基于转录组数据藏波罗花WRKY转录因子鉴定与表达分析

为了解在藏波罗花中转录因子WRKY基因家族的结构和组织特异性表达情况,进而为其特征成分萜类化合物的合成与调控提供一定的借鉴,本研究从藏波罗花的转录组数据中共鉴定出了38个WRKY转录因子,进行生物信息学分析。根据WRKY转录因子保守结构域将38个藏波罗花WRKY转录因子分为三类（Ⅰ,Ⅱ,Ⅲ）,长度为115～623。表达量分析结果表明,有16个转录因子在叶和根中差异表达,其中10个在叶中显著上调,6个在根中显著上调。选取了5条WRKY转录因子进行qRT-PCR验证,结果与转录组数据相一致,表明转录组数据可靠。这些在藏波罗花中不同组织之间的差异WRKY转录因子可能在应对极端环境和介导组织特异性次生代谢产物方面发挥作用,这也为后续探究植物在应对高海拔极端环境和次生代谢产物的合成与积累提供一定的帮助。     

豹猫转录组从头组装及组织特异性表达分析

豹猫Prionailurus bengalensis是亚洲分布最广的食肉动物之一,国家二级重点保护野生动物.本研究对豹猫6个组织(大脑、心脏、肾脏、肝脏、肺和骨骼肌)进行了转录组测序,共获得51.4 Gb的原始数据.使用Trinity进行从头组装,最终获得了369246条转录本,其平均长度为1465 bp,转录本N50...     

基于高通量转录组测序的草鱼雌雄性腺差异表达基因分析

草鱼雌性个体生长优势明显高于雄性,挖掘精巢和卵巢差异表达的功能基因对草鱼生殖调控具有重要的价值及应用前景。采用Illumina HiSep 2500转录组测序技术分别对12月龄的3尾雌性草鱼和3尾雄性草鱼的性腺组织的mRNA进行测序分析,精巢和卵巢共得到8 363个差异表达基因。与卵巢相比,精巢上调基因6 446个(77%),下调基因1 917个(23%),差异明显。将上述差异基因在Nr、GO和KEGG数据库进行比对,获得48种Go功能注释分类和313条KEGG代谢通路。分析发现,Go功能分类中"绑定"功能富集差异基因的数量最多,占总数的15.8%;KEGG代谢通路中"信号转导"通路富集差异基因的数量最多,占总数的10.2%。结合转录本数据,挑选与草鱼性腺发育相关的关键差异基因Dmrt1、amh、foxl2、cyp19a1a进行分析,研究显示,与卵巢相比,Dmrt1和amh基因在草鱼精巢中极显著高表达(p0.01);与精巢相比,cyp19a1a和foxl2基因在草鱼卵巢中显著高表达(p0.05),与转录组数据分析结论一致,说明Dmrt1和amh基因与早期草鱼精巢发育调控相关,cyp19a1a和foxl2基因与早期草鱼卵巢发育调控相关。本实验为进一步了解草鱼性别基因的调控机理提供数据依据。     

小麦根系响应高盐胁迫的时序转录组分析

小麦是重要的粮食作物,盐胁迫严重影响小麦的生长发育,小麦的耐盐机制尚不完全清楚.本研究以济麦19为材料,采用NaCl处理和时序RNA测序技术(time course RNA-seq)分析小麦根系响应高盐胁迫的分子机制.与盐胁迫0 h相比,不同处理时间下小麦根中响应盐胁迫的差异表达基因总数为5526个.通过Gene On...     

地果的转录组学分析

为探究地果(Ficus tikoua)的遗传变异特征,利用Illumina HiSeq-2500平台获取地果的基因表达谱。结果表明,共获得了197 362个转录本,总长度和平均长度分别为114 072 125和577 bp。使用BlastX和BlastN共注释了139 992个转录本(占总数的70.93%)。从3个品种叶片和茎的6对样品中,分别鉴定了12 397、12 340、10 373、94 431、71 830和44 465个差异表达基因,以及注释了126、129、125、134、138和137条代谢途径。这将有助于理解地果的遗传特征,以及不同组织中代谢途径的变化。     

