基于转录特征的水稻WRKY转录因子功能注释

转录水平的变化是转录因子功能发挥的重要体现形式,高通量测序技术的发展和应用揭示了丰富的转录数据,对转录数据的深度分析有助于基因的注释和功能研究。本文以水稻WRKY转录因子家族为对象,在总结WRKY基因功能的基础上,对生物和非生物胁迫、发育、营养和激素处理等不同生物学过程中的转录数据进行了系统的整理和挖掘,获得了不同反应中转录变化的特定WRKY基因清单,丰富了水稻WRKY转录因子家族成员的注释信息,以期这些信息为后续的功能研究提供有价值的参考。

基于cDNA微阵列数据对乳酸菌生长及应激代谢转录组特征的研究

应用本实验室编写的perl程序包将GEO数据库中乳酸菌生长、应激代谢相关实验数据和相应的基因组数据整合,构建易于本地查询和访问的MySQL数据库,利用该数据库研究乳酸菌在生长、衰亡及应对不利环境(如胆盐、过氧化氢、碳氮源饥饿)时的转录组特征,并利用MEME软件寻找到了各种情况下部分协同表达基因前导序列的共有motif,即潜在的调控蛋白作用位点。结果表明:除了胆汁和草酸铵对嗜酸乳杆菌细胞代谢特征的影响具有较高的一致性外,面对其他压力环境乳酸菌细胞的应激模式具有较大的差异,有着各自受到特异性诱导表达或抑制的基因群,但在各种情况下翻译(J)、脂类运输和代谢(I)、细胞内运输和分泌(U)相关因的转录水平变化都比较显著,特别是蛋白质翻译相关基因变化最为明显,说明细胞主要通过调控蛋白质翻译相关基因的种类和表达量来对细胞的状态进行调整。

碱胁迫下“鲟龙1号”(Huso dauricus♀×Acipenser schrenckii♂)鳃组织结构变化及转录表达特征

为探究碱胁迫下杂交鲟“鲟龙1号”(Huso dauricus♀×Acipenser schrenckii♂)的响应机制,本研究设置了淡水组(3.13~3.20 mmol/L)与胁迫组(14.4~16.67 mmol/L),采集鳃进行了组织病理分析,并利用转录组测序技术,进一步分析转录组表达特征。结果显示:高碱胁迫下“鲟龙1号”鳃组织出现明显损伤,鳃小片末端发生肿胀、卷曲和融合,鳃上细胞出现增生和膨大。转录组测序共获得80422条Unigene,富集到GO数据库的差异表达基因为371个,其中87个基因表达上调,284个基因表达下调,富集到KEGG的代谢通路主要为矿物吸收、近曲小管碳酸氢盐重吸收和胃酸分泌等通路,其中近曲小管碳酸氢盐重吸收为主效应途径,该途径的Nhe3、Atp1a、Atp1b和Gdha基因显著表达下调。综上所述,碱度为14.4~16.67 mmol/L的条件下,“鲟龙1号”幼鱼已发生鳃组织损伤,且矿物质重吸收、钾/钠离子转运与排氨等功能受到影响。

基于外周血转录组定性特征的活动性结核识别标志构建

目的基于单个样本内基因表达值的相对高低秩序关系(REOs)构建识别活动性结核的基因对标志。方法以来自4 个数据集的75 例活动性结核及69 例潜伏感染者的外周全血样本为训练集, 分别提取上述两类样本基因表达谱中高度稳定的REOs模式;然后筛选出二者间REOs模式发生逆转的基因对集合, 并根据每个基因对在两类样本间发生逆转的程度将其降序排列;最后选取分类准确率最高的前k 个基因对作为标志, 进行独立数据集验证。结果在训练集中筛选出一个由7对基因组成的标志, 即7-GPS;该标志应用多数投票规则区分活动性结核与潜伏感染样本的准确率为88.89%, 区分活动性结核与正常样本的准确率为90.09%;在来自不同检测平台的混合验证数据中, 其区分活动性结核与潜伏感染样本的受试者工作特征曲线下面积(AUC)为0.914(95%CI:0.881~0.948), 区分活动性结核与正常样本的AUC为0.934(95%CI:0.904~0.964);另外, 该标志中的4 个基因ETV7、BATF2、ANKRD22与CARD17P倾向于在活动性结核外周血样本中高表达, 且其表达值与临床抗结核治疗时间显著相关。结论 7-GPS标志的分类效能良好, 适合对单个外周血样本进行个体化分析, 具有一定的应用潜力。

