Identification of novel juvenile-hormone signaling activators via high-throughput screening with a chemical library

Juvenile hormone (JH) is an insect-specific hormone that regulates molting and metamorphosis. Hence, JH signaling inhibitors (JHSIs) and activators (JHSAs) can be used as effective insect growth regulators (IGRs) for pest management. In our previous study, we established a high-throughput screening (HTS) system for exploration of novel JHSIs and JHSAs using a Bombyx mori cell line (BmN_JF&AR cells) and succeeded in identifying novel JHSIs from a chemical library. Here, we searched for novel JHSAs using this system. The four-step HTS yielded 10 compounds as candidate JHSAs; some of these compounds showed novel basic structures, whereas the others were composed of a 4-phenoxyphenoxymethyl skeleton, the basic structure of several existing JH analogs (pyriproxyfen and fenoxycarb). Topical application of seven compounds to B. mori larvae significantly prolonged the larval period, suggesting that the identified JHSAs may be promising IGRs targeting the JH signaling pathway.     

马尾松叶表微生物多样性

为发掘对林木生长发育有利的优良微生物资源,并筛选适合叶表微生物的收集方法,以马尾松针叶为试验材料,分别用悬摇法和超声波法收集马尾松叶表微生物,用扩增子高通量测序技术、MUSCLE和Qiime软件研究马尾松叶表微生物的多样性。结果表明:扫描电镜观测结果显示,马尾松针叶表面定殖有大量微生物,包括真菌(菌丝及孢子)和细菌。扩增子高通量测序结果表明,马尾松叶表微生物物种丰富,包含细菌运算分类单位(OTUs)490个,真菌OTUs 1273个。马尾松叶表细菌以未分类的蓝细菌属(unidentified_Cyanobacteria)(36.53%)、未分类的拜叶林克氏菌属(unidentified_Beijerinckia)(28.60%)、鞘氨醇单胞菌属(Sphingomonas)(2.35%)为优势属;叶表真菌以枝孢属(Cladosporium)(2.45%)、拟盘多毛孢属(Pestalotiopsis)(0.92%)、无头孢菌属(Capnobotryella)(0.91%)为优势属。针对叶表细菌多样性的研究表明,悬摇法和超声波法均有较高的物种检出度;在叶表真菌多样性的研究中超声波法优于悬摇法,但超声波法样品间数据变异性较大,测定结果不稳定。     

大蒜根腐病根际土壤真菌群落结构及多样性分析

研究旨在解析根腐病发生时大蒜根际土壤真菌群落结构,明确大蒜根腐病与根际土壤真菌群落多样性变化的关系,探明根腐病发生的微生态机制,为病害的防控提供理论基础。以新疆连续3年的大蒜根腐病发病田的根际土壤为研究对象,采用高通量测序方法对大蒜根际土壤总DNA的真菌ITS区序列进行大规模测序分析。结果表明,与健康对照相比,在门的水平上,子囊菌门和接合菌门真菌是大蒜根腐病根际土壤的优势真菌类群,其中高水平的子囊菌门菌群与病害发生有着密切的关系。在属的水平上,注释获得137个属,其中19个属与大蒜根腐病有较大的相关性。镰刀菌属真菌数量在病土中高于健康对照。随着连作年限的延续,根腐病的发生愈发严重,土壤中真菌群落的Shannon指数、Simpson指数、真菌属的数量逐年降低,大蒜根腐病的发生与植株根际真菌群落组成及多样性密切相关,土壤真菌类群的平衡和多样性改变是根腐病发生的一大诱因。     

