基于全长转录组测序的掌叶大黄R1-MYB基因家族鉴定分析

作为MYB (v-myb avian myeloblastosis viral oncogene homolog)转录因子家族的一大类, R1-MYB (MYBrelated)家族在植物生长发育、环境胁迫及激素信号转导等方面发挥重要的调节作用。本研究基于全长转录组测序分析,系统筛选掌叶大黄R1-MYB家族基因,分析其编码蛋白的理化、结构域及分子进化等特性,利用RNA-seq进行基因组织表达分析,并检测RpMYB24响应激素、非生物胁迫的表达模式。结果表明,共鉴定到掌叶大黄49个R1-MYB家族基因,主要编码热稳定的亲水蛋白,大部分定位于细胞核。各蛋白无规卷曲和α螺旋所占比例较大,存在MYB家族标志性的W型保守氨基酸。掌叶大黄R1-MYB家族成员分布于CCA1 (circadian clock associated 1)-like、I-box (GATAAG)-like、CPC (CAPRICE)-like、TRF (telomere repeat binding factor)-like和TBP (TATA binding protein)-like 5个亚组,且CCA1-like数量...     

三七PnMYB1R1基因的克隆、亚细胞定位与不同胁迫下的表达分析

MYB转录因子是植物中最大的转录因子家族之一,在植物信号转导、生长发育和植物防御反应中起到重要作用。本研究以三七(Panax notoginseng)为材料,设计特异引物,克隆了三七中1R-MYB转录因子PnMYB1R1基因,并进行氨基酸序列分析、原核表达和纯化、亚细胞定位、转录活性分析、组织特异性分析及非生物胁迫下的诱导表达分析。PnMYB1R1基因开放阅读框(ORF)长738 bp,编码245个氨基酸,蛋白分子质量27.0 kD。序列分析及系统进化树结果表明PnMYB1R1包含1个保守的R3结构域,属于1R-MYB型转录因子中的TRF-like类蛋白。构建原核表达载体pET28a-PnMYB1R1并在大肠杆菌BL21 (DE3)菌株中成功表达PnMYB1R1重组蛋白,纯化得到可溶性重组蛋白。亚细胞定位实验结果表明PnMYB1R1定位于植物细胞的细胞核中。转录活性分析显示PnMYB1R1转录因子具有转录激活活性。实时荧光定量PCR结果表明PnMYB1R1基因在三七主根中表达量最高,茎、叶次之,须根中表达量最低。盐、低温及干旱胁迫均能够影响主根、须根、茎、叶中PnMYB1R1基因的表达...     

基于转录组的不同产地党参糖基转移酶鉴定及分析

潞党参为山西道地药材,其药效成分党参多糖等含量显著高于其他产地党参[Codonopsis pilosula(Franch.) Nannf.]。糖基转移酶是党参主要药效成分党参多糖和党参苷Ⅰ等合成的关键酶,本研究基于不同产地党参转录组数据,对糖基转移酶基因进行GO及KEGG功能注释、保守结构域分析、进化树分析及表达模式分析,为探索潞党参道地性形成机制提供理论依据。本研究共筛选鉴定出98个糖基转移酶基因,分属于GT family 1、GT family 2、GT family 90等家族。GO功能注释发现多属于分子功能中的催化活性条目。KEGG功能注释发现党参糖基转移酶主要参与聚糖生物以及萜类和聚酮类化合物代谢。其中, GT family 1的42个糖基转移酶基因保守结构域为:[X]-W-[2X]-Q-[3X]-[LH]-[5X]-[FLTHCGWNS]-[2X]-E-[4X]-[GVP]-[4X]-P-[4X]-Q-[2X]-[NAK]。通过与拟南芥的糖基转移酶序列比对分析,党参糖基转移酶主要位于拟南芥UGT73、72、85分支中。基因表达模式分析发现党参CpUGT73AH2在潞党参植株...     