基于单细胞RNA测序分析百草枯诱导小鼠大脑帕金森样改变的转录组特征

[背景]百草枯(PQ)是世界上使用最广泛的除草剂之一,是帕金森病(PD)的危险因素之一,但其致PD发病的机制不甚清楚。单细胞RNA测序(scRNA-seq)技术可从基因层面研究细胞异质性,为探究PQ致PD发病机制提供深入见解。[目的]通过scRNA-seq分析PQ染毒小鼠大脑细胞分群情况,以及与PD样改变相关细胞亚群参与的生物学过程,为揭示PQ致小鼠大脑PD样改变的机制研究提供线索。[方法]6只雄性6周龄C57BL/6小鼠随机分为对照组和实验组,每组3只,分别以0(生理盐水)、10.0 mg·kg^(-1) PQ腹腔注射染毒,每隔2 d 1次,连续10次注射造模;染毒结束后,取小鼠大脑,进行scRNA-seq。根据不同细胞类型的基因表达特征进行细胞分群,利用生物信息学工具筛选PD相关细胞亚群,并对其特征基因进行基因注释(GO)富集分析、京都基因与基因组百科全书(KEGG)通路富集分析以及基因集富集分析(GSEA)、蛋白互作网络分析、转录因子预测等。最后,对PD相关细胞亚群在PQ处理组和对照组间的差异基因进行GO和KEGG分析等,分析这些基因可能参与的生物学过程。[结果]测序数据符合质控标准,共获取55779个细胞,所有细胞降维分析结果显示其可以被进一步分为37个聚类,包括5种主要的细胞类型;根据每个亚群排名前20特征基因KEGG分析,筛选出特异表达的Cluster 33亚群(多巴胺能神经元)与PD显著相关。GO分析结果显示该亚群生物学功能方面主要富集神经递质转运和对神经递质的调控,GSEA分析结果显示酪氨酸代谢通路和脑组织神经活动配体-受体相互作用通路显著富集,转录调控网络分析结果显示差异表达转录因子有39个。进一步分析PQ处理对该亚群的影响,结果表明PQ处理影响多巴胺神经元亚群代谢通路、内吞和Ras相关蛋白1(Rap1)信号通路和丝裂原活化蛋白激酶(MAPK)信号通路等;GO分析显示差异基因与离子运输、突触组装调节等生物学过程有关,并参与了细胞质和突触等细胞组分的形成。[结论]研究初步绘制了PQ暴露后小鼠大脑单细胞转录组图谱,并筛选出特异表达的Cluster33亚群(多巴胺能神经元)与PD显著相关,其生物学功能改变可能是PQ致小鼠大脑PD样改变的机制之一。

葡萄VvWRKY70基因生物信息学及表达特性分析

WRKY是植物特有的转录因子,在多种生物和非生物胁迫中发挥作用。基于RNA-seq技术,课题组前期研究发现,SO_(2)熏蒸结合低温保鲜过程中多个功能未知的葡萄WRKY基因差异表达。经氨基酸序列同源性比对,研究选择与AtWRKY70氨基酸序列相似度最高的VvWRKY70进行生物信息学及表达特性分析。生物信息学分析结果表明,VvWRKY70基因启动子区含有W-box元件及茉莉酸甲酯、水杨酸等激素响应元件;编码蛋白由322个氨基酸组成,分子质量约36.57 ku,属亲水性蛋白;该蛋白丝/苏氨酸含量丰富;二级结构主要由无规卷曲和α-螺旋组成,N端含有高度保守的WRKY结构域,C端为C_(2)HC型锌指结构,属WRKY第Ⅲ亚家族;预测显示,其主要定位于细胞核中,且在进化上较为保守。qRT-PCR分析表明,VvWRKY70在玫瑰香葡萄根、茎、叶、花蕾、果中均有表达;葡萄果实VvWRKY70受SO_(2)和灰霉菌诱导上调表达,低温处理组下调表达。综上可见,VvWRKY70可能作为转录因子参与调节植株生理和果实发育过程,并在葡萄果实采后SO_(2)保鲜过程中发挥作用,调节果实贮藏期间的生物和非生物胁迫应答。

基于转录组特征基因反向匹配方法发现抗流感病毒化合物BIX02189

流感病毒是RNA病毒,分甲、乙、丙、丁4型,其中甲型流感病毒和乙型流感病毒可引发人类急性呼吸道疾病,全球每年约30万患者死于流感感染。流感病毒的生命周期高度依赖宿主,靶向宿主因子已经成为抗病毒药物研究的重要策略。本研究通过转录组特征基因反向匹配(transcriptome signature reversion,TSR)方法,计算获得干预多宿主因子的抗流感病毒化合物列表,评价列表中化合物体外抗流感病毒活性,最终获得活性化合物BIX02189。结果显示,BIX02189具有广谱抗流感病毒活性,抗甲流病毒H1N1(A/Puerto Rico/8/1934)和乙流病毒(B/江西新建/BV/39/2008)的半数有效浓度(half maximal effective concentration,EC_(50))分别为17.1和9.4μmol·L^(-1),其中抗甲流活性优于利巴韦林(97.9μmol·L^(-1))。转录组间无监督学习相似性成簇分析显示,BIX02189的抗流感病毒主要机制可能通过干预Raf/MEK/ERK通路,实现对流感病毒颗粒的生成和释放的阻断。

