龟纹瓢虫幼虫体内主要共生菌种类分析

共生菌和昆虫是互利共生的关系，它参与宿主生殖、代谢、解毒等生理过程。龟纹瓢虫为农田生态系统重要天敌昆虫，同时具有取食植物花粉的习性。为明确龟纹瓢虫体内共生菌的种类与群落结构，通过Illumina MiSeq平台对龟纹瓢虫幼虫体内共生菌的16S rDNA V3～V4区进行高通量测序，生物信息学方法分析共生菌的种类和多样性。结果显示，共获得3910个OTUs，长度为360～480 bp的序列占99.54%； OTU注释结果表明，龟纹瓢虫共生菌包括37个门，75个纲，143个目，261个科，605个属；变形菌门Proteobacteria（51.84%）、厚壁菌门Firmicutes（21.81%）、放线菌门Actinobacteria（11.35%）、蓝藻菌门Cyanobacteria（7.06%）、拟杆菌门Bacteroidetes（4.49%）为龟纹瓢虫幼虫的5个优势种群。在属水平上葡萄球菌Staphylococcus（16%）、不动杆菌属Acinetobacter（8.54%）、Paracocccus（4.93%）、假单胞菌Pseudomonas（3.59%）、Boechera gunnisoniana（3.34%）、棒状杆菌属Corynebacterium（2.18%）、阪崎肠杆菌Cronobacter（2.04%）、考克氏菌属Kocuria（1.50%）、链球菌属Streptococcus（1.15%）和芽胞杆菌Bacillus（1.11%）为优势菌群。本研究初步明确了龟纹瓢虫幼虫体内共生菌种群组成与结构，为通过共生菌提高龟纹瓢虫人工繁殖效率提供参考，同时为转基因植物对龟纹瓢虫的安全评价提供新的思路。     

Chronic effects of different fertilization regimes on nirS-type denitrifier communities across the black soil region of Northeast China

Denitrification is one of the major processes causing nitrogen loss from arable soils.This study aimed to investigate the responses of nir S-type denitrifier communities to different chronic fertilization regimes across the black soil region of Northeast China.Soil samples were collected from sites located in the north(NB),middle(MB),and south(SB)of the black soil region of Northeast China,each with four chronic fertilization regimes:no fertilizer(No F),chemical fertilizer(CF),manure(M),and chemical fertilizer plus manure(CFM).Methods of quantitative polymerase chain reaction(q PCR)and Illumina Mi Seq sequencing were applied to assess the abundance and composition of denitrifier communities by targeting the nir S gene.The results showed that the M and CFM regimes significantly increased the abundances of nir S-type denitrifiers compared with No F at the three locations.The majority of nir S sequences were grouped as unclassified denitrifiers,and the different fertilizers induced little variation in the relative abundance of known nir S-type denitrifier taxa.Over 90%of the sequences were shared among the four fertilization regimes at each location,but none of the abundant operational taxonomic units(OTUs)were shared among the three locations.Principal coordinate analysis(PCo A)revealed that the communities of nir S-type denitrifier were separated into three groups that corresponded with their locations.Although similar fertilization regimes did not induce consistent changes in the nir S-type denitrifier communities,soil p H and NO-3-N content simultaneously and significantly influenced the structure of nir S-type denitrifier communities at the three locations.Our results highlight that geographical separation rather than chronic fertilization was the dominant factor determining the nir S-type denitrifier community structures,and similar chronic fertilization regimes did not induce consistent shifts of nir S-type denitrifier communities in the black soils.     