LAL途径特异性转录因子研究进展

链霉菌可产生许多具有重要应用价值的次级代谢产物,如抗生素、免疫调节剂、抗肿瘤药物等,它们已经广泛应用于临床医药中。次级代谢产物的生物合成受到转录因子的调控,转录因子通过控制结构基因的转录激活或阻遏解除,决定了表达方式和程度。Lux R家族大ATP结合调控因子(large ATP-binding regulators of the Lux R family,LAL)是一类新的次级代谢途径转录因子,由900~1000个氨基酸残基组成,它的N-端具有Walker A和Walker B模体的ATP/GTP结构域,C-端具有保守的Lux R家族螺旋-转角-螺旋模体的DNA结合域。已在40余种次级代谢产物生物合成基因簇中发现LAL,本文对LAL途径特异性转录调控的研究进展进行综述。     

转录因子PITX2的研究进展

PITX2是属于Bicoid家族的转录因子,并且与另外两个高度同源的蛋白PITX1和PITX3构成了PTX亚家族。本文对PITX2基因和蛋白质的结构,表达与功能,与Wnt／β-catenin信号途径的关系以及突变所致遗传性疾病及其分子机制等进行了综述。     

响应ABA胁迫的甘草bZIP转录因子的系统筛选与分析

甘草为我国最常用的大宗药材之一,主要分布在干旱、半干旱区域,有着重要的经济和生态价值。bZIP转录因子在植物生物或非生物胁迫响应、生长发育、次生代谢产物合成等方面具有重要的调控作用。本研究利用Illumina高通量测序技术对不同浓度脱落酸(ABA)处理的甘草转录组进行测序,并基于已发表的甘草全基因组数据,以拟南芥基因组中发现的bZIP序列为参照,鉴定了甘草中的bZIP转录因子。之后筛选ABA依赖bZIP转录因子基因作为候选基因,对其编码蛋白理化性质、结构以及外源ABA胁迫下基因的表达模式进行分析。共鉴定到69个甘草bZIP转录因子家族基因,命名为GubZIP1-69,并根据它们与拟南芥bZIP的同源相似性分为A～I以及S这10个亚家族。通过计算69个GubZIPs基因在不同外源ABA胁迫下的相对表达量,筛选出可能参与ABA信号通路的调控基因,即GubZIP1、GubZIP5、GubZIP8、GubZIP30、GubZIP33和GubZIP56。外源ABA胁迫下候选GubZIPs基因的表达模式分析结果显示,与0 mg·L^-1浓度ABA处理下相比, 25 mg·L     

E2F转录因子在细胞周期调控中作用的研究进展

目前研究表明多种细胞因子参与细胞周期调控,其中Ｅ2Ｆ转录因子家族已被证明是重要的调控环节之一[1]。它们通过调节控制细胞ＤＮＡ合成及与细胞增殖有关的基因表达而起作用,同时其本身的活性又受另一些在细胞周期进程中有重要作用的蛋白所调控。这样调节Ｅ2Ｆ家族的蛋白,Ｅ2Ｆ转录因子及它们的靶基因就形成了一条对细胞增殖有调节作用的途径。现对Ｅ2Ｆ转录因子在细胞周期调控中的作用及Ｅ2Ｆ家族与细胞凋亡及肿瘤的关系进行综述。1　Ｅ2Ｆ转录因子家族的组成　　Ｅ2Ｆ转录因子最初作为腺病毒Ｅ2启动子的活化剂被发现[2],目前已发现5个不同…     

甘蓝AP2/ERF转录因子的克隆和生物信息学分析

摘 要 目的：运用电子克隆的方法获得甘蓝中AP2/ERF转录因子基因。 方法: 以拟南芥的AP2/ERF转录因子作为探针,对甘蓝的EST数据库进行搜索,应用相关软件进行序列拼接组装和延长。结果: 克隆出甘蓝两个AP2/ERF B3亚族转录因子BoAP2/ERF1和BoAP2/ERF2。通过生物信息学方法,对两条序列编码蛋白质的氨基酸组成,亲水性/疏水性、理化性质、亚细胞定位、高级结构和空间构型等方面进行了预测和分析。结论:BoAP2/ERF1和BoAP2/ERF2基因由371和352个氨基酸组成、相对分子质量为40.85kDa和39.44kDa的蛋白质,定位于细胞核。序列分析结果显示,该蛋白可能具有信号转导和胁迫响应应答等功能。     