基于单细胞转录组测序解析小鼠肝泡型棘球蚴病肝脏细胞转录谱特征

目的从单细胞水平探究多房棘球蚴感染小鼠晚期阶段肝脏组织微环境细胞组成及其转录谱特征。方法收集2只多房棘球蚴感染BALB/c小鼠(6~8周龄)肝脏病灶旁组织和配对远端肝组织进行单细胞转录组测序。利用R软件Seurat包对获得的数据进行质量控制、多样本整合和批次效应校正,应用统一流形逼近与投影(uniform manifold ap⁃proximation and projection,UMAP)算法进行细胞聚类,根据经典标记基因注释细胞类型。通过差异基因表达分析筛选各细胞类型的差异表达基因,进行基因本体论(Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)功能富集分析,预测细胞生物学作用。结果对来自多房棘球蚴感染小鼠肝脏病灶旁和远端肝组织的43710个细胞进行了分析,归类为11种细胞类型:中性粒细胞、T细胞、巨噬细胞、粒细胞⁃单核细胞祖细胞、B细胞、浆细胞、嗜碱性粒细胞、肝星状细胞、内皮细胞、肝细胞、血小板。T细胞是组织微环境中占比最高的免疫细胞,包括5种CD4+T细胞、2种CD8+T细胞和磷酸抗原反应γδT细胞。与病灶远端肝组织相比,病灶旁肝组织中的CD4+辅助性T细胞和CD4+细胞毒性T细胞比例降低、辅助性T细胞2(Th2细胞)比例明显增高。Th2细胞差异表达基因主要与免疫系统负调控过程相关,CD4+细胞毒性T细胞高表达基因与免疫系统激活相关。结论通过单细胞转录组测序揭示了多房棘球蚴感染小鼠肝组织微环境中的细胞组成和分布差异,病灶旁肝组织中Th2细胞升高可能与免疫抑制性微环境形成有关。

Structural Characteristics and Molecular Mechanism of Hepatitis B Virus Reverse Transcriptase

Hepatitis B virus (HBV), a typical member of the Hepadnaviridae family, is responsible for infections that cause B-type hepatitis which leads to severe public health problems around the world. The small enveloped DNA-containing virus replicates via reverse transcription, and this unique process is accomplished by the virally encoded reverse transcriptase (RT). This multi-functional protein plays a vital role in the viral life cycle. Here, we provide a summary of current knowledge regarding the structural characteristics and molecular mechanisms of HBV RT. Improved understanding of these processes is of both theoretical and practical significance for fundamental studies of HBV and drug discovery.

Molecular identification based on ITS sequences for Kappaphycus and Eucheuma cultivated in China

The systematic classification of the Eucheumatoideae is difficult because of their variable morphology and interpretation of reproductive structures. Kappaphycus and Eucheuma specimens cultivated on the Hainan and Fujian coast of China were introduced from Vietnam, the Philippines and Indonesia. Combined with morphological characteristics, all Kappaphycus and Eucheuma cultivated strains were identified by internal transcribed spacer (ITS) sequences. The phylogenetic tree was constructed using neighbor-joining and maximum likelihood methods. The results indicate that different ITS sequence lengths occurred in the different genera and species. An obvious difference in morphology could be found in the protuberance shape between Kappaphycus and Eucheuma. The protuberance in Eucheuma was thorn-like and in Kappaphycus was wartlike or papillate. Their ITS sequence lengths differed significantly in nucleotide variation rates up to 58.55%-63.90%. All nucleotide variations occurred in the ITS1 and ITS2 regions except for five nucleotide transversions in the 5.8S rDNA region. In addition, the difference was at the branches among congeneric species. Kappaphycus sp. had branches with small buds, while K. alvarezii did not have such a feature. The nucleotide variation rates varied from 7.02% to 7.48% among species; within the same species of the clades it was <1.20%. Eucheumatoideae algae cultivated in China consisted of three clades, K. alvarezii, Kappaphycus sp., and E. denticulatum. The results indicate that ITS sequence analysis was an effective way for identification of interspecies and intraspecies phylogenetic relationships and might provide a clue for molecular identification of algal Eucheumatoideae.