转录组分析转录因子AtbHLH68调控细胞壁发育的分子机制

细胞壁是植物细胞特有的结构,在参与形态建成、水分和营养物质运输以及抵御生物和非生物胁迫中发挥重要作用。前人研究表明AtbHLH68作为bHLH蛋白的第10亚家族成员,在拟南芥茎维管组织中表达。为探究其在细胞壁发育方面的分子机制,本研究建立了雌二醇诱导pER8-AtbHLH68拟南芥表达系统,实时荧光定量PCR(RT-qPCR)结果显示,10μmol/L雌二醇处理4 h能够有效诱导AtbHLH68的表达,并且随着诱导时间的增加AtbHLH68的m RNA水平进一步积累,处理8 h后达到对照组29倍。利用转录组测序分析得到了在拟南芥茎中AtbHLH68诱导表达后产生的差异基因。与对照组相比,10μmol/L雌二醇处理8 h后,共得到差异基因2 334个,其中831个基因显著上调,1 503个基因显著下调。显著富集的GO词条主要与细胞壁组分、防御响应、果胶修饰与降解以及激素响应有关。KEGG分析表明DEGs参与了果胶修饰或木质素生物合成代谢等过程。这些结果表明参与以上过程的差异基因可能被AtbHLH68直接或间接调控,从而导致拟南芥茎细胞壁组分相关基因表达量的变化。该研究为阐明转录因子Atb...     

金耳菌丝及子实体的转录组差异分析

为探讨金耳菌丝体时期和子实体时期基因表达变化,利用高通量测序技术进行RNA-Seq分析.差异基因分析显示,两个时期共有10 992个差异表达基因,其中在子实体时期上调、下调的基因数分别为9 988和1 004个.GO功能聚类分析显示,差异表达基因主要富集在催化活性、结合、细胞过程、代谢过程、细胞以及细胞组分.KEGG富...     

刺猎蝽属种间转录组比较分析

【目的】建立刺猎蝽属Sclomina 3种的转录组数据库,分析近缘种间转录组表达差异,探讨在转录组水平的种间趋异情况。【方法】采用Illumina HiSeq^TM4000高通量测序平台进行刺猎蝽属齿缘刺猎蝽S.erinacea、兴仁刺猎蝽S.xingrensis和广西刺猎蝽S.guangxiensis 3种转录组测序;利用Nr, Swiss-Prot, COG/KOG和KEGG数据库进行基因功能注释;对种间差异表达基因(differentially expressed genes, DEGs)进行GO功能注释和KEGG通路富集分析。【结果】齿缘刺猎蝽、兴仁刺猎蝽和广西刺猎蝽转录组测序组装获得42 215个unigenes。21 117个基因在上述4个数据库中得到注释(各数据库注释基因数为：Nr, 20 522; Swiss-Prot, 15 550; COG/KOG, 13 969; KEGG,10 850)。兴仁刺猎蝽vs齿缘刺猎蝽转录组有3 390个DEGs (803个上调,2 587个下调),兴仁刺猎蝽vs广西刺猎蝽转录组有12 543个DEGs (6 63...     

泽兰实蝇雌雄成虫转录组及差异分析

[目的]泽兰实蝇Procecidochares utilis Stone是入侵杂草紫茎泽兰Eupatorium adenophorum Spreng的重要的专食性天敌,已成为控制紫茎泽兰的重要因子.本研究旨在获得泽兰实蝇转录组,并深入研究泽兰实蝇雌雄成虫差异表达基因.[方法]利用Illumina高通量测序技术对泽兰实蝇雌雄成虫进行转录组测序和生物信息分析.[结果]总共获得29 147条unigenes,平均长度为457 bp,同时将所得序列注释到七大数据库进行比对,共获得19 384条unigenes注释结果.分析发现11 331条差异表达基因;与雄成虫相比,雌成虫有2 640条unigenes表达量上调,8 691条unigenes表达量下调.[结论]本研究获得了泽兰实蝇转录组,为今后泽兰实蝇性别相关研究提供了序列支持.     