不同耕作方式下黑土微生物群落对干湿交替的响应

以吉林德惠市黑土长期田间定位实验地土壤为研究对象,通过室内模拟培养,采用高通量测序方法(16S rRNA)研究免耕和垄作土壤微生物群落对不同频率和强度的干湿交替处理的响应。结果表明:干湿交替显著降低免耕土壤中微生物群落的多样性,且频率越高干旱强度越大多样性降低越显著;但干湿交替对垄作土壤的微生物多样性影响不显著。与对照相比,干湿交替显著增加免耕土壤中浮霉菌门(Planctomycetes)和疣微菌门(Verrucomicrobia)的相对丰度,显著降低免耕和垄作土壤中Saccharibacteria菌门和Parcubacteria菌门的相对丰度。无论是免耕还是垄作条件下,干湿交替频率的不同导致土壤微生物群落结构产生显著差异,而干湿交替强度的不同对土壤微生物群落结构没有显著影响。研究结果为预测干旱气候对黑土生态功能的影响提供了理论基础。     

基于Illumina高通量测序的泡桐转录组研究

利用新一代高通量测序技术平台Illumina Solexa对泡桐进行转录组测序和数据de novo组装,并对得到的unigene进行功能注释、分类及代谢通路分析.结果表明,N50值为2 278 bp、平均长度为1 410 bp的泡桐unigene共有188 019条,将其与NCBI的Nr和Swiss-Prot数据库比对后发现,分别有120 808条和85 880条unigene与其他物种的基因具有同源性.利用COG数据库可将有关的41 914条泡桐unigene分成25类,KEGG数据库分析发现共有43 553个unigene参与215种代谢通路.找到与木质素合成相关的泡桐unigene.     

Changes in bacterial community and abundance of functional genes in paddy soil with cry1Ab transgenic rice

A field experiment involving cry1Ab transgenic rice(GM) and its parental non-cry1Ab rice(M) has been on-going since 2014. The diversity of the bacterial communities and the abundance of the microbial functional genes which drive the conversion of nitrogen in paddy soil were analyzed during the growth period of rice in the fifth year of the experiment, using 16 S rRNAbased Illumina Mi Seq and real-time PCR on the amoA, nirS and nirK genes. The results showed no differences in the alpha diversity indexes of the bacterial communities, including Chao1, Shannon and Simpson, between the fields cultivated with line GM and cultivar M at any of the growth stages of rice. However, the bacterial communities in the paddy soil with line GM were separated from those of paddy soil with cultivar M at each of the growth stages of rice, based on the unweighted Uni Frac NMDS or PCoA. In addition, the analyses of ADONIS and ANOSIM, based on the unweighted Uni Frac distance, indicated that the above separations between line GM and cultivar M were statistically significant(P0.05) during the growth season of rice. The increases in the relative abundances of Acidobacteria or Bacteroidetes, in the paddy soils with line GM or cultivar M, respectively, led to the differences in the bacterial communities between them. At the same time, functional gene prediction based on Illumina Mi Seq data suggested that the abundance of many functional genes increased in the paddy soil with line GM at the maturity stage of rice, such as genes related to the metabolism of starch, amino acids and nitrogen. Otherwise, the copies of bacterial amo A gene, archaeal amo A gene and denitrifying bacterial nir K gene significantly increased(P0.05 or 0.01) in the paddy soil with line GM. In summary, the release of cry1Ab transgenic rice had effects on either the composition of bacterial communities or the abundance of microbial functional genes in the paddy soil.     

烤后健康烟叶和霉烂烟叶真菌群落结构分析

为了解烤后健康烟叶和霉烂烟叶真菌群落多样性,采用Illumina MiSeq测序平台对烤后健康与霉烂烟叶样品上真菌群落的ITS1-ITS2区域PCR扩增片段进行测序并分析。健康烟叶组(MJ)和霉烂烟叶组(MB)2类样品真菌主要为子囊菌门(Ascomycota)、担子菌门(Basidiomycota)、接合菌门(Zygomycota)等6个门25纲58目187科260个属的394个真菌分类单元(OTUs)。在属水平上,健康烟叶组(MJ)优势真菌为链格孢属(Alternaria),其次为曲霉属(Aspergillus)、新凸轮孢菌属(Neocamarosporium)、赤霉属(Gibberella)、支顶孢属(Acremonium);霉烂烟叶组(MB)优势真菌为曲霉属(Aspergillus),其次为青霉属(Penicillium)和根霉属(Rhizopus)。Alpha指数表明,健康烟叶组(MJ)真菌群落丰富度与多样性均高于霉烂烟叶组(MB)。71.06%的OTUs归属于8个功能类群,其中腐生菌是主要功能类群,研究结果揭示了烤后健康烟叶和霉烂烟叶真菌群落结构,为制定相应的防治方案提供参考依据。     

