Molecular cloning and expression analysis of interleukin - 1β from Japanese sea perch(Lateolabrax japonicus )

The technology of homology cloning and anchored PCR was used to clone the IL-1β gene from the Japanese sea perch （Lateolabrax iaponicus）. The full-length cDNA of sea perch IL-1β was 1 310 bp, including a 5＇ untranslated regiop （UTR） of 136 bp, a 3＇ UTR ot 430 bp, and an ORF of 774 bp encoding a polypeptide of 258 amino acids with an estimated molecular mass of 29.31 kDa. The searches for nucleotides and protein sequence similarities with the BLAST analysis indicated that the deduced amino acid sequence of sea perch IL-1β was homological to the IL-1β in other fish species and even the mammalian. Conserved signature sequences of the IL-1β gene family were found in the sea perch IL-1β deduced amino acid sequence. Temporal expressions of the IL-1β gene in LPS or iridovirus challenged group and in control group were measured by the semi-quantitative RT-PCR. The mRNA transcripts of IL-1β could be detected in head-kidney, spleen, liver, gill and heart of the healthy individuals, and the expression level of IL-1β in head-kidney, spleen and gill was higher than that in liver and heart, but it was hard to be detected in the brain. After being stimulated by the LPS or iridovirus, the IL-1β expression in most of examined tissues was up-regulated, and also could be detected in the brain. These results indicated that the expression of sea perch IL-1β was constitutive and could be up-regulated by immune effector stimulation. Therefore the sea perch IL-1β could play a critical role in the host-pathogen interaction.

Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) (P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) (P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

Effect of Indoor Concentrate Feeding vs. Outdoor Grazing on the Expression of Genes Involved in Muscle Growth and Nutrient Content in Japanese Black Steer Muscle

The objective of the present study was to investigate the effect of outdoor grazing on the expression of genes involved in muscle growth and the nutrient contents of skeletal muscle in steers. Ten Japanese Black steers were divided into two groups: grazing (GR) and concentrate (CT) groups. Crude protein, extractable lipid, moisture, fatty acid, cooking loss and Warner Bratzler shear force in muscle tissue were analyzed. The gene expression of myosin heavy chain (MyHC) isoform (2a, 2x and slow), myostatin, follistatin, peroxisome proliferator-activated receptor γ2 (PPARγ2), CCAAT/enhancer binding protein α (C/EBPα), heat shock protein (HSP) 27 and HSP40 in skeletal muscles was evaluated at the end of fattening. Decreases in MyHC-2a and MyHC-2x (fast-twitch fiber type) expression in the longissimus lumborum (LL) muscle were detected in the GR group compared with the CT group;in contrast, an increase in MyHC-slow (slow-twitch fiber type) expression was shown in the GR group. These results suggest that grazing initiated muscle fiber type conversion to slow-twitch from fast-twitch. A decrease in extractable lipid content was observed in the GR group in the LL and semitendinosus (ST) muscles. Crude protein content in the LL muscle in the GR group was higher than in the CT group. MyHC expression in LL muscle in the GR group was greater than in the CT group. A decrease in myostatin and PPARγ2 gene expression was detected in the GR group compared with the CT group in both muscles. Expression of C/EBPα in LL muscle in the GR group was lower than in the CT group. These results suggest that grazing steers at the end of fattening may lead to an increase in protein content and a decrease in fat accumulation in LL and/or ST muscles by regulation of myostatin, MyHC, PPARγ2 and C/EBPα gene expression.