山羊卵泡发育相关基因的筛选及分析

【背景】山羊第一卵泡波中的优势卵泡（dominant follicles, DF）和从属卵泡（subordinate follicles, SF）是整个卵泡发育过程中最为关键的两个阶段。随着卵泡的进一步发育,最终DF可能发育成为成熟卵泡,直到排卵;SF将走向闭锁,其中颗粒细胞的凋亡是导致卵泡发生闭锁的关键因素。然而目前对促进卵泡的优势化或导致其闭锁的分子机理尚不清楚。【目的】通过对山羊第一卵泡波中DF和SF颗粒细胞进行高通量测序,旨在筛选影响卵泡发育的关键基因,为深入探究卵泡发育的调控机制提供理论依据。【方法】选取10只1岁龄健康的贵州白山羊分别注射前列腺素F_2α,使其同期发情,此后每天用B超检测并记录卵泡的生长情况,发情3 d后,统一屠宰并采集第一卵泡波中DF (直径4.5—6 mm)与SF (直径3 —4.5 mm),分别分离其中的颗粒细胞,提取总RNA、构建文库后通过Illumina Hiseq 2500平台进行测序。利用FastQC对测序产出raw reads进行质量评估并经过过滤后,获得品质较高的clean reads;使用Trinity对得到的clean reads进行重新组装,从而获得unigenes;使用CLC Genomics Workbench将unigenes与山羊RefSeq数据库进行比对获得mRNA;使用DESeq2 软件对获得的mRNA进行差异表达分析;分别采用goseq和kobas软件对得到的差异表达基因进行GO分析及KEGG信号通路分析;最终通过qRT-PCR对筛选出的可能影响卵泡发育的关键基因进行验证。【结果】分别对测序得到的raw reads进行过滤后,在DF颗粒细胞中获得43 217 934条clean reads,占raw reads的比例为95.19%;SF颗粒细胞中获得40 766 348条clean reads,占raw reads的比例为95.35%。将得到的unigenes与山羊的RefSeq 数据库进行比对后,共得到33 896条带有注释的转录本,再通过设定FPKM>1, q value<0.05,共在两种卵泡颗粒细胞中获得13 644个基因。设定参数：FPKM≥1,SF-FPKM/DF-FPKM>1,P<0.05,获得695个差异表达mRNA,其中233个在SF颗粒细胞中表达显著上调,462个表达显著下调;对所获得695个差异表达mRNA进行GO功能富集分析,共分为三大类42组：其中生物学过程占47.6%,细胞组分占47.6%,分子功能占4.8%;KEGG信号通路分析,发现20条通路,其中与核糖体通路相关的基因富集最为显著。通过在Genecard中进行功能分析后,筛选6个可能与山羊卵泡发育密切相关的基因,其中PRLR、PTX3、RGN在SF颗粒细胞中表现为上调;DKK3、ALDH1A2、RARRES1则表现为下调。qRT-PCR显示PRLR、RGN、DKK3、ALDH1A2、RARRES1的表达趋势与高通量测序结果一致,且RGN在从属卵泡颗粒细胞中的表达量极显著地高于优势卵泡（P<0.01）;DKK3、ALDH1A2、RARRES1在优势卵泡颗粒细胞中的表达量极显著地高于从属卵泡（P<0.01）。【结论】DKK3、ALDH1A2、RARRES1和RGN在优势卵泡和从属卵泡中表达量存在极显著差异,推测在山羊卵泡发育过程中可能促进卵泡的优势化或导致闭锁,对深入探究卵泡发育的调控机制具有重要意义。     

苹果花脸病田间病情发展及ASSVd在组培条件下的传播

为了明确苹果锈果类病毒（apple scar skin viroid，ASSVd）引起的苹果花脸病在田间的病情发展情况以及ASSVd在组培条件下的传播方式，2012—2015年对河北省顺平南神南村3个果园进行持续调查和检测。结果显示，果园A、B、C的显症率分别从3.00%、19.35%和10.00%上升至10.00%、34.41%和22.00%，带毒率分别从11.00%、20.43%和14.00%上升至23.00%、37.63%和30.00%，带毒率和显症率都逐年增加，而且带毒率大于显症率，即有些带毒果树不显症。发病果树田间分布有明显的发病中心。通过高通量测序发现显症果树ASSVd vsiRNA reads数是带毒未显症reads数的4.08倍，同时实时荧光定量PCR检测发现显症树ASSVd病毒积累量显著高于带毒未显症树。以携带ASSVd的组培苗和无毒组培苗为试材，利用RT-PCR检测明确了ASSVd可通过伤口沾染带毒汁液、组培剪污染、含毒培养基、根系接触进行传播，其中根系接触传染率较高。     

Improvement,identification, and target prediction for miRNAs in the porcine genome by using massive,public high-throughput sequencing data

Despite the broad variety of available microRNA (miRNA) research tools and methods, their application to the identification, annotation, and target prediction of miRNAs in nonmodel organisms is still limited. In this study, we collected nearly all public sRNA-seq data to improve the annotation for known miRNAs and identify novel miRNAs that have not been annotated in pigs (Sus scrofa). We newly annotated 210 mature sequences in known miRNAs and found that 43 of the known miRNA precursors were problematic due to redundant/missing annotations or incorrect sequences. We also predicted 811 novel miRNAs with high confidence, which was twice the current number of known miRNAs for pigs in miRBase. In addition, we proposed a correlation-based strategy to predict target genes for miRNAs by using a large amount of sRNA-seq and RNA-seq data. We found that the correlation-based strategy provided additional evidence of expression compared with traditional target prediction methods. The correlation-based strategy also identified the regulatory pairs that were controlled by nonbinding sites with a particular pattern, which provided abundant complementarity for studying the mechanism of miRNAs that regulate gene expression. In summary, our study improved the annotation of known miRNAs, identified a large number of novel miRNAs, and predicted target genes for all pig miRNAs by using massive public data. This large data-based strategy is also applicable for other nonmodel organisms with incomplete annotation information.     

