012 microsatellites were identified, of which mono‐, di‐, tri‐, tetra‐, penta‐, and hexa‐nucleotide repeats were 181 (6.0%), 1379 (45.8%), 1064 (35.3%), 220 (7.3%), 73 (2.4%) and 95 (3.2%) respectively. The AC/GT and GAT/ATC motifs were the most frequent motif types of di‐nucleotide and tri‐nucleotide repeats respectively. To validate the newly identified simple sequence repeat markers, 15 primer pairs were randomly selected to test in 24 individuals of A. adenophora, and all of these primer sets could amplify PCR products of expected size. Of them, eight loci displayed polymorphism with the number of alleles per locus ranging from two to four and values for observed and expected heterozygosities ranging from 0.042 to 1 and from 0.082 to 0.551, with the averages of 0.578 and 0.371 respectively. These novel microsatellites should be useful for investigation of the genetic structure and genetic diversity between the invasive and native populations of this species.     

Effect of dietary supplementation of probiotic on performance and intestinal microflora of Chinese soft‐shelled turtle (Trionyx sinensis)

This experiment was conducted to study the effect of dietary supplementation of probiotic on performance and intestinal microflora of Chinese soft‐shelled turtle. Healthy Chinese soft‐shelled turtles (6000 pieces with average weight of 5 g) were selected and randomly divided into two groups: control and treatment with three replicates each. The control was fed on normal basal diet, whereas treatment was fed on basal diet supplemented with 1 × 10⁸ cfu.g^?1 Bacillus subtilis preparation. The results showed that the final mean weight of treatment (265.84 ± 3.48 g) was significantly higher (P < 0.05) than that of control (240.45 ± 4.32 g). There was significant difference (P < 0.05) in FCR, and DGs were obtained in probiotics fed group as compared to control. The level of sucrase, maltase, amylase, lipase and ATPase of treatment was significantly higher (P < 0.05) than that of control. Pyrosequencing showed that the dietary supplementation of probiotic Bacillus strain decreased microbial diversity. Phylum Firmicutes was dominant in both groups. Compared with the control, Firmicutes was increased, while others were decreased in treatment group. The results indicated that dietary supplementation of probiotic can improve growth performance, digestibility and intestinal microflora in Chinese soft‐shelled turtle.     

Impact of polyculture ecosystems of Ctenopharyngodon idellus with Eriocheir sinensis on bacterial communities in the sediments of Dongping Lake in China: Focus on optimization of polyculture modes

This study investigated the influence of polyculture ecosystems of grass carp (Ctenopharyngodon idellus) and Chinese mitten crab (Eriocheir sinensis) on the bacterial community of sediments in the Lake Dongping in China, using the Illumina HiSeq Sequencing approach. The bacterial community diversity was reduced to different extent due to the polyculture of animals. The bacterial community composition changed significantly among the ecosystems of control (without any animals cultured), G1C1 (with the highest animal biomass: C. idellus, 2 ind/m²; E. sinensis, 1 ind/m²) and G2C2 (with the lowest biomass: C. idellus, 1 ind/m²; E. sinensis, 0.5 ind/m²). Relative abundance increase for sulphide‐oxidizing bacteria and decrease for sulphate‐reducing bacteria were detected with more quantities of fish–crab cultured in ecosystems. Variations in the concentration of sediment sulphate mainly accounted for the shifts of both the bacterial diversity and community composition. Lastly, G2C2 was recommended as the optimal polyculture ecosystem that could be used in the Lake. The results suggested negative impacts of polyculture ecosystems of C. idellus with E. sinensis on the bacterial diversity but positive impacts on the community composition and indicated the role of total biomass of animas in shaping the bacterial community in the sediments of lake ecosystems.     

利用Illumina MiSeq测序平台分析肉鸡盲肠微生物多样性

为对肉鸡盲肠微生物的多样性进行探索,选取15只饲养在相同条件下的科宝-500肉鸡,利用Illumina MiSeq测序平台与Qiime等16srRNA序列分析工具在22日龄时采集盲肠内容物进行研究。结果显示:1)测序共获得359 158条有效序列与3 210个OTU,并且由稀释曲线可以证明此次测序结果比较全面的覆盖了肉鸡盲肠微生物群落。2)各样品菌群之间存在差异,得到序列的数量以及菌群多样性差别较大。3)通过核心菌群分析可知,4个共享菌群在门水平上分别为:拟杆菌门Bacteroidetes(70.0%±12.1%)、厚壁菌门Firmicutes(25.4%±11.2%)、变形菌门Proteobacteria(4.3%±3.7%)和蓝菌门Cyanobacteria(0.3%±0.5%)。4)有益菌群如乳酸杆菌属Lactobacillus、双歧杆菌属Bifidobacterium虽然存在于盲肠微生物中,但相对丰度不高。5)PICRUSt分析盲肠菌群对应的基因功能发现:功能转运、嘌呤代谢、DNA修复重组蛋白、氧化磷酸化和甲烷代谢等基因功能丰度最高。由此可见,个体差异虽然对肉鸡盲肠微生物结构影响较大,但是仍然存在不少共性。