Cloning and Expression of PKR Gene of Ctenopharyngodon idellus Induced by PolyI:C in vitro

[Objective] The study aimed at cloning PKR gene from Ctenopharyngodon idellus induced by PolyI：C in vitro,so as to provide foundation for study on the anti-virus genes of C.idellus.[Method] By referring to the PKR gene sequences of zebra fish（AJ852023.1） and Carassius auratus（AY293929.1） in Genbank,three pairs of degenerate primers were designed with Primer Premier 5.0 software;in vitro C.idellus kidney cells（CIK） were treated with 100 μg/ml of Poly I：C for 12,36 and 48 h,and then total RNA of the cells treated was extracted for amplifying the PKR gene by RT-PCR.[Result] The PKR gene was amplified from the cells treated with Poly I：C for 36 and 48 h,but not from the cells treated for 12 h;in addition,the expression level increased with the processing time.Part of the amplified sequence of C.idellus shared the homology of 100% and 81.48% with the sequences of carp and zebra fish separately.[Conclusion] Part of the PKR gene sequence was cloned successfully from C.idellus.Moreover,we have proved that PolyI：C induction is effective for PKR protein expression,which will provide reference for treating viral diseases of C.idellus.

Cloning, Sequencing, and Characterization of Porcine Sterol Regulatory Element Binding Proteins-1c Gene

Primers were designed according to the known sequences of Sterol Regulatory Element Binding Proteins-1c (SREBP-1c) genes of human, rat and pig. RT-PCR was then used to amplify porcine SREBP-1c gene with total RNA of spleen tissue. A 760 bp segment of cDNA was cloned and sequenced. Homogeneous comparison showed that the sequence of porcine SREBP-1c had 99.9% and 99.1% homogeneity with the two known partial mRNA sequences of porcine SREBP-1c gene. The complete cDNA was obtained mainly based on the known partial sequences, which has 3 830 bp, encoding 1 151 amino acids with a calculated molecular weight of 121 479 Da. It is the first time that we get complete encode sequence of porcine SREBP-1c gene. The complete cDNA sequence has high homogeneity with SREBP-1c gene of other species. A characteristic structure of HLH (Helix-Loop-Helix) and four transmembrane segments were found in the amino acids. The sequence had been submitted to GenBank (Accession No.NM_-214157).

Molecular Cloning and Sequence Analysis of C4H Gene of Mangifera indica L.

Cinnamate-4-hydroxylase( C4H) is a key enzyme in phenylpropanoid pathway in plants. Its activity and abundance directly affect the biosynthesis of flavonoids and aromatic compounds. In this study,degenerate primers were designed according to previously reported C4 H gene sequences to clone C4H cDNA sequence with 3'and 5'RACE-PCR from mango( Mangifera indica L). The full-length cD NA of M. indica C4H is 1 680 bp long. Its open reading frame( ORF)is 1 518 bp,encoding a protein of 505 amino acids with a predicted molecular weight of 58. 08 kDa. The isoelectric point of the predicted protein is 9. 52. Functional prediction showed that this gene is mainly located in mitochondria. In addition,the tertiary structure of the protein was built using SWISS-MODEL,and the results showed that the protein has three possible conformations. Phylogenetic analysis based on C4H protein sequences revealed that M. indica has a close genetic relationship with olive( Canarium album) and cocoa( Theobroma cacao). By analyzing the expression level of C4H gene in three colored mango cultivars,we found that that the expression level of C4 H gene in Guifei( with red peel) was the highest,and that in Guiqi( with green peel) was the lowest. The results provide a theoretical basis for studying the molecular mechanism of anthocyanin biosynthesis and C4H's impact on the color of mango fruit.

Cloning and RNA interference analysis of the salivary protein C002 gene in Schizaphis graminum

The full-length c DNA of functionally-unknown salivary protein C002 in Schizaphis graminum was cloned using rapid amplification of c DNA ends（RACE） and designated as Sg C002（Gen Bank accession no. KC977563）. It is 767 bp long and encodes a protein of 190 amino acid residues with a predicted mass of 21.5 k Da and a predicted cleavage site of N-terminal signal peptide between the 24 th and the 25 th residues. Sg C002 is specifically expressed in salivary gland with the highest level at the 2nd instar. Introducing Sg C002-specific 476-si RNA, but not 546-si RNA to aphids through artificial diet significantly suppressed Sg C002 expression. Silencing Sg C002 gene led to lethality of the aphid on wheat plants, but not on pure artificial diet. Our study demonstrated that artificial diet-mediated RNAi can be a useful tool for research on the roles of genes in aphid salivary gland, and also provided new insights into the characteristics of C002 in wheat aphids.

cDNA Cloning, Bioinformatic and Tissue-specific Expression Analysis of Porcine JARID1C Gene