藏东南林芝地区典型农业土地利用方式对土壤微生物群落特征的影响

为研究藏东南林芝地区土壤微生物群落对农业土地利用方式的响应特征及其关键影响因素,可深入认识农业土地利用对该区域生态系统稳定性的影响,并为该地区农业土地资源合理利用提供科学依据。采集藏东南林芝地区2种典型农业土地利用方式(农田及放牧草地)土壤样品,以自然森林土壤样品为对照,利用磷脂脂肪酸法(PLFA)和MiSeq高通量测序分析不同土地利用方式下土壤微生物数量、多样性和群落组成的差异性,并结合土壤理化指标探讨影响土壤微生物群落组成和结构的关键因素。结果表明:与自然森林相比,农田和放牧草地土壤中微生物PLFA总量降低了38.7%~51.8%,其中革兰氏阳性细菌、革兰氏阴性细菌、放线菌和真菌的生物量分别降低了26.1%~47.6%、40.0%~61.1%、44.1%~60.6%和5.2%~31.3%。农田种植显著降低土壤真菌的多样性,其丰富度、香农多样性相比于森林土壤分别降低了53.0%和71.4%,而细菌群落的丰富度、香农多样性则显著降低了15.7%和5.1%。农田种植显著增加土壤细菌中放线菌门(A ctinobacteria)的相对丰度,但是显著降低了浮霉菌门(Planctomycetes)、绿弯菌门(Chloroflexi)和厚壁菌门(Firmicutes)的相对丰度,放牧草地则显著促进拟杆菌门(Bacteroidetes)和蓝藻门(Cyanobacteria)的生长。农业土地利用显著提高土壤真菌群落中担子菌门伞菌纲(A garicomycetes)的优势度,其在农田和放牧草地土壤中的相对丰度分别是自然森林土壤的23倍和19倍,而子囊菌门(A scomycota)和接合菌门(Zygomycota)的相对丰度则分别降低了67.7%~89.6%和58.7%~67.4%。基于距离矩阵的冗余分析表明,土壤细菌和真菌的群落结构在3种土地利用方式下有显著差异。土壤微生物的数量、多样性和群落组成受到土壤pH值、土壤有机碳、土壤C/N等理化性质的显著影响,并且土壤真菌对环境因子改变的响应比细菌敏感。研究表明,典型农业土地利用方式导致藏东南林芝地区土壤微生物群落的数量和多样性相比于自然植被显著降低,并改变细菌和真菌的群落组成和结构,而且真菌对农业土地利用方式的响应比细菌敏感。     

Bacterial diversity in soils subjected to long-term chemical fertilization can be more stably maintained with the addition of livestock manure than wheat straw