东北黑土nirS型反硝化细菌群落和网络结构对长期施用化肥的响应

【目的】探明长期施用氮磷钾化肥对东北黑土农田土壤nirS型反硝化细菌群落和网络结构的影响,为更加合理的肥料配施提供理论依据。【方法】基于农业农村部黑龙江耕地保育与农业环境科学观测实验站平台,选取不施肥(NoF)、氮肥(N)、磷肥(P)、钾肥(K)、氮钾肥(NK)、氮磷肥(NP)、磷钾肥(PK)、氮磷钾肥(NPK)8个施肥处理,借助荧光定量PCR、Illumina MiSeq高通量测序和分子生态网络技术,分析东北黑土nirS型反硝化细菌丰度、群落及网络结构,及影响反硝化细菌群落变异的主要环境因子。【结果】1)长期施用氮肥均在显著增加nirS基因拷贝数的同时,降低了nirS型反硝化细菌的群落多样性,而磷、钾肥对其影响并不显著。2)Proteobacteria是所有处理中的优势反硝化细菌门,相对丰度为16.96%~27.34%,且氮肥的施用促进了隶属于该菌门中Bradyrhizobium的生长。3)PCoA分析结果显示,8个施肥处理nirS型反硝化细菌群落主要分成施氮肥和不施氮肥两组,说明长期氮肥的施用显著改变了东北黑土反硝化细菌的群落结构。结合Mantel test的结果可知,土壤pH是影响nirS型反硝化细菌群落改变的主要因素。4)分别构建施氮肥和不施氮肥的反硝化细菌分子生态网络,发现施氮肥和不施氮肥网络结构存在很大差异,长期施用氮肥明显简化了nirS型反硝化细菌的网络结构,同时使其网络结构稳定性降低,易受外界环境扰动。【结论】尽管施用化学氮肥有利于土壤养分的增加,但其土壤nirS型反硝化细菌群落及网络结构发生了较大改变,而磷、钾肥的施用对反硝化细菌群落无显著影响。本试验结果为进一步研究东北黑土区农田土壤反硝化微生物对不同施肥管理的响应机制提供了重要科学依据。     

辽河干流河岸带植物及微生物多样性研究

为明确辽河保护区河岸带生物多样性恢复现状以促进河岸带生态系统的健康管理,本研究通过植被现场调查和土壤微生物高通量测序分析,对辽河干流23个采样点的植物多样性和微生物多样性进行测定,结果表明:辽河干流23个采样点的植被Shannon-Weiner多样性指数平均为1.227,平均植被覆盖率达到74.19%;河岸带土壤微生物Shannon-Weiner多样性指数平均为9.55;其优势度大于1%的细菌属于11个门、32个纲、40个属;人类活动干扰、放牧情况以及土地利用类型是影响河岸带植物和微生物多样性的关键因子。目前辽河保护区河岸带生物多样性恢复状态较好,但日渐增多的人类活动对河岸带生物多样性的恢复产生不利影响,因此建议尽量减少封育区内的人类活动、放牧情况等,以加快辽河保护区河岸带植物及土壤微生物多样性的恢复进程。     

Effects of different molecular weights of chitosan on methane production and bacterial community structure in vitro

As a new feed additive, chitosan has been shown in recent years to have a certain role in reducing methane emissions from the gastrointestinal tracts of ruminants. However, the effects of chitosan with different molecular weights on rumen fermentation, methane production and bacterial community structure are not yet clear. A basal diet without chitosan served as the control(CTL), and the treatment diets were supplemented with chitosan with different molecular weights: 1 000(1K), 3 000(3K), 5 000(5K), 50 000(5W) and 200 000(20W) dry matter(DM). Six fermentation units per treatment were established. Gas chromatography was used to measure the concentrations of methane, H_2 and volatile fatty acids(VFAs). The bacterial 16S rRNA genes were sequenced with an Illumina MiSeq platform and analysed to reveal the relative abundances of bacterial community taxa. The results showed that the propionate proportion was significantly increased by the addition of chitosan with different molecular weights(P0.05), while methane production and the acetate proportion were significantly decreased(P0.05). The relative abundances of Rikenellaceae_RC9_gut_group and Prevotellaceae_UCG_003 were significantly increased in the 3K chitosan group compared with the CTL group, whereas the relative abundance of Ruminococcaceae_NK4A214_group was significantly decreased(P0.05). Correlation analyses of the relative abundances of the bacterial genera showed that Prevotella was positively related to propionate production(P0.05). In conclusion, 3K chitosan could reduce methane production by replacing fibrolytic bacteria(Firmicutes and Fibrobacteres) with amylolytic bacteria(Bacteroidetes and Proteobacteria) in the bacterial community structure.     

添加芽孢杆菌对草鱼池塘中真核微生物的影响

为了研究添加芽孢杆菌对池塘中真核微生物群落结构和理化因子的影响,采用高通量测序技术分析了实验组(添加芽孢杆菌池塘)与对照组(普通池塘)水体真核微生物群落结构,同时分析了两组池塘的水体理化指标。结果表明:8、9月实验组池塘水体中TN、NH_4~+-N、NO_3~--N含量显著低于对照组(P0.05)。水体中梅尼小环藻(Cyclotella meneghiniana)、红囊藻(Hedriocystis)、蓝隐藻(Chroomonas)、丝孢酵母属(Trichosporon)和小环藻属(Cyclotella)真核微生物丰度显著高于对照组(P0.05)。实验组池塘水体真核微生物Chao1指数和Shannon指数显著高于对照组(P0.05)。实验结果证实:通过向池塘添加芽孢杆菌,可以改变水体中真核微生物群落的结构,从而实现对池塘理化因子的调节。研究结果对于降低水产养殖尾水对水域环境的污染具有一定的意义。