Jumonji, AT-rich interactive domain 1C （JARID1C） protein belongs to the highly conserved ARID protein family, which is involved in chromatin remodeling and transcriptional regulation during cell growth, differentiation, and development. In humans, this gene plays a vital role in normal brain development and function. Using an in silico approach in combination with 5＇ rapid amplification of cDNA ends （5＇ RACE）, the full-length cDNA of JARIDIC （GenBank accession No. EF139241） from porcine ovary, which contains 5,908 bp nucleotides, with an open reading frame （ORF） of 4,548 bp, has been cloned. The putative porcine JARID 1C protein, which is located in the nucleus, encodes 1,516 amino acids with a molecular weight of 170 kDa and a pI of 5.44. Bioinformatic prediction indicates that the protein contains several conserved domains： a JmjN domain, an ARID domain, a JmjC domain, a C5HC2 zinc finger domain, and a PHD zinc finger domain. Similarity comparisons for nucleic and amino acid sequences reveal that the porcine JARID1C protein shares a high identity with its dog, mouse, rat, and human counterparts. The phylogenetic tree of the JARID1 subfamily proteins has been constructed to reveal the evolutionary relationship of various species. Real-time PCR analysis shows that the JARIDIC gene is expressed in various tissues, but at different levels. The expression levels of this gene are higher in the brain and gonad than in other tissues, suggesting that the JARID1C protein plays a role in porcine brain and gonad functions.

Cloning and Expression of C4B Gene in Siji Goose

[Objective] This study aimed to clone C4B gene in Siji goose and detect its expression level in different tissues. [Method] cDNA sequence of C4B gene was cloned with RACE-PCR method. Amino acid sequences in multiple species were aligned in GenBank, and a phylogenetic tree was constructed for homology analysis. [Result] C4B gene in Siji goose shared relatively high homology with chicken and quail; Siji goose C4B gene was expressed highly in liver and lung of adult geese and expressed lowly in epididymis, seminiferous duct, brain, kidney, testis, heart, oviduct and smal intestine. [Conclusion] In the present study, mRNA expression lev-el of C4B gene in different tissues and organs of Siji goose was determined by flu-orescence quantitative PCR, which provided basis for rapid diagnosis of specific an-imal diseases.

Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97

AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied. RESULTS: Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10). CONCLUSION: Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.

Cloning and Expression of a Chitinase Gene from Serratia marcescens Strain C8-8

A 1 692 bp long chitinase-encoding ch/A gene was cloned from the genomic DNA of Serrat/a marcescens strain C8-8 by PCR, which was speculated to en- code a 563 aa long polypeptide chain with molecular weight of about 60.9 kD. Homolog analysis showed that the chiA gene sequence cloned from C8-8 shared the highest similarity with cMA sequences from Serrat/a maresscens strains 141 （ DQ 990373.1 ） and 14041 （ DQ 493896. 1 ）, which reached 99%. Domain analysis showed that N-termlnal （23 aa） of the chiA gene cloned from C8-8 harbored typical signal peptide sequence, while C-telminal harbored the other two domains, in- eluding the PKD region （73 aa） and chitinase catalytic region （387 aa）. The PCR fragment was digested with restriction endonucleases and cloned into plasmid pET28a. The recombinant plasmid pET＇28a-ch/A was firstly transformed into Escherichia coli DI-I5 , and then transformed into expression host E. coli DH3 to express ch/A gene. The recombinant strain DH3 chiA could produce transparent hydrolysis circles on the colloidal chitin plate induced by isopropyl-l-thiogalactopyranoside （IFrG）. SDS-PAGE electrophoresis analysis showed that, a protein with relative molecular weight of about 60 kD was expressed by the recombinant strain DH3 chiA, which was consistent with the except molecular weight. After initial purification, biological activity test showed that the recombinant expression product could hydrolyze chitin, which produced transparent hydrolysis circles on the colloidal chitin plates. Results indicated that chiA gene from Serrat/a marcescens strain C8-8 had biological functions and could be utilized as a potential biological control factor.

Molecular Cloning of a Novel cDNA From Mus Muscular BALB/c Mice Encoding Glycosyl Hydrolase Family 1: A Homolog of Human Lactase-Phlorizin Hydrolase

Objective To study the mechanism of lactose intolerance （LI） by cloning the mouse lactase cDNA and recombining a vector. Methods Total murine RNA was isolated from the small intestine of a 4-week-old BALB/c mouse （δ）. Crene-specific primers were designed and synthesized according to the cDNA sequences of lactase-phlorizin hydrolase （LPH） in human, rat, and rabbit. A coding sequence （CDS） fragment was obtained using RT-PCR, and inserted into a clone vector pNEB-193, then the cDNA was sequenced and analyzed using bioinformatics. Results The cDNA from the BALB/c mouse with 912 bp encoding 303 amino acid residues. Analysis of the deduced amino acid sequence using bioinforrnatics revealed that this cDNA shared extensive sequence homology with human LPH containing a conserved glycosyl hydrolase family 1 motif important for regulating lactase intolerance. Conclusion BALB/c mouse LPH cDNA （GenBank accession No： AY751548） provides a necessary foundation for study of the biological function and regulatory mechanism of the lactose intolerance in mice.