镉胁迫后旱柳转录组变化分析

随着重金属镉(Cd)应用范围的扩大,由此引发的土壤镉污染问题日益严重。以具有植物恢复潜力的旱柳Salix matsudana作为研究对象,探究不同浓度的Cd (2.5 mg/L, 50 mg/L)胁迫后旱柳无性系1 d、7 d和30 d后基因表达与代谢通路的变化。转录组测序结果表明:共获得102 595个非冗余基因(Unigenes),相同浓度不同时间的差异基因总数为26 623个和32 154个;相同时间不同浓度的差异基因总数为8 550个、3 444个和11 428个。从中筛选得到与Cd胁迫响应密切相关的基因25个,其中金属硫蛋白、ABC转运蛋白、锌和锰转运蛋白等基因的表达不仅会随着Cd胁迫浓度变化而且同时受到胁迫时间的改变而发生改变;油菜素内酯合成通路的ROT3和黄酮类化合物合成通路的FLS、F3H均明显上调。此外Cd胁迫引起旱柳在代谢过程、细胞过程、膜、细胞器、细胞、细胞部分、催化活化和结合蛋白这8个方面发生改变,参与这些GO条目的差异表达基因数随着Cd浓度和胁迫时间的增加而增加。并对转录组信息的可靠性用RT-PCR和酶活性生理实验数据进行了验证。文中通过转录组测序分析旱柳Cd胁迫后的响应机制,从而为旱柳修复土壤Cd污染提供理论指导。     

A Simple,Highly Efficient Plating Method for Trypanosomatids1

A simple technique for plating trypanosomatids includes use of plates with lower agar concentrations than those usually prescribed for plating bacteria and a simple system to prevent dehydration due to the long incubation time needed for formation of visible colonies. Consistently high plating efficiency was thus achieved. Colonies from Herpetomonas samuelpessoai and Crithidia deanei were clearly distinguishable from each other; their individual characteristics varied with plating conditions.     

A Highly Efficient Method for Construction of Rice Artificial MicroRNA Vectors

Artificial microRNA (amiRNA) has become a powerful tool for gene silencing in plants. A new method for easy and rapid construction of rice artificial miRNA vector is described. The procedure involved modification of the pCAMBIA1300-UR vector by insertion of a ‘vector modification fragment’. This was prepared from the precursor of Os-amiR528 by eliminating the central miRNA-containing region while simultaneously creating an AfeI restriction site. The fragment was then introduced to the destination vector to produce a multipurpose ‘Highly Efficient gene Silencing Compatible vector’ (HESC vector). AfeI was used to produce linearized HESC vectors, and a blunt end PCR product that included amiRNA sequence was cloned into this site by a single ligation reaction to create the completed amiRNA vector. Tests showed that the method was highly efficient, and greatly reduced the time needed for vector construction and resulted in a DNA sequence identical to that of the current method, making it particularly suitable for use in a systems biology approach to functional genomic research.     

黄秋葵花和果荚转录组测序及类黄酮代谢差异表达分析

采用Illumina Hi seq 2500转录组高通量测序,构建黄秋葵花和果荚转录组文库,并利用测序评估、差异基因功能注释等生物信息学方法进行分析。研究结果表明：(1)分别获得黄秋葵花和果荚有效数据7.12 Gb和8.14 Gb,碱基百分比(Q30)均达到91.0%以上。(2)获得差异表达基因(DEGs)1 336个,其中上调基因319个,下调基因1 017个。(3)获得功能注释的基因有1 131个,GO将455注释基因归成41个功能小类,主要涉及代谢过程、催化活性、单一生物过程和细胞过程等过程;KOG注释了472个DEGs,涉及23个功能分类,其中与次生代谢直接相关过程O和Q类别获得111个注释结果;KEGG将372个DEGs注释到80条代谢通路中,获得F3H、F3′5′H、DFR、ANR、ANS、GT、LAR共10个关键差异基因,其中F3H、DFR在黄秋葵花中表现上调效应,F3′5′H、ANR、LAR在黄秋葵果荚中表现显著上调效应,ANS、GT则分别在花和果荚中均有上调或下调效应。(4)实时定量PCR分析显示,其中7个差异表达基因得到的相对表达量与转录组表达谱分析趋势完全一致。(5)类黄酮代谢途径分析表明,黄秋葵花通过F3H、DFR、ANS、GT途径将NAR催化为生成花青素苷;果荚则将NAR通过F3′5′H将催化为DHM,后在FLS催化下生成黄酮醇类物质等;部分NAR在F3H、 DFR催化下,生成无色飞燕草素苷元,再分别在ANS、LAR作用下,进入花青素苷元和原花青素合成途径。该研究结果丰富了黄秋葵转录组信息,为黄秋葵类黄酮物质纯化和功能利用提供参考依据。     

DEqMS: A Method for Accurate Variance Estimation in Differential Protein Expression Analysis

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Highlights

• •DEqMS is a method for statistical analysis of quantitative MS-data.
• •Variance estimates based on the actual MS-data structure.
• •Improved statistical power and accuracy in protein differential analysis.
• •DEqMS is available as a user-friendly R package in Bioconductor.