转录因子FoxO1在神经元凋亡中的作用

Forkhead转录因子（Fox蛋白家族）是2000年才正式统一命名的一个新的转录因子家族．自20世纪90年代首次在果蝇中发现该基因以来,其家族已先后发现100多个Fox基因。该家族的共同特征是具有一个长110个氨基酸的保守的DNA结合结构域,称为Fox（Forkheadbox）结构域,含有一个螺旋-转角－螺旋基序,     

抑制 BHK21细胞 STYK1/NOK基因的转录组分析 #br#

目的 转录组分析抑制丝氨酸-苏氨酸-酪氨酸激酶 1（STYK1/NOK）后仓鼠肾正常细胞 BHK21的基因表 达,并分析其可能作用的信号通路。方法 BHK21细胞分为空载体组（转染 6 µg空载体 pBs/U6）、低转染浓度组（2 µg si-STYK1/NOK质粒+4 µg空载体 pBs/U6）和高转染浓度组（6 µg si-STYK1/NOK质粒）。转染 48 h后,Western blot 检测 STYK1/NOK蛋白表达,基于检测结果,选取空载体组和高转染浓度组提取总 RNA进行转录组测序。将测序所 得的序列进行过滤比对,获得差异表达基因后,应用 R软件进行生物功能（GO）分析和 KEGG信号通路分析。应用 STRING蛋白质互作数据库中的互作关系进行差异基因蛋白互作网络的分析。采用 qRT-PCR验证关键差异表达基 因亚甲基四氢叶酸脱氢酶 2（Mthfd2）、转录激活因子 5（Atf5）,ⅡA分泌型磷脂酶 A2（Pla2g2a）、6-磷酸果糖-2-激酶/ 果糖-2,6-双磷酸酶 3（Pfkfb3）的表达。CCK-8法检测空载体组和高转染浓度组细胞增殖情况。结果 Western blot 结果表明高转染浓度组明显抑制 STYK1/NOK蛋白表达。高转染浓度组与空载体组相比,共发现差异表达基因 44 个,包括 19个上调基因和 25个下调基因。GO富集分析发现差异表达基因主要影响生物调控、细胞进程、代谢调控、 细胞生物过程、细胞、细胞部分、胞外区、配体和催化活性。KEGG通路分析发现差异表达基因主要集中作用于免疫 系统、癌症、细胞周期、信号转导和氨基酸代谢等方面。qRT-PCR结果表明,高转染浓度组Mthfd2、Atf5的相对表达量 显著上调（P＜0.05）,Pfkfb3、Pla2g2a的相对表达量显著下调（P＜0.05）,与测序结果相一致。细胞增殖实验表明高转 染浓度组细胞增殖明显高于空载体组（P＜0.01）。结论 抑制 BHK21细胞 STYK1/NOK表达后,致使 BHK21细胞原 癌基因表达增强,抑癌基因表达减少,促进细胞增殖。     

Study of GPR81, the lactate receptor, from distant species identifies residues and motifs critical for GPR81 functions

Receptors from distant species may have conserved functions despite significant differences in protein sequences. Whereas the noncritical residues are often changed in distant species, the amino acids critical in receptor functions are often conserved. Studying the conserved residues between receptors from distant species offers valuable information to probe the roles of residues in receptor function. We identified two zebrafish receptors (zGPR81-1 and zGPR81-2) that show approximately 60% identity to human GPR81, GPR109a, and GPR109b but respond only to l-lactate and not to the GPR109a ligands. Protein sequence comparison among zebrafish GPR81s, mammalian GPR81s, GPR109a, and GPR109b identified a common structure (six Cys residues at the extracellular domains that potentially form three disulfide bonds) in this subfamily of receptors. In addition, a number of residues conserved in all GPR81s but not in GPR109s have been identified. Furthermore, we identified a conserved motif, C165-E166-S167-F168, at the second extracellular loop of GPR81. Using site-directed mutagenesis, we showed that Arg71 at the transmembrane domain 2 is very critical for GPR81 function. In addition, we demonstrated that the C165-E166-S167-F168 motif at the second extracellular loop is critical for GPR81 function, and the conserved six Cys residues at the extracellular regions are necessary for GPR81 function. It is important to mention that for those residues important for GPR81 function, the corresponding residues or motifs in GPR109a are also critical for GPR109a function. These findings help us better understand the interaction between lactate and GPR81 and provide useful information for GPR81 ligand design.     

Roles of conserved intracellular sequences regions for the proper expression of dopamine D2 and D3 receptors

The dopamine D(2) receptor and D(3) receptor (D(2)R, D(3)R) have high homology in both their amino acid composition and signaling pathways. Virtually all signaling pathways reported thus far overlap between the two receptors with the exception that the D(3)R signals are 2 approximately 5 times less efficient than D(2)R. Previous studies have suggested that conformational constraints of D(3)R might be responsible for the poor coupling with the G protein. To this hypothesis, point mutations were introduced into some of the conserved regions between D(2)R and D(3)R, and their effects on receptor expression were investigated. Among the four conserved intracellular receptor regions examined (TTT motif in the 1(st) intracellular loop, SS motif in the 2(nd) intracellular loop, YxxL and TxxS/xS motifs in the 3(rd) intracellular loop), a mutation of the Thr-Thr-Thr (TTT) motif in the first intracellular loop or the LxxY motif in the 3(rd) intracellular loop markedly decreased the level of D(3)R expression compared with D(2)R. The TTT motif was further mutated individually or in combination to test which residue plays a critical role on the expression of the receptor proteins. Different amino acids between D(2)R and D(3)R in the 1(st) intracellular loop were exchanged to determine if the adjacent amino acid residues are responsible for the differences between D(2)R and D(3)R. The first two threonine residues become more important when the individual threonine residue is mutated. However, all three intact threonine residues are essential for proper expression of the receptor proteins. The neighboring sequences around the triplet threonine residues in the 1(st) loop of D(3)R are not important for proper positioning of the receptor proteins on the plasma membrane. It was concluded that D(2)R has a more flexible overall conformation that can accept mutated residues in the intracellular region than D(3)R, which might be partly responsible for the quantitative differences in the signaling efficiency between D(2)R and D(3)R.     

Classification of spider neurotoxins using structural motifs by primary structure features. Single residue distribution analysis and pattern analysis techniques.

In recent years the data on the novel structures of spider toxins have been greatly increasing. The sequence data should be classified. We introduced two primary structure analysis techniques-single residue distribution analysis (SRDA) and pattern analysis for classifying spider polypeptide toxins with molecular weight less than 10kDa. For multiple sequence alignment, we also introduced three novel sequence representation formats named as a simple record, motif record and a pattern record, which can be useful for large-scale analysis of structures. About 300 sequences of spider toxins were analyzed and nine primary structure motifs were identified. New classification of spider toxins was proposed on the basis of previously described principal structural motif (PSM) and extra structural motif (ESM) [Kozlov, S.A., Malyavka, A.A., McCutchen, B., Lu, A., Schepers, E., Herrmann, R., Grishin, E.V., 2005. A novel strategy for the identification of toxin-like structures in spider venom. Proteins 59 (1), 131-140]. Five main structural classes were revealed, and for putative ion channel inhibitors from the most numerous classes 1, 2, and 3, five-digital personal ID numbers were introduced. A reference table with simple, motif and pattern representation sequence formats was created for all analyzed structures.     

