Microsatellites are highly polymorphic markers which have been used in a wide range of genetic studies.In recent years, various sources of next-generation sequencing data have been used to develop new microsatellite l...Microsatellites are highly polymorphic markers which have been used in a wide range of genetic studies.In recent years, various sources of next-generation sequencing data have been used to develop new microsatellite loci, but compared with the more common shotgun genomic sequencing or transcriptome data, the potential utility of RAD-seq data for microsatellite ascertainment is comparatively under-used.In this study, we employed MiddRAD-seq data to develop polymorphic microsatellite loci for the endangered yew species Taxus florinii. Of 8,823,053 clean reads generated for ten individuals of a population, 94,851(~1%) contained microsatellite motifs. These corresponded to 2993 unique loci, of which 526(~18%) exhibited polymorphism. Of which, 237 were suitable for designing microsatellite primer pairs, and 128 loci were randomly selected for PCR validation and microsatellite screening. Out of the 128 primer pairs, 16 loci gave clear, reproducible patterns, and were then screened and characterized in 24 individuals from two populations. The total number of alleles per locus ranged from two to ten(mean=4.875), and within-population expected heterozygosity from zero to 0.789(mean = 0.530),indicating that these microsatellite loci will be useful for population genetics and speciation studies of T. florinii. This study represents one of few examples to mine polymorphic microsatellite loci from ddRAD data.展开更多
OCT4, a member of the POU family of gene products, is an octamer motif-binding transcription factor. As it is known to play a crucial role in cancer processes including proliferation, invasion, and chemoradioresistanc...OCT4, a member of the POU family of gene products, is an octamer motif-binding transcription factor. As it is known to play a crucial role in cancer processes including proliferation, invasion, and chemoradioresistance, it is important to identify the direct targets of OCT4 in living cancer cells. Here, chromatin immunoprecipitation-sequencing （ChlP-seq） was used to identify OCT4 binding sites in glioblastoma cancer cells. The results showed that 5438 OCT4 binding sites were localized in the glioblastoma cancer genome and that these sites contained a consensus sequence TTTkswTw （k=T or G, s=C or G, w=A or T）, which occurred 3931 times in 2312 OCT4 binding regions. Furthermore, binding motifs of some other transcription factors were identified in OCT4 binding regions. Our results provide a valuable dataset for understanding gene regulation mechanisms underlying the function of OCT4 in glioblastoma cancer.展开更多
The lifetime of G. biloba is very long, and its growth is relatively slow. However, little is known about growth-related genes in this species. We combined mRNA sequencing (RNA-Seq) with bulked segregant analysis (BSA...The lifetime of G. biloba is very long, and its growth is relatively slow. However, little is known about growth-related genes in this species. We combined mRNA sequencing (RNA-Seq) with bulked segregant analysis (BSA) to fine map significant agronomic trait genes by developing polymorphism molecular markers at the transcriptome level. In this study, transcriptome sequencing of high growth (GD) and low growth (BD) samples of G. biloba half-sib families was performed. After assembling the clean reads, 601 differential expression genes were detected and 513 of them were assigned functional annotations. Single nucleotide polymorphism (SNP) analysis identified SNPs associated with 119 genes in the GD and BD groups;58 of these genes were annotated. Two Homeobox-leucine zipper protein genes were up-regulated in the GD group compared with the BD group;therefore, these are very likely related to high growth of G. biloba. This study provides molecular level data that could be used for seed selection of high growth G. biloba half-sib families for future breeding programs.展开更多
【目的】评价低温下苦瓜种子的发芽能力,鉴定芽期低温响应基因并分析其表达模式,为探明苦瓜种子低温发芽分子调控机制提供理论指导。【方法】以6份苦瓜种质种子为材料,设置15℃的发芽温度,调查发芽率、发芽势、发芽指数、胚根长度、活...【目的】评价低温下苦瓜种子的发芽能力,鉴定芽期低温响应基因并分析其表达模式,为探明苦瓜种子低温发芽分子调控机制提供理论指导。【方法】以6份苦瓜种质种子为材料,设置15℃的发芽温度,调查发芽率、发芽势、发芽指数、胚根长度、活力指数、平均发芽时间、平均发芽速度和种子萌发指数的变化,利用隶属函数法和聚类分析进行发芽能力综合评价及分类,选取发芽能力强和弱的种质进行转录组测序,获得差异基因后进行GO功能注释和KEGG富集分析,从中挖掘低温响应相关候选基因,利用荧光定量PCR进行验证,同时分析候选基因在苦瓜不同组织(芽、根、茎、叶)和不同发芽时期(0,7,14 d)的表达差异,探讨低温对苦瓜发芽的影响及相关基因的调控作用。【结果】低温处理下,不同种质苦瓜种子发芽指标出现差异,且各指标受影响程度不同;利用综合隶属函数值和聚类分析发现,苦瓜种质MC15和CH02发芽能力较强,ZK54和ZK90发芽能力中等,BG19和YW1发芽能力较弱。对MC15和BG19进行转录组测序,共获得3062个差异基因,GO功能注释发现参与基因较多的有与胁迫、应激与防御响应,质膜和胞外区,DNA结合以及酶和蛋白活性相关的条目等;KEGG富集分析发现“植物激素信号转导”“淀粉和蔗糖代谢”“光合作用-天线蛋白”“二萜生物合成”等途径的差异基因数较多,富集因子较高;结合转录组测序、基因克隆、序列比对和荧光定量PCR分析得出,基因McLEAL和McDOG1L-3可能参与芽期低温响应,两者编码的氨基酸序列与葫芦科作物的序列相似性较高,且均在芽中特异性表达,表达趋势与转录组结果一致。在发芽0,7和14 d 3个时期中,2个基因的表达量在发芽能力较强的苦瓜种质MC15和CH02中呈下降趋势,在发芽能力较弱的苦瓜种质BG19和YW1中呈先降后升趋势。【结论】低温导致苦瓜种子发芽出现种质间及指标间差异,MC15和CH02的发芽能力较强,可用于后续种质创新和生产利用;2个低温响应相关基因McLEAL和McDOG1L-3的表达存在组织特异性,且随发芽进程出现种质间差异和时间效应,推测两者可能协同调控低温下苦瓜的延迟发芽。展开更多
目的·探究白介素-1β(interleukin-1β,IL-1β)在预测口腔颌面间隙感染(oral and maxillofacial space infection,OMSI)严重程度的作用,并探索影响IL-1β释放的关键机制、关键细胞亚群及OMSI患者免疫细胞间通信网络。方法·选...目的·探究白介素-1β(interleukin-1β,IL-1β)在预测口腔颌面间隙感染(oral and maxillofacial space infection,OMSI)严重程度的作用,并探索影响IL-1β释放的关键机制、关键细胞亚群及OMSI患者免疫细胞间通信网络。方法·选取2023年1月至2023年11月就诊于上海交通大学医学院附属第九人民医院口腔外科的OMSI患者共62例,包括中度感染20例、重度感染21例和极重度感染21例,采用Logistic回归分析影响极重度感染的危险因素,绘制受试者操作特征曲线(receiver operating characteristic curve,ROC)评估上述指标预测极重度感染的能力。分别选取2例中度、重度和极重度患者的外周血单个核细胞(peripheral blood mononuclear cell,PBMC),结合2名健康对照(GSE224198)进行单细胞RNA测序(single-cell RNA sequencing,scRNA-seq)分析,明确促炎关键细胞亚群及关键基因随着感染程度加重的变化趋势,通过CellChat分析细胞间通信。通过实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,qPCR)及Westernblotting验证PBMC中炎性小体激活水平。结果·与中度和重度感染患者相比,极重度感染患者降钙素原(procalcitonin,PCT)(P<0.05)和IL-1β(P<0.05)水平显著升高。Logistic回归显示IL-1β是极重度感染的独立危险因素(OR=1.814,95%CI 1.256~2.621,P=0.002)。联合IL-1β和PCT预测极重度感染的ROC曲线下面积(area under the curve,AUC)为0.943。scRNA-seq显示,感染加重过程中单核细胞NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor family pyrin domain-containing 3,NLRP3)基因、IL1B基因表达持续上调,中间型单核细胞是表达IL1B基因的主要细胞亚群。IL-1Β-IL-1R信号通信、趋化因子配体(C-C motif chemokine ligand,CCL)和细胞间黏附分子(intercellular adhesion molecule,ICAM)信号通信在单核细胞中显著增强;T细胞与单核细胞间巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF)信号通信亦显著增强。随着感染的加重,外周血NLRP3和IL1B的mRNA水平逐渐升高,NLRP3、caspase-1 p20、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、IL-1β蛋白表达水平持续升高。结论·联合入院IL-1β和PCT水平可有效预测OMSI极重度感染。感染患者PBMC中NLRP3炎性小体激活。IL-1β的升高与中间型单核细胞密切相关,同时单核细胞介导的IL-1Β-IL-1R、CCL、ICAM信号通信及T细胞介导的MIF信号通信共同促进炎症反应。展开更多
基金funded by the National Natural Science Foundations of China (31370252, 41571059)the National Key Basic Research Program of China (2014CB954100)supported by the China Scholarship Council for one-year study at the Aberystwyth University,UK
文摘Microsatellites are highly polymorphic markers which have been used in a wide range of genetic studies.In recent years, various sources of next-generation sequencing data have been used to develop new microsatellite loci, but compared with the more common shotgun genomic sequencing or transcriptome data, the potential utility of RAD-seq data for microsatellite ascertainment is comparatively under-used.In this study, we employed MiddRAD-seq data to develop polymorphic microsatellite loci for the endangered yew species Taxus florinii. Of 8,823,053 clean reads generated for ten individuals of a population, 94,851(~1%) contained microsatellite motifs. These corresponded to 2993 unique loci, of which 526(~18%) exhibited polymorphism. Of which, 237 were suitable for designing microsatellite primer pairs, and 128 loci were randomly selected for PCR validation and microsatellite screening. Out of the 128 primer pairs, 16 loci gave clear, reproducible patterns, and were then screened and characterized in 24 individuals from two populations. The total number of alleles per locus ranged from two to ten(mean=4.875), and within-population expected heterozygosity from zero to 0.789(mean = 0.530),indicating that these microsatellite loci will be useful for population genetics and speciation studies of T. florinii. This study represents one of few examples to mine polymorphic microsatellite loci from ddRAD data.
基金supported by the Ministry of Science and Technology, China(Nos. 2004CB518707, 2006DFA32950, 2006AA02Z4A2, 2006AA02A303, 2007DFC30360, and 2008DFA11320)the National Natural Science Foundation of China (No. 81101580)
文摘OCT4, a member of the POU family of gene products, is an octamer motif-binding transcription factor. As it is known to play a crucial role in cancer processes including proliferation, invasion, and chemoradioresistance, it is important to identify the direct targets of OCT4 in living cancer cells. Here, chromatin immunoprecipitation-sequencing （ChlP-seq） was used to identify OCT4 binding sites in glioblastoma cancer cells. The results showed that 5438 OCT4 binding sites were localized in the glioblastoma cancer genome and that these sites contained a consensus sequence TTTkswTw （k=T or G, s=C or G, w=A or T）, which occurred 3931 times in 2312 OCT4 binding regions. Furthermore, binding motifs of some other transcription factors were identified in OCT4 binding regions. Our results provide a valuable dataset for understanding gene regulation mechanisms underlying the function of OCT4 in glioblastoma cancer.
文摘The lifetime of G. biloba is very long, and its growth is relatively slow. However, little is known about growth-related genes in this species. We combined mRNA sequencing (RNA-Seq) with bulked segregant analysis (BSA) to fine map significant agronomic trait genes by developing polymorphism molecular markers at the transcriptome level. In this study, transcriptome sequencing of high growth (GD) and low growth (BD) samples of G. biloba half-sib families was performed. After assembling the clean reads, 601 differential expression genes were detected and 513 of them were assigned functional annotations. Single nucleotide polymorphism (SNP) analysis identified SNPs associated with 119 genes in the GD and BD groups;58 of these genes were annotated. Two Homeobox-leucine zipper protein genes were up-regulated in the GD group compared with the BD group;therefore, these are very likely related to high growth of G. biloba. This study provides molecular level data that could be used for seed selection of high growth G. biloba half-sib families for future breeding programs.
文摘【目的】评价低温下苦瓜种子的发芽能力,鉴定芽期低温响应基因并分析其表达模式,为探明苦瓜种子低温发芽分子调控机制提供理论指导。【方法】以6份苦瓜种质种子为材料,设置15℃的发芽温度,调查发芽率、发芽势、发芽指数、胚根长度、活力指数、平均发芽时间、平均发芽速度和种子萌发指数的变化,利用隶属函数法和聚类分析进行发芽能力综合评价及分类,选取发芽能力强和弱的种质进行转录组测序,获得差异基因后进行GO功能注释和KEGG富集分析,从中挖掘低温响应相关候选基因,利用荧光定量PCR进行验证,同时分析候选基因在苦瓜不同组织(芽、根、茎、叶)和不同发芽时期(0,7,14 d)的表达差异,探讨低温对苦瓜发芽的影响及相关基因的调控作用。【结果】低温处理下,不同种质苦瓜种子发芽指标出现差异,且各指标受影响程度不同;利用综合隶属函数值和聚类分析发现,苦瓜种质MC15和CH02发芽能力较强,ZK54和ZK90发芽能力中等,BG19和YW1发芽能力较弱。对MC15和BG19进行转录组测序,共获得3062个差异基因,GO功能注释发现参与基因较多的有与胁迫、应激与防御响应,质膜和胞外区,DNA结合以及酶和蛋白活性相关的条目等;KEGG富集分析发现“植物激素信号转导”“淀粉和蔗糖代谢”“光合作用-天线蛋白”“二萜生物合成”等途径的差异基因数较多,富集因子较高;结合转录组测序、基因克隆、序列比对和荧光定量PCR分析得出,基因McLEAL和McDOG1L-3可能参与芽期低温响应,两者编码的氨基酸序列与葫芦科作物的序列相似性较高,且均在芽中特异性表达,表达趋势与转录组结果一致。在发芽0,7和14 d 3个时期中,2个基因的表达量在发芽能力较强的苦瓜种质MC15和CH02中呈下降趋势,在发芽能力较弱的苦瓜种质BG19和YW1中呈先降后升趋势。【结论】低温导致苦瓜种子发芽出现种质间及指标间差异,MC15和CH02的发芽能力较强,可用于后续种质创新和生产利用;2个低温响应相关基因McLEAL和McDOG1L-3的表达存在组织特异性,且随发芽进程出现种质间差异和时间效应,推测两者可能协同调控低温下苦瓜的延迟发芽。