猪瘟病毒C株表位突变毒株的构建及拯救

本研究旨在突变猪瘟病毒C株WH303位点,构建感染性克隆,为猪瘟病毒C株标记疫苗的研究提供候选毒株。本试验以猪瘟病毒C株为研究材料,经重叠PCR方法突变WH303位点,再分别连接至C株和C-flag株全长感染性克隆。体外转录RNA,转染PK15细胞,细胞连续传代完成病毒的拯救和增殖。结果显示,经特异性限制内切酶酶切反应验证,感染性克隆构建成功。经RT-PCR、过氧化物酶单层细胞试验检测,表明病毒成功拯救。病毒细胞传代至17代,检测到病毒,表明病毒可以稳定传代。综上,本研究成功构建了C株WH303突变感染性克隆MC和MC-flag,并成功拯救MC株,得到可以稳定传代的病毒株。

褐飞虱凝集素基因NlCTL4的克隆及功能分析

C型凝集素(C-type lectins,CTLs)是广泛存在于昆虫体内一类含糖识别结构域的蛋白超家族,通常作为模式识别受体,在识别外来病原微生物并启动昆虫先天免疫过程中发挥重要功能。本研究克隆并鉴定了一条褐飞虱CTL编码基因NlCTL4(GenBank登录号:OQ032531),其c DNA序列全长936 bp,编码311个氨基酸。序列分析显示,Nl CTL4蛋白N端存在一段由21个氨基酸残基组成的信号肽,C端仅具有一个典型的糖识别结构域,属于S型CTLs。系统发育分析表明,Nl CTL4与半翅目其他昆虫的CTLs聚为一支,其中与玻璃翅叶蝉CTL亲缘关系最近。q RT-PCR分析显示,Nl CTL4基因在褐飞虱不同发育阶段和不同组织中均有表达。Nl CTL4在5龄若虫期的表达量最高,且在高龄若虫期(4~5龄)的表达量显著高于低龄若虫(1~3龄),但在雌、雄成虫中其表达量无显著差异。Nl CTL4在褐飞虱雌成虫肠道中表达量最高,脂肪体次之,在血淋巴中表达量最低。大肠杆菌(革兰氏阴性细菌)、金黄色葡萄球菌(革兰氏阳性细菌)和金龟子绿僵菌(丝状病原真菌)均能显著诱导Nl CTL4的表达水平,其中大肠杆菌和金黄色葡萄球菌在诱导36 h时Nl CTL4的表达量达到顶峰,金龟子绿僵菌接种24 h时的诱导水平最高。RNAi分析结果显示,抑制Nl CTL4表达后褐飞虱5龄若虫存活率及对金龟子绿僵菌侵染的抵御能力显著降低。本研究结果初步证实了褐飞虱Nl CTL4在宿主生长发育和免疫防御过程中发挥重要调控作用,且在开发褐飞虱RNAi抗虫和病原真菌防虫技术中具有重要的潜在应用价值。

A New Furost-20(22)-ene Oligoglycoside from Asparagus cochinchinensis

A new furost-20(22)-ene oligoglycoside named as aspacochioside C was isolated from the roots of Asparagus cochinchinensis (Lour.) Merr.. Its structure was elucidated to be 26-O-b- D-glucopyranosyl-(25S)-5b-furost-20(22)-en-3b,26-diol-3-O-a-L-rhamnopyranosyl-(14)-b-D- glucopyranoside on the basis of spectroscopic techniques including 1D and 2D NMR experiments.

Molecular Cloning of a Glycyrrhiza uralensis F.Aquaporin GuPIP1 Up-regulated in Response to Drought,Salt and ABA Stress

A PCR-bosed homologous cloning strategy was used to identify aquaporin genes from the roots of Chinese licorice （ Glycyrrhiza uralertsis F. ）. A 1236 bp cDNA with 870 bp open reading frame encoding a 290 amino acids aquaporin ortholog, GuPIP1, was successfully cloned and characterized. The deduced GuPIP1 protein contains six putative transmembrane domains; two conserved NPA motifs as well as the MIP and PIP family signature sequences. A rabbit polyelonal antibody against N-terminal peptide of GuPIP1 corresponded to a 31 kDa GuPIP1 protein on Western blot of plasma membrane preparation of root tissue. RT-PCR and Western blot analysis indicated the expression of GuPIP1 in the root, leaf, and stem tissues. Thus far, GuPIP1 is the first Glycyrrhiza uralensis F. aquaporin that has been identified at a molecular level. Quantitative real-time PