Maternal Deprivation Enhances Behavioral Vulnerability to Stress Associated with miR-504 Expression in Nucleus Accumbens of Rats

Objective

In this study, the effect of maternal deprivation (MD) and chronic unpredictable stress (CUS) in inducing depressive behaviors and associated molecular mechanism were investigated in rats.

Methods

Maternal deprivation was established by separating pups from their mothers for 6 hours daily from postnatal day 1 to day 14. Chronic unpredictable stress was established by water deprivation, elevated open platform, food deprivation, restraint stress and electric foot shock. The depressive behaviors were determined by use of sucrose preference test and forced swim test.

Results

Rats in MD/CUS group exhibited lower sucrose preference rate, longer immobility time, and lighter body weights than rats in other groups (MD/control, non-MD/CUS and non-MD/control group). Meanwhile, higher miR-504 expression and lower dopamine receptor D1 (DRD1) and D2 (DRD2) expression were observed in the nucleus accumbens of rats in the MD/CUS group than in the other three groups. MiR-504 expression correlated negatively with DRD1 gene expression and sucrose preference rate in the sucrose preference test, but correlated positively with immobility time in forced swim test. Both DRD2 mRNA and protein expression correlated negatively with immobility time in forced swim test.

Conclusion

These results suggest that MD enhances behavioral vulnerability to stress during adulthood, which is associated with the upregulation of miR-504 and downregulation of DRD2 expression in the nucleus accumbens.     

Applying Reversible Mutations of Nodulation and Nitrogen-Fixation Genes to Study Social Cheating in Rhizobium etli-Legume Interaction

Mutualisms are common in nature, though these symbioses can be quite permeable to cheaters in situations where one individual parasitizes the other by discontinuing cooperation yet still exploits the benefits of the partnership. In the Rhizobium-legume system, there are two separate contexts, namely nodulation and nitrogen fixation processes, by which resident Rhizobium individuals can benefit by cheating. Here, we constructed reversible and irreversible mutations in key nodulation and nitrogen-fixation pathways of Rhizobium etli and compared their interaction with plant hosts Phaseolus vulgaris to that of wild type. We show that R. etli reversible mutants deficient in nodulation factor production are capable of intra-specific cheating, wherein mutants exploit other Rhizobium individuals capable of producing these factors. Similarly, we show that R. etli mutants are also capable of cheating inter-specifically, colonizing the host legume yet contributing nothing to the partnership in terms of nitrogen fixation. Our findings indicate that cheating is possible in both of these frameworks, seemingly without damaging the stability of the mutualism itself. These results may potentially help explain observations suggesting that legume plants are commonly infected by multiple bacterial lineages during the nodulation process.     

The Mitochondrial Genome of Paramphistomum cervi (Digenea), the First Representative for the Family Paramphistomidae

We determined the complete mitochondrial DNA (mtDNA) sequence of a fluke, Paramphistomum cervi (Digenea: Paramphistomidae). This genome (14,014 bp) is slightly larger than that of Clonorchis sinensis (13,875 bp), but smaller than those of other digenean species. The mt genome of P. cervi contains 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions (NCRs), a complement consistent with those of other digeneans. The arrangement of protein-coding and ribosomal RNA genes in the P. cervi mitochondrial genome is identical to that of other digeneans except for a group of Schistosoma species that exhibit a derived arrangement. The positions of some transfer RNA genes differ. Bayesian phylogenetic analyses, based on concatenated nucleotide sequences and amino-acid sequences of the 12 protein-coding genes, placed P. cervi within the Order Plagiorchiida, but relationships depicted within that order were not quite as expected from previous studies. The complete mtDNA sequence of P. cervi provides important genetic markers for diagnostics, ecological and evolutionary studies of digeneans.     

白蚁-共生微生物系统降解木质纤维素研究进展

开发利用木质纤维素材料能显著增加地球上可再生资源的储备量。白蚁分布广泛,常见于热带和亚热带地区,它们借助细菌、古细菌、真菌等肠道微生物和原生动物协同降解食物中的木质纤维素,在生态系统的碳、氮循环中发挥着十分重要的作用。本文概括了近年来白蚁肠道微生物研究的进展,特别是近年来已被证明的肠道微生物在木质纤维素降解方面的作用,以期为后续研究木质纤维素的降解提供参考信息。     

侵染中华蜜蜂6日龄幼虫的蜜蜂球囊菌的微小RNA差异表达谱及调控网络

【目的】蜜蜂球囊菌(Ascosphaeraapis,简称球囊菌)是专性侵染蜜蜂幼虫的致死性真菌病原。MicroRNA(miRNA)作为一类重要的基因表达调控因子,能够广泛参与真菌及其宿主的相互作用过程。本研究通过比较分析球囊菌孢子(AaCK)和侵染中华蜜蜂(Apis cerana cerana,简称中蜂) 6日龄幼虫肠道内的球囊菌(AaT)的smallRNA(sRNA)组学数据对球囊菌的差异表达miRNA(differentiallyexpressed miRNA,DEmiRNA)、靶mRNA及二者间的调控网络进行全面解析,旨在揭示miRNA介导的球囊菌对中蜂幼虫的侵染机制。【方法】对于球囊菌侵染的中蜂6日龄幼虫肠道的small RNA-seq (sRNA-seq)数据,利用BLAST工具连续比对东方蜜蜂(Apiscerana)和球囊菌的参考基因组筛滤得到AaT的sRNA组学数据。分别将AaCK和AaT的sRNA组学数据比对miRBase数据库,对球囊菌侵染宿主前后miRNA的数量和结构特征进行分析。联用RNAhybrid+svm_light、Miranda和TargetScan软件预测AaCK vs AaT比较组中DEmiRNA的靶mRNA,进而利用相关生物信息学软件对上述靶mRNA进行GO分类和KEGG代谢通路富集分析。通过Cytoscape软件对DEmiRNA-mRNA调控网络进行可视化。利用Stem-loop RT-PCR、RT-qPCR和分子克隆验证测序结果的可靠性。【结果】在AaCK和AaT中分别鉴定到380和387个miRNA。结构特征分析结果显示,AaCK和AaT的mi RNA皆集中分布在18–25 nt,且首位碱基主要偏向于U。AaCKvsAaT比较组共有270个DEmiRNA,包含155个上调miRNA和115个下调miRNA,分别靶向结合6091和6145个mRNA。GO分类结果显示,上述靶mRNA主要涉及代谢进程、细胞进程、应激反应等15个生物学进程;细胞、细胞组分、细胞器等12个细胞组分;催化活性、结合、转运子活性等11个分子功能。KEGG代谢通路富集分析结果显示,上述靶mRNA富集在123条代谢通路,参与对氨基酸代谢、碳水化合物代谢以及核苷酸代谢等物质代谢,氧化磷酸化、硫代谢、氮代谢等能量代谢,以及MAPK和Hippo等信号通路的调控。球囊菌DEmiRNA与靶mRNA之间存在复杂的调控关系,其中miR-29-x、miR-250-x、miR-4968-y、miR-11200-x、novel-m0023-5p、novel-m0130-5p和novel-m0135-5p等DEmiRNA可靶向结合与球囊菌的半胱氨酸蛋白酶、DNA甲基化转移酶以及几丁质酶相关的mRNA;此外,miR-7-x、miR-9-z、miR-319-y和miR-5951-y等同时参与调控MAPK信号通路;进一步分析发现,miR-250-x同时参与对DNA甲基化转移酶、MAPK信号通路及其他酶类合成与代谢途径的调控,并可能参与球囊菌与中蜂6日龄幼虫之间的跨界调控。通过Stem-loopRT-PCR和RT-qPCR验证了4个DEmiRNA的差异表达,并利用分子克隆和Sanger测序证实miR-7-x的序列与测序结果一致。【结论】本研究解析了侵染中蜂6日龄幼虫的球囊菌的miRNA差异表达谱及DEmiRNA的调控网络,揭示了球囊菌DEmiRNA可能通过调控病原的物质和能量代谢、增殖、毒力、信号通路及相关mRNA参与对中蜂幼虫的侵染过程。miR-7-x、miR-250-x、novel-m0023-5p等关键DEmiRNA有望作为白垩病治疗的新型分子靶点。     

铜绿假单胞菌cntRLMN操纵子介导锌离子摄取的功能鉴定

【目的】本研究以铜绿假单胞菌PAO1 (Pseudomonas aeruginosa PAO1,菌种编号ATCC15692)为对象,研究cntRLMN在锌离子摄取中的功能。【方法】在ΔznuBC的基础上,以同源重组的方法构建了cntRLMN的各种突变菌株,通过质粒接合转移的方法构建其互补菌株及lacZ转录融合报告菌株,运用β-半乳糖苷酶酶活检测研究了Zur蛋白对cntRLMN的转录调控,凝胶阻滞实验(EMSA)检验Zur蛋白与cnt启动子及cnt启动子的突变片段的体外结合,并进一步通过生长曲线分析对cntRLMN中cntR、cntL、cntN等基因的锌离子摄取功能进行了分析和鉴定。最终,通过构建大蜡螟幼虫的侵染模型来研究cntRLMN对铜绿假单胞菌毒力发挥的影响。【结果】lacZ转录融合的酶活分析显示cntRLMN受Zur蛋白的负调控,其表达以Zur蛋白依赖的方式受锌离子饥饿的诱导;EMSA实验的结果显示cntRLMN的启动子可以与His-Zur结合形成DNA-蛋白质复合体,结合位点为GCGTTATAGTATATCAT;生长曲线和大蜡螟幼虫侵染实验的分析结果显示ZnuBC和CntRLMN的功能存在互补性,仅znuBC和cntRLMN双缺失突变时菌株在限锌培养条件下的生长和对大蜡螟幼虫的毒性才受到显著抑制,说明CntRLMN代表另一种独立的锌离子摄取系统。【结论】cntRLMN是受Zur直接负调控的另一种独立的铜绿假单胞菌锌离子摄取系统,对铜绿假单胞菌毒力的发挥起重要作用。     

烟草青枯病劳尔氏菌与拮抗菌对根系分泌物的竞争作用

[目的]研究青枯病病原菌与拮抗菌的营养特性及其对烟草根系分泌物的响应差异,对提高拮抗菌定殖能力、有效生物防控烟草青枯病具有非常重要的意义。[方法]本研究通过筛选与鉴定贵州烟区青枯病病原菌株及拮抗菌株,通过Biolog表型芯片技术分别检测病原菌与拮抗菌的特征性碳、氮源,利用气质联用(GC-MS)检测烟草主栽品种K326根系分泌物的主要物质,在此基础上进行病原菌与拮抗菌对其利用能力、利用强度以及共培养的研究。[结果]经鉴定,分离、筛选到的病原菌株和拮抗菌株分别为青枯劳尔氏菌(Ralstonia solawacearum)和枯草芽孢杆菌(Bacillus subtilis);在含量为0.01μg/mL以上的根系分泌物中,12种物质的含量从高到低排序为:果胶>葡萄糖>木糖>阿拉伯糖>半乳糖>核糖>蔗糖>苯甲酸>果糖=D-甘露醇>棕榈酸>富马酸,果胶含量最高且明显高于其他物质;拮抗菌(LX4)对碳源利用能力高于病原菌(Rs)的碳源有阿拉伯糖、木糖和核糖,分别是病原菌利用能力的1.22、1.95和2.17倍;前12 h拮抗菌利用果糖强度高于病原菌,不同碳源共培养24 h后LX4对gfp-Rs(绿色荧光蛋白标记后的青枯病病原菌)抑制率为18.34%(阿拉伯糖)、53.23%(木糖)、63.53%(核糖)和52.09%(果糖)。[结论]拮抗菌对烟草根系分泌物的利用不及病原菌,但在特定碳源条件下拮抗菌能够利用根系分泌物中的某些碳源产生某种拮抗物质抑制病原菌,拮抗菌与病原菌之间同时存在利用性竞争和干扰性竞争关系,研究结果为进一步研究烟草青枯病的生物防控提供了新的理论依据。     

相容溶质四氢嘧啶与羟基四氢嘧啶的代谢调控研究进展

四氢嘧啶(Ectoine)及其衍生物羟基四氢嘧啶(5-hydroxyectoine,5-HE)是嗜盐微生物胞内合成的一类能够抵抗外界高盐胁迫的相容溶质,具有细胞、细胞膜、蛋白质和核酸的保护作用,可抵抗高盐、高温、冷冻和干燥等极端环境因素的刺激,从而倍受关注。本文对不同类型微生物Ectoine/5-HE(Ects)生物合成代谢、分解代谢以及吸收/转运系统涉及的调控机制进行综述,以期为Ects合成产量的提升与高效积聚策略的优化,提供一定的理论参考依据。     

一株寒地高效解无机磷细菌的分离鉴定及拮抗作用

【目的】从北方寒地种植的不同农作物根际土壤中分离高效解磷的细菌,为微生物制剂和磷肥的开发提供适于本地区的优良菌种。【方法】通过初筛和复筛从26株解磷菌中筛选获得一株高效解磷细菌,对其进行生理生化和分子生物学鉴定,同时采用钼蓝比色法测定解磷能力。采用平板对峙法测定拮抗植物病原菌能力。【结果】通过筛选后获得的菌株B51-7经鉴定为伯克霍尔德菌属。菌株在发酵液中可溶性磷含量最高达到832.74 mg/L,同时具有很强的广谱抑菌作用,抑菌率最高为89.71%,可以显著促进水稻生长。【结论】菌株B51-7是一株具有生物防治作用的高效解磷细菌,可应用于生物菌肥和生防制剂中。     

顺反式肉桂醛对肉源隆德假单胞菌生物被膜和致腐性的抑制作用

【目的】为比较反式和顺式肉桂醛对肉源假单胞菌生物被膜和致腐性的影响。【方法】通过平板计数测定两种肉桂醛对隆德假单胞菌的最小抑菌浓度(MIC),结晶紫法、珠涡流法、激光共聚焦显微镜观察、福林法等检测亚抑菌浓度肉桂醛处理下隆德假单胞菌生物被膜形成、运动性和胞外酶活性变化。荧光定量RT-PCR检测肉桂醛对隆德假单胞菌粘附lapA、鞭毛fliC、蛋白酶aprX和脂肪酶lip基因表达量的影响。【结果】反式和顺式肉桂醛对隆德假单胞菌的MIC分别为200μg/mL和225μg/mL,1/8 MIC、1/4MIC、1/2MIC亚抑菌浓度肉桂醛显著降低隆德假单胞菌生物被膜结晶紫和粘附性,其中1/2MIC反式和顺式肉桂醛处理下被膜分别减少60.27%和52.05%,菌体粘附降低56.35%和61.10%。亚抑菌浓度肉桂醛显著减少被膜厚度,反式肉桂醛还能显著杀灭被膜菌。且肉桂醛能显著抑制菌体的泳动性,反式肉桂醛对生物被膜和泳动性的抑制效果更强。肉桂醛还能抑制隆德假单胞菌蛋白酶和脂肪酶活性,其中1/2MIC反式和顺式肉桂醛处理下菌体蛋白酶分别减少61.90%和76.19%,脂肪酶降低40.17%和47.01%。且发现肉桂醛显著降低lapA、fliC、aprX和lip表达量,其中1/2MIC反式和顺式肉桂醛分别降低4个基因表达量至对照组的0.05–0.16和0.02–0.12倍。【结论】两种亚抑菌浓度肉桂醛异构体显著抑制隆德假单胞菌生物被膜和致腐性,其中反式肉桂醛对生物被膜抑制较强,而顺式肉桂醛更有效地降低致腐酶活性,其与肉桂醛下调相应基因表达密切相关。