Quantitative Real-Time Reverse Transcription-PCR Assay for the Expression of Tob mRNA in Human Colorectal Cancer

OBJECTIVE Tob is a member of Tob/BTG antiproliferative family. To date, Tob expression in human carcinoma using clinical specimens has not been studied in depth except for lung carcinoma and thyroid carcinoma. This study is the first to investigate the expression levels of Tob gene in human colorectal cancer tissues, and their corresponding para-cancerous tissues. The correlation of expression of the Tob gene with clinicopathological characteristics of colorectal cancer was also analyzed. METHODS Quantitative real time RT-PCR was used to detect the expression of Tob mRNA in 31 colorectal cancers. RESULTS Compared with normal tissues, up-regulation of Tob mRNA was observed in 31 colorectal cancer tissues （P = 0.020）. The expression level of Tob at Dukes C ＋ D phase was higher than Dukes A ＋ B phase, and the difference was significant （P 〈 0.05）. However, in this study, it was found that the expression of Tob mRNA was not related with age, gender, and pathological type of colorectal cancer. CONCLUSION The up-regulation of Tob may be closely associated with tumorigenesis of colorectal carcinoma.

mRNA Expression of Chemokine Receptors on Peripheral Blood Mononuclear Cells and Correlation with Clinical Features in Systemic Lupus Erythematosus Patients

Objective To investigate the expressions of chemokine receptors and interleukin （IL） receptors on the peripheral blood mononuclear cells （PBMCs） from systemic lupus erythematosus （SLE） patients and their correlations with clinical features as well as SLE disease activity index （SLEDAI）. Methods The mRNA expressions of chemokine receptors and IL receptors on PBMCs of 93 SLE patients and 30 healthy controls were detected by reverse transcription-polymerase chain reaction, including CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CXCR3, CXCR5, CX3CR1, XCR1, IL-4R, and IL-10R. The clinical features of SLE patients were recorded. The correlations of chemokine receptors and IL receptors mRNA expressions with clinical features as well as SLEDAI were assayed using linear regression analysis. Results The level of CCR5 mRNA in SLE patients （including active and inactive SLE） was signifi- cantly higher than that in healthy controls （P〈0.05）, and there was no significant difference between active and inactive patients in this respect （P〉0.05）. CX3CR1 mRNA expression significantly increased from healthy control to inactive SLE to active SLE in sequence. The others （except for CCR8, CXCR3, and IL-1 OR） in active SLE patients weresignificantly higher than those in both inactive SLE patients and healthy controls （all P〈0.05）. There were positive correlations between SLEDAI and CCR2 （r=0.424, t=4.313, P〈0.001）, CCR3 （r=0.518, t=5.410, P〈0.001）, CCR4 （r=0.376, t=3.851, P〈0.001）, CCR6 （r=0.457, t=4.513,P〈0.001）, CXCR5 （r=0.455, t=4.629, P〈0.001）, CX3CR1 （r=0.44-5, t=4.523, P〈0.001）, as well as XCRI （r=0.540, t=5.445, P〈0.001）. And CCR5 mRNA expression level was positively correlated with IL-4R mRNA （r=0.313, t=2.353, P〈0.05）. The patients with myositis and cutaneous vasculitis simultaneously showed lower levels of CCR5 and CX3CRI, and CCR5 expression was negatively correlated with the scores of SLEDAI in SLE cases accompanied by photosensitivity （r=0.426, t=- 2.155, P〈0.05）. Conclusion Increased expressions of CCR5 and CX3CRI on PBMCs may be indicators in clinical survey for SLE.

Transcriptome analysis of three cotton pests reveals features of gene expressions in the mesophyll feeder Apolygus lucorum

The green mirid bug Apolygus lucorum is an agricultural pest that is known to cause damage to more than 150 plant species. Here, we report the transcriptomes ofA. lucorum at three different developmental stages （the second and fifth instar nymphs and adults）. A total of 98,236 unigenes with an average length of 1,335nt was obtained, of which 50,640 were annotated, including those encoding digestive enzymes and cytochrome P450s. Comparisons with cotton bollworm and cotton aphid transcriptomes revealed distinct features ofA. lucorum as a mesophyll feeder. The gene expression dynamics varied during development from young nymphs to adults. The high-quality transcriptome data and the gene expression dynamics reported here provide valuable data for a more comprehensive understanding of the physiology and development of mirid bugs, and for mining targets for their control.

Sequence signatures of genes with accompanying antisense transcripts in Saccharomyces cerevisiae

Recent studies have found many antisense non-coding transcripts at the opposite strand of some protein-coding genes.In yeast,it was reported that such antisense transcripts play regulatory roles for their partner genes by forming a feedback loop with the protein-coding genes.Since not all coding genes have accompanying antisense transcripts,it would be interesting to know whether there are sequence signatures in a coding gene that are decisive or associated with the existence of such antisense partners.We collected all the annotated antisense transcripts in the yeast Saccharomyces cerevisiae,analyzed sequence motifs around the genes with antisense partners,and classified genes with and without accompanying antisense transcripts by using machine learning methods.Some weak but statistically significant sequence features are detected,which indicates that there are sequence signatures around the protein-coding genes that may be decisive or indicative for the existence of accompanying antisense transcripts.