Genome-wide identification and transcriptome-based expression analysis of sox gene family in the Japanese flounder Paralichthys olivaceus

Sox genes are transcription factors that ubiquitously exist in animal species, and share a conserved high mobility group(HMG) box. They play important biological roles in diverse developmental processes, such as sex determination and differentiation, chondrogenesis, neurogenesis, and early embryonic development. In this study, we identified 25 sox genes from genome and transcriptome of Japanese flounder Paralichthys olivaceus. These s ox genes could be mainly classified into seven subfamilies(B1, B2, C, D, E, F, and K), and each subfamily exhibited a relatively conserved gene structure. Besides, subfamilies A and G were found exclusively in human and mouse, and sox 32 in subfamily K only existed in teleosts. Compared with other mammals, some s ox genes in teleosts had two duplicates. The loss, duplication, and divergence of sox genes during evolution provided an evidence for whole-genome duplication that occurred in the radiation of teleosts. The expression of Japanese flounder sox genes was also analyzed by FPKM value. Our results showed that certain s ox genes exhibited obviously tissue-specific and spatio-temproal expression. Especially, gonal-basied expression analysis uncovered that s ox7 and s ox2 were ovary-biased, and s ox8 b was testis-biased. Moreover, sox10 a was expressed specifically in ovary, and sox8 a in testis. Therefore this study provide a solid foundation for future functional and evolutionary analysis of sox genes in Japanese flounder.

A Brief Study on the Application of Euphemistic Expressionsin Business Negotiations

This paper will introduce euphemistic expression, business negotiation, and some euphemistic expressions with different sentence patterns according to different situations in business negotiations. Application of euphemistic expressions can not only reduce language slip, increase language techniques, display the virtue of personality, but also soften the tension of climate, and establish a favorable, cooperating re lationship. It can importantly increase the likeliness of the negotiation's success.

日本鳗鲡TBK-1基因的克隆及免疫刺激的表达模式

利用PCR技术克隆了日本鳗鲡(Anguilla japonica)TBK-1基因(AjTBK-1),其开放阅读框为2193 bp,编码731个氨基酸。序列结构分析结果显示,AjTBK-1含有4个保守结构域,分别为氨基端激酶结构域,泛素样结构域和羧基端两个卷曲-螺旋结构域。系统发育分析表明,鱼类与四足类的TBK-1各自聚为一枝。实时定量PCR(qPCR)结果显示,AjTBK-1在日本鳗鲡各组织中均有表达。Poly I:C刺激6 h后,日本鳗鲡脾脏组织中AjTBK-1的上调倍数最高,为对照组的1.63倍;迟缓爱德华氏菌(Edwardsiella tarda)感染24 h后,日本鳗鲡肝脏组织中AjTBK-1的上调倍数最高,为对照组的2.2倍:表明AjTBK-1参与了日本鳗鲡抗病毒、抗细菌免疫反应应答。

隐喻视角下的汉日“眼”类惯用语的对比研究

惯用语是广大民众思维智慧的结晶,“眼”是人体最重要的器官之一,在汉语和日语的人体词研究中均排首位,并各自产生大量与“眼”相关的惯用语。以汉语和日语中含有“眼”的隐喻惯用语为研究对象,首先通过定量分析找出彼此的共性,再以认知语言学的隐喻和转喻为理论基础,根据“眼”的实体隐喻目标域的不同,分别在容器域、意识域、情感域、能力域和思维域这五个目标域范围内以语料为基础对“眼”的实体隐喻进行具体分析,了解“眼”在汉日语惯用语中的语义扩展方式上的异同。研究得知:汉语和日语惯用语中“眼”的隐喻有诸多相似之处便是源于两国人民对“眼”认知思维的相似性;与此同时,由于中日地理环境、审美心理、价值观的不同,导致惯用语中“眼”的隐喻表达也存在很大的差异。

日语中的委婉语学习

日语委婉语表达是日语语言特征之一,也是日语中一种十分常见的表达形式,日本人的生活中处处使用委婉语,既反映了日本人委婉的性格特征,也反映了日本自古以来的文化特点。日语委婉语与日本的地理位置、历史背景,以及日本人的生活习惯、社交习惯、民族文化等有着密不可分的关系。在日语中,委婉语有多种表达方式,使用频率较高,对日语学习者而言,委婉语的学习在日语学习中占据了重要的地位。根据塞尔提出的间接言语行为理论,委婉语是其典型代表。该文将分析日语中的委婉表达,并对其简单进行分类,以期对委婉语的学习和研究提供帮助。

中日语言前置表现对比研究

文章设置7个特定场面和1个非特定场面,对中日语言中的前置表现进行了实例对比分析,总结出两国语言在前置表现使用上的异同。相同之处:中日语言中存在的前置表现均能体现信息传达者对信息接受者的某种关照,并且「对传达性」关照型前置表现的使用频率均高于「对人」关照型。不同之处:汉语中前置表现的使用频率不及日语。汉语中前置表现的使用频率不及日语的原因,从文化角度来看是因为二者文化特征不同:日本人重视“内”与“外”,形成的是委婉文化,表达情感时讲究委婉;而中国人既重视传统礼仪也喜欢直来直去,表达情感时还存在一定的“默认一致”心理倾向。从语言角度来看是因为二者语言类型不同:日语属于黏着语,前置表现中经常使用的「ガ/ケド节」在语言表达中起着重要作用;而汉语属于孤立语,其孤立语的特性决定了汉语前置表现的表达形式没有日语统一性高,定型化表达也没有日语多。

Comparative transcriptome analyses reveal candidate genes regulating wood quality in Japanese larch(Larix kaempferi)

We studied the molecular mechanism of the quality traits of wood formation in larch.We used the immature latewood cells of two Japanese larch(Larix kaempferi)clones with significant differences in density and in microfibrillar angle(MFA)as materials to analyze their gene expression profiles.A total of 1735 differentially expressed genes were detected in immature latewood cells of the two clones,among which,971 were up-regulated and 764 were down-regulated.Digital gene expression profiling analysis revealed that genes encoding transcription factor members NAC66 and R2R3-MYB4,microtubule-associated protein,actin-related protein,cell wall protein members,arabinogalactan protein,Fasciclin-like arabinogalactan protein and glycine-rich protein,and several cell-wall-synthesis genes affected wood density and MFA by regulating latewood formation at transcriptional level.Our study results represent a basis for selection of quality traits and genetic improvement of larch wood.

Development of an Indirect ELISA Using Recombinant Truncated Envelope Glycoprotein for Detection of Antibodies against Japanese Encephalitis Virus

[ Objective] To develop an indirect ELISA assay for detecting antibodies against envelope glycoprotein （ E protein） of Japanese encephalitis virus （JEV）. [ Method] Specific primers were designed according to JEV sequences published in the GenBank. The cDNA of JEV E gene （about 1 000 10p） was amplified by the RT-PCR with the specific primers. After sequencing analysis, the E gene was cloned into pET30a expression vector and expressed in E. coli BL21 （DE3） with the induction of IPTG. After denaturation, purification and renaturation, the recombinant protein was analyzed by the SDS-PAGE and the westem blotting. An indirect ELISA was developed to detect antibodies against JEV. [ Result] The E protein was mainly expressed in inclusion body. With the purified E protein, the indirect ELISA was developed and displayed good specificity, sensitivity and repeatability, [ Conclusion]The developed ELISA using the truncated E protein as antigen is a simple, convenient and rapid serological method for diagnosis, monitoring antibody level and epidemiological investigation of JEV.

Study on DNA Immunization by Recombinants Encoding Japanese Encephalitis Virus prME and E Proteins

To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by liposome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E). BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01 strains (10 5 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected in HepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using anti-E. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization, and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE. It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of pJE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.

Methylation Status of the Follistatin Gene at Different Development Stages of Japanese Flounder(Paralichthys olivaceus)

Follistatin （Fst） is a hyperplasia factor that plays a crucial role in muscle development. DNA methylation, a significant process, regulates gene expression. The aim of our study is to examine the DNA methylation and expression patterns ofFst gene at five different development stages of Japanese flounder （stage A, 7 dph; stage B, 90 dph; stage C, about 180 dph; stage D, about 24 months; stage E, about 36 months）. The muscle tissue of Japanese flounder was obtained at different development stages in dais experiment. DNA methylation levels in the promoter and exon 2 of Fst were determined by bisulfite sequencing, and the relative expression of the Fst gene at the five stages was measured by quantitative PCR. The results showed that the lowest methylation level was at stage A and the highest methylation level was at stage B. Moreover, the highest expression level of the Fst gene was observed at stage A. The mRNA abundance was negatively correlated with DNA methylation level. Three CpG islands in the promoter region and three CpG islands in exon 2 of Fst were found in the binding sequence of the putative transcription factor. These results offered a theoretical basis for the mechanism of Fst gene regulation to muscle development at different development stages.

日本七鳃鳗转化生长因子βⅠ型受体基因(L-Tgfbr1)的克隆与表达分析

作为无颌类脊椎动物的现存代表之一,日本七鳃鳗(Lampetrajaponica)是研究免疫系统起源与进化的重要模型。为了探究TGF-β(Transforming growth factor beta)信号通路在日本七鳃鳗免疫调节中的功能,本研究利用PCR技术克隆了日本七鳃鳗TGF-βⅠ型受体基因(L-Tgfbr1)的编码序列,开放阅读框长度为1335 bp,编码444个氨基酸残基。L-Tgfbr1蛋白含有已知的TGF-βⅠ型受体分子的主要功能结构域,其中位于胞内的丝/苏氨酸激酶催化结构域保守性较高。系统进化树分析表明,L-Tgfbr1处于脊椎动物Tgfbr1蛋白的底端进化枝上,表明其在Tgfbr1进化史中具有原始性地位。实时定量PCR结果发现,L-Tgfbr1在心脏等组织中的转录水平较高。利用脂多糖注入七鳃鳗激活其先天性免疫应答,L-Tgfbr1在肾脏、鳃、髓小体、肝脏、白细胞、口腔腺中的转录水平呈现一过性的迅速上调。利用免疫印迹进一步验证了脂多糖免疫24 h时后L-Tgfbr1在白细胞中的蛋白表达水平显著上调。利用免疫荧光染色发现L-Tgfbr1蛋白主要分布于七鳃鳗白细胞和髓小体细胞的细胞质中。以上结果表明L-Tgfbr1及其介导的TGF-β通路可能在七鳃鳗免疫调控中发挥重要功能。

日本学生汉语第一人称代词表达特点

文章以第一人称代词可表达的六种作者身份(行为发起者、行为承受者、范围界定者、情感表达者、观点持有者、等价判断者),考察日本汉语学习者和中国汉语母语者在记叙文语料中第一人称代词及其作者身份的表达特点。结果显示,日本汉语学习者过度使用第一人称代词,具体表现为:在“我(们)”作为行为发起者、“我”作为范围界定者/观点持有者/情感表达者的作者身份上,存在显著的使用过度;而在“我”作为行为承受者的作者身份上存在显著的使用不足。文章最后进一步探讨这一现象的具体成因并提出相关教学建议。

日语学位论文中的句末认识情态使用特征研究——基于中日语料库对比视角

句末认识情态是表明作者的写作态度和观点的重要表达方式。该研究基于日语专业博士毕业生的博士论文和日本本族语者的博士论文,自建语料库,对比分析了中日博士学位论文中句末认识情态使用特征,发现:(1)中国日语学习者与日本本族语者相比,在“断定·推量”“或然性”“证据性”“知觉·思考动词等”4类句末认识情态的使用上均存在显著差异;(2)日语学习者与本族语者一样倾向于集中地使用思考动词和推量表达等少数几个句末表达;(3)日语学习者在博士论文撰写过程中存在句末情态多用、少用的问题。其中「であろう(大概)」的多用和「と考えられる(一般认为)」的少用现象最为显著。该研究结果对日语论文写作及其教学有指导意义。

晋察冀抗日根据地妇女运动与妇女解放的实践探索——以晋察冀抗日根据地妇女的社会舆论表达为中心

晋察冀抗日根据地妇女积极参与生产,勇于参政。随着经济地位、政治地位和社会地位的提升,促进了她们群体意识的形成与发展,推动了晋察冀抗日根据地妇女的社会舆论表达。她们提倡妇女解放和生产、婚姻解放,宣传妇女参政和妇女教育,参与抗日宣传表达等。通过妇女组织引领社会舆论,通过开展多种形式活动扩大了舆论传播力,同时也推动了自身解放。

跨文化交流背景下中日语言文化差异对翻译的影响——以配虑表达和敬语为例

随着全球化进程的加快,跨文化交流越来越频繁,翻译实践不仅要重视语法和句子结构的准确性,更需要理解不同文化之间的细微差别。语言是文化的一部分,承载着一个国家的历史、地理、人文、行为意识、风俗习惯等丰富的内涵,任何一种语言都已深深留下民族的烙印,必定有其独特性。日本的民族特性孕育出日语的暧昧性和距离性,这两大特点体现在日本人日常生活交际活动中的配虑表达和敬语上。对于语言文化差异,翻译时稍有疏忽,便有失之毫厘,谬以千里的可能。该文从配虑表达和敬语两个方面分析中日语言文化差异对翻译的影响,并探讨解决对策。

蟹类原肌球蛋白基因的克隆与表达

通过分子生物学技术方法,分别克隆得到锯缘青蟹(Scylla serrata)、中华绒螯蟹(Eriocheir sinensis)和三疣梭子蟹(Portunus trituberculatus)的原肌球蛋白基因序列。测序结果表明,三个基因的序列长度均为855 bp,编码284个氨基酸残基,三者氨基酸序列同源性为99.3%。三种蟹的原肌球蛋白基因序列与GenBank中其他甲壳类动物的原肌球蛋白具有很高的同源性。将锯缘青蟹的原肌球蛋白基因与pGEX-4T-3载体连接后,经IPTG诱导得到分子量约为61 ku的融合表达蛋白。通过与甲壳类过敏患者血清的免疫印迹反应,证实融合表达的原肌球蛋白具有过敏原性,表明原肌球蛋白是蟹类的主要过敏原之一。该融合蛋白有望用于甲壳类食物过敏诊断试剂的开发与应用。

乙型脑炎病毒贵州分离株E基因原核表达及其免疫原性的研究

收集某猪场1例发病种公猪的睾丸病料,处理后采用细胞培养与乳鼠脑内接毒相结合的方法盲传并结合RT-PCR方法分离鉴定,命名为乙型脑炎病毒(JEV)贵州分离株(GZ株),然后根据GenBank登录的日本乙型脑炎病毒SA14和SA14-14-2株全基因序列设计并合成1对扩增E基因的特异性引物,用RT-PCR方法扩增出JEV贵州分离株E基因,并将E基因克隆到pMD19-T载体上,构建重组质粒pMD19-T-E,经双酶切鉴定、测序及序列分析鉴定后,再将E基因亚克隆到pET-32a(+)原核表达载体上,成功构建乙脑病毒E基因原核表达质粒pET-32a-E,将构建好的原核表达质粒pET-32a-E转化至宿主菌BL21(DH3)中,诱导表达目的蛋白,表达产物经SDS-PAGE电泳和Western blot分析,得到了约59ku条带,与预期大小相符,进一步提取、纯化和浓缩目的蛋白,并将其加入弗氏佐剂后免疫小鼠,免疫后采血检测抗体。结果显示:乙型脑炎病毒贵州分离株E基因原核表达目的蛋白能诱导小鼠产生一定抗体水平,该研究为乙型脑炎亚单位疫苗的研究奠定了基础。