基于高通量测序的青叶胆转录组研究

目的 探讨青叶胆中獐牙菜苦苷生物合成的遗传基础。方法 采用Illumina Hiseq 2500高通量测序技术,对青叶胆全草进行转录组测序。结果 共获得68 643个unigene,平均长度901 bp,共有42 954条unigene注释到NR,SwissProt,COG,KOG,GO,Pfam数据库中,獐牙菜苦苷生物合成的3个阶段中,有43条unigene参与中间体的生成,3条unigene参与萜类骨架合成,25条unigene可能参与萜类最后的修饰。结论 首次对青叶胆转录组进行测序分析,获得了可能参与獐牙菜苦苷生物合成的候选基因,为后续功能基因的挖掘奠定了基础。     

Sequencing and phylogenetic analysis of neurotoxin gene from an environmental isolate of Clostridium sp.: comparison with other clostridial neurotoxins

A Clostridium sp. isolated from intestine of decaying fish exhibited 99% sequence identity with C. tetani at 16S rRNA level. It produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) as well as tetanus antitoxin. The gene fragments for light chain, C-terminal and N-terminal regions of the heavy chain of the toxin were amplified using three reported primer sets for tetanus neurotoxin (TeNT). The neurotoxin gene fragments were cloned in Escherichia coli and sequenced. The sequences obtained exhibited approximately 98, 99 and 98% sequence identity with reported gene sequences of TeNT/LC, TeNT/HC and TeNT/HN, respectively. The phylogenetic interrelationship between the neurotoxin gene of Clostridium sp. with previously reported gene sequences of Clostridium botulinum A to G and C. tetani was examined by analysis of differences in the nucleotide sequences. Six amino acids were substituted at four different positions in the light chain of neurotoxin from the isolate when compared with the reported closest sequence of TeNT. Of these, four were located in the β15 motif at a solvent inaccessible, buried region of the protein molecule. One of these substitutions were on the solvent accessible surface residue of α1 motif, previously shown to have strong sequence conservation. A substitution of two amino acids observed in N-terminal region of heavy chain were buried residues, located in the β21 and β37 motifs showing variability in other related sequences. The C-terminal region responsible for binding to receptor was conserved, showing no changes in the amino acid sequence.Nucleotide sequence data reported are available in the EMBL database under the accession numbers CAI79619, AM076940, and CAI79618.’     

Two new rhamnopyranosides of neolignans from Sanguisorba officinalis

Two new rhamnopyranosides of neolignans, (7S,8R)-4,9,5',9'-tetrahydroxy-3,3'-dimethoxy-8-O-4'-neolignan-7-O-α-l-rhamnopyranoside (1) and (7S,8R)-4,9,9'-trihydroxy-3,3',5'-trimethoxy-8-O-4'-neolignan-7-O-α-l-rhamnopyranoside (2), together with a known compound (7S,8R)-4,7,9,9'-tetrahydroxy-3,3'-dimethoxy-8-O-4'-neolignan (3), were isolated from the 80% EtOH extract of the roots of Sanguisorba officinalis. Their structures were characterized by spectroscopic analysis including 1D NMR, 2D NMR, and HR-ESI-MS, and chemical method.     

Identification of a drug target motif: an anti-tumor drug NK109 interacts with a PNxxxxP

The synthetic compound NK109 shows anti-tumor effects against a number of human cancer cell lines. The mechanism of action is thought to involve the inhibition of DNA topoisomerase II. However, NK109 also exhibits potent anti-tumor activities against doxorubicin-, cisplatin- and etoposide-resistant human cell lines. This paper describes target validation of NK109 using biotinylated NK109 and a T7 phage library screening procedure. Phage particles displaying an affinity for NK109 were isolated and the DNA sequence determined. The amino acid sequences of selected peptides, and the results of mutation experiments by alanine scanning, confirmed that the binding target motif of NK109 is PNxxxxP. In silico analysis of the interaction between NK109 and the peptide, by docking simulation and molecular dynamics, supported this conclusion. The PNxxxxP motif exists in the C2 domain of protein kinase Calpha. NK109 was confirmed to bind the C2 domain from surface plasmon resonance analysis. Furthermore, NK109 moderately inhibited protein kinase C activity in vitro. Our results show that the anti-tumor activity of NK109 stems from interactions with multiple protein targets.     