     

A Low-Temperature Additive-Involved Leaching Method for Highly Efficient Inorganic Perovskite Solar Cells

Inorganic CsPbI₃ perovskite with an optical bandgap ranging from 1.67 to 1.75 eV is a promising light-harvesting material as a top cell in tandem solar cells, but its high fabrication temperature can damage the middle layers or the bottom subcells. Here, an additive-involved leaching method to fabricate CsPbI₃ perovskite films is demonstrated, which can decrease the preparation temperature to 100 °C. The CsPbI₃ perovskite films with high crystallinity are achieved by a solution assisted reaction between DMAPbI₃ and Cs₄PbI₆ with the leaching of DMA⁺, Cs⁺, and I⁻. The as-prepared CsPbI₃ perovskite films exhibit much superior stability compared to their high-temperature counterparts. As a result, a power conversion efficiency of over 16% is obtained, and the unencapsulated device maintains over 93% of the initial efficiency after aging for 30 days in air with a relative humidity of 10%.     

A Simple and Efficient Method for DNA Purification from Samples of Highly Clotted Blood

Rapid purification of DNA from samples of highly clotted blood is a challenging problem due to the difficulty in recovering and dispersing blood clots. We developed a new method for discarding the serum-separator gel and rapidly dispersing the blood clots. A special disposable tip was inserted into the serum-separator gel so that the serum-separator gel could be discarded. The blood clot obtained was dispersed into small pieces through a copper mesh (pore size, 250 μm) in a special dispersing instrument by centrifugation. After lysis of red blood cells and white blood cells, genomic DNA was concentrated and desalted by isopropanol precipitation. The mean yield of DNA purified from a 0.3-ml blood clot was 22.70 μg in 173 samples of clotted blood cryopreserved for 1 month, and 19.02 μg in 1,372 samples of clotted blood cryopreserved for >6 months. DNA samples were successfully performed through polymerase chain reaction, real time polymerase chain reaction, and melt curve analysis. Their quality was comparable with that purified directly from EDTA-anticoagulated blood. The new method overcomes the difficulties in recovering and dispersing blood clots, allowing efficient purification of DNA from samples of highly clotted blood.     

A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells

The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs “mismatch nucleases” T7E1 or “Surveyor” that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an “all-in-one” CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation.     

钩苞大丁草高通量转录组测序及差异表达分析

该研究采用Illumina Hi-Seq2500高通量测序技术,对叶背有纤维和无纤维发育的两组钩苞大丁草(Gerbera delavayi Franch.)叶片样品的cDNA进行转录组测序,分析其叶背毡毛纤维发育机理。测序结果得到了108 694条单基因序列,进一步筛选得到了1 605条差异表达基因,838条差异表达基因在GO数据库具有功能定义,512条差异表达基因在COG分类体系中具有详细的蛋白功能释义,315条差异表达基因注释到了KEGG数据库中。其中,氨基糖和核苷酸糖代谢途径中控制纤维素合成的相关基因,苯丙烷类生物合成途径中控制木质素合成的相关基因,以及激素信号转导途径中控制生长素信号转导的相关基因表达量下调;激素信号转导途径中控制细胞分裂素、脱落酸信号转导的相关基因表达量上调。研究结果在一定程度上丰富了钩苞大丁草的基因信息,并为后续的遗传改良提供了基础数据。     

一种简单高效的克隆水稻端粒相关序列的方法

采用盒式PCR扩增水稻端粒相关序列,将扩增产物与T载体连接,转化大肠杆菌,再通过PCR对获得的转化子进行鉴定.应用此方法成功地从水稻中克隆到多个端粒相关序列.