基于Illumina MiSeq技术比较二种多脂鱼在腌干过程中的菌相变化

通过比较研究多脂红肉鱼(蓝圆鲹)和白肉鱼(带鱼)腌干加工中菌相的变化规律,以探讨加工过程对菌相的影响并寻找具有抗氧化作用的优势菌。在腌干加工过程中采用Illumina平台的MiSeq技术比较分析了两种多脂鱼的菌相变化情况。结果显示,两种鱼的菌相主要分布在拟杆菌门、变形菌门;在科水平上,初始原料的蓝圆鲹和带鱼分别含7个和15个科的细菌,带鱼包括了蓝圆鲹的所有菌群,肠杆菌科作为共同的优势菌,在蓝圆鲹和带鱼中分别占47%和26%。从腌制开始,两种鱼的菌群数都大量减少,弧菌和芽孢杆菌科作为共同优势菌,前者平均占蓝圆鲹和带鱼的40.3%和42.2%,后者则平均占16.7%和13.3%。原料中,蓝圆鲹和带鱼都包含了肠杆菌科、假单胞菌、弧菌科和希瓦氏菌科这4种腐败菌,加工阶段,两种鱼的优势腐败菌都为弧菌科。乳酸菌包括链球菌科和乳杆菌科,仅出现在带鱼中。研究表明,在腌干加工中,带鱼的细菌减少程度大于蓝圆鲹,总体上均呈现下降趋势,两种鱼含共同的菌群和优势菌,却表现出明显的差异。腌干后两种鱼的腐败菌大大减少,说明腌干加工有利于降低鱼类腐败的可能性。可选择带鱼作乳酸菌的分离以进行后续的抗氧化研究。     

花蜜化学成分及其生态功能研究进展

目的植物蜜腺基因能够调控其分泌产物花蜜中的化学组成,从而影响植物对传粉者的吸引力,本研究主要揭示腾冲红花油茶蜜腺发育与分泌过程中其基因表达的变化情况。方法采用高通量Illumina Hiseq测序手段对花蕾期蜜腺与泌蜜高峰期蜜腺进行转录组测序,并对测序数据进行相关分析研究,探讨蜜腺发育与分泌过程中的调控基因。结果在蜜腺发育与分泌过程中共检测到58 488个差异基因,其中表达上调的基因有1 602个,表达下调的基因有693个。GO分析结果表明：被注释生物学过程的差异表达基因有3 096个,其中富集差异基因数量较多的生物学过程为胞内过程、生物调节过程和新陈代谢过程;富集于分子功能的差异表达基因有1 280个,其中富集差异基因数量较多的分子功能为连接功能、催化活性和载体活性。Pathway显著性富集分析发现：有2 147个差异表达基因参与了292条代谢途径,其中富集差异基因数量最多的途径是代谢途径(941个,占43.83%),主要是参与糖代谢、氨基酸代谢等过程。结论蜜腺发育及花蜜分泌过程中大多数差异表达基因与花蜜化学组成的调控过程有关。     

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.     

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.     

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。     

小豆和绿豆茎基感病部位真菌群落结构研究

为了解小豆和绿豆茎基感病部位的真菌组成,采用Illumina高通量测序对小豆和绿豆茎基感病部位的真菌群落结构进行分析。结果表明,小豆和绿豆6个茎基感病部位真菌群落样品测序优化后共得到231个OTU （operational taxonomic units）,共同OTU为97个。2种作物茎基感病部位真菌群落中共同的优势菌门为子囊菌门（Ascomycota）,优势菌纲为粪壳菌纲（Sordariomycetes）,优势菌目为肉座菌目（Hypocreales）,优势菌科为从赤壳科（Nectriaceae）。对优势菌属分析发现,小豆和绿豆茎基感病部位镰刀菌属（Fusarium）处于绝对优势,相对丰度分别为32.94%和18.38%。Alpha多样性指数结果表明,绿豆茎基感病部位的真菌群落丰度高于小豆茎基感病部位的真菌群落丰度,但二者真菌群落多样性相似,为进一步研究小豆和绿豆茎基感病部位真菌群落的致病机理提供了理论依据。