基于转录组学研究岩藻黄素在扰动剪切应力对EPCs炎性及增殖影响

目的 在扰动剪切应力（Oscillatory shear stress, OSS, ±4 dynes/cm2, 1 Hz）作用下内皮祖细胞（Endothelial progenitor cells, EPCs）转录组学数据生物信息学分析的基础上,探讨岩藻黄素（Fucoxanthin, FUCO）对OSS作用下的EPCs炎性、增殖以及细胞周期等生物活性改变的影响作用。方法 采用Flexcell flow STR-4000流体系统模拟体内扰动流剪切应力环境;利用Illumina高通量测序平台对相应处理的EPCs进行转录组测序分析,并对筛选到的差异蛋白编码基因进行功能以及通路分析;利用实时荧光定量PCR技术（RT-qPCR）验证相应生物信息学数据;Western blot（WB）检测筛选基因-前列腺素内过氧化物合酶2（PTGS2）蛋白表达;采用EdU和PI染色分别进行EPCs增殖和周期检测。结果 与静止对照组相比,共计差异表达基因1066个,涉及细胞增殖和死亡、感染性疾病等信号转导通路;GO功能分析显示,该类基因功能主要影响的过程有细胞及细胞组分、细胞进程、细胞器及生物进程等生物活动;根据P值<0.05且Fold change（FC）>2的条件,筛选显著差异基因数17个,并经RT-qPCR以及WB验证,显示PTGS2 mRNA差异表达最为显著（P<0.001）,PTGS2蛋白表达差异明显（P<0.05）;进一步的结果显示,与OSS组EPCs相比,FUCO处理的OSS组EPCs中PTGS2 mRNA表达显著降低（P<0.001）,其蛋白表达被抑制（P<0.05）,并且FUCO处理过的OSS组EPCs增殖活性以及DNA合成能力增强。结论 结合转录组数据基础,提示FUCO能够显著降低OSS引发的EPCs炎症反应,尤其明显抑制炎性因子PTGS2表达;促进EPCs细胞增殖和 DNA合成,这为FUCO临床干预EPCs进而完善心血管疾病干细胞治疗提供理论依据。     

Genetic variation and identification of cultivated Fallopia multiflora and its wild relatives by using chloroplast matK and 18S rRNA gene sequences

FALLOPIA MULTIFLORA (Thunb.) Harald . has been widely and discriminatingly used in China for the study and treatment of anemia, swirl, deobstruent, pyrosis, insomnia, amnesia, atheroma and also for regulating immune functions. However, there is still confusion about the herbal drug's botanical origins and the phylogenetic relationship between the cultivars and the wild relatives. In order to develop an efficient method for identification, a molecular analysis was performed based on 18 S rRNA gene and partial MATK gene sequences. The 18 S rRNA gene sequences of F. MULTIFLORA were 1809 bp in length and were highly conserved, indicating that the cultivars and the wild F. MULTIFLORA have the same botanical origin. Based on our 18 S rRNA gene sequences analysis, F. MULTIFLORA could be easily distinguished at the DNA level from adulterants and some herbs with similar components. The MATK gene partial sequences were found to span 1271 bp. The phylogenetic relation of F. MULTIFLORA based on the MATK gene showed that all samples in this paper were divided into four clades. The sequences of the partial MATK gene had many permutations, which were related to the geographical distributions of the samples. MATK gene sequences provided valuable information for the identification of F. MULTIFLORA. New taxonomic information could be obtained to authenticate the botanical origin of the F. MULTIFLORA, the species and the medicines made of it.