Addition of organic matter such as livestock manures and plant residues is a feasible practice to mitigate soil degradation caused by long-term application of chemical fertilizers, and the mitigation is largely mediated though activities of the soil-dwelling microorganisms. However, the roles of different kinds of organic matter in maintaining bacterial community structure have not been assessed in a comparative manner. In this study, 454 pyrosequencing of 16S rRNA gene was employed to compare the bacterial community structure among soils that had been subjected to 30 years of NPK fertilization under six treatment regimes: non-fertilization control, fertilization only, and fertilization combined with the use of pig manure, cow manure or low- and high-level of wheat straws. Consistent with expectation, long-term application of NPK chemical fertilizers caused a significant decrease of bacterial diversity in terms of species richness (i.e. number of unique operational taxonomic units (OTU)), Faith's index of phylogenetic diversity and Chao 1 index. Incorporation of wheat straw into soil produced little effects on bacterial community, whereas addition of either pig manure or cow manure restored bacterial diversity to levels that are comparable to that of the non-fertilization control. Moreover, bacterial abundance determined by quantitative PCR was positively correlated with the nutritional status of the soil (e.g., nitrate, total nitrogen, total carbon, available phosphorus); however, bacterial diversity was predominantly determined by soil pH. Together, our data implicate the role of livestock manures in preventing the loss of bacterial diversity during long-term chemical fertilization, and highlight pH as the major deterministic factor for soil bacterial community structure.     

零换水条件下养殖水体中碳氮比对生物絮团形成及团头鲂肠道菌群结构的影响

本试验旨在运用454焦磷酸测序技术同时结合PCR-变性梯度凝胶电泳(DGGE)技术分析零换水条件下养殖水体中碳氮比对生物絮团形成及团头鲂(Megalobrama amblycephala)肠道菌群结构的影响。试验共设置5组,其养殖水体中碳氮比分别为8(CN8组,设为对照组)、12(CN12组)、16(CN16组)、20(CN20组)、24(CN24组),每组设置3个水泥池(重复),每个水泥池投放团头鲂幼鱼20尾。养殖期为8周,每隔1周采集水样1次,检测养殖水体中氨氮、亚硝酸盐氮及生物絮团含量。养殖8周后,每个水泥池分别随机挑选3尾鱼无菌采集盲肠内容物,运用454焦磷酸测序技术分析不同碳氮比零换水养殖条件下团头鲂肠道菌群结构的变化。结果表明:当碳氮比≥16时,形成的生物絮团可以有效调节水质,降低水体中的氨氮和亚硝酸盐氮含量。随着碳氮比由8升高到16和20,肠道中放线菌门(Actinobateria)含量变化不显著(P0.05),厚壁菌门(Firmicutes)含量显著增加(P0.05),梭杆菌门(Fusobateria)和变形菌门(Proteobacteria)含量显著减少(P0.05)。对厚壁菌门进行深入分析发现,在纲水平上,与对照组相比,CN16、CN20和CN24组团头鲂肠道中梭菌纲(Clostridia)含量显著减少(P0.05),而芽孢杆菌纲(Baccilli)含量显著增加(P0.05)。DGGE指纹图谱分析表明,养殖水体中碳氮比能够影响团头鲂的肠道菌群的多样性。CN8组和其他组的相似性系数最低,仅为0.43,而CN20和CN24组的相似性系数最高,达到0.96。由此可见,零换水条件下养殖水体的碳氮比影响了生物絮团的形成量,改变了团头鲂肠道菌群结构和多样性。以团头鲂肠道菌群结构为评价指标,零换水条件下团头鲂养殖水体中适宜碳氮比为16~20。     

454焦磷酸测序技术对不同猪种肠道菌群差异的分析（英文）

454焦磷酸测序用于分析比较巴马香猪(BAMA)、黑香猪(BLACK)与外三元猪(CROSS)的肠道菌群差异。结果表明:三个猪种的肠道细菌多样性与丰度各有不同,差异较大。威克氏菌属、丛毛单胞菌属与光岗菌属等病原菌在BAMA肠道内检测到,而在BLACK与CROSS肠道内未检测到。BAMA肠道中丝状杆菌属与瘤胃球菌属等纤维素分解菌的多样性与丰度远大于在BLACK与CROSS肠道中的分布量。BAMA肠道内高含量的潜在病原菌与纤维分解菌表明BAMA对恶劣环境的适应性与抗病力较强,以及耐粗饲性强,容易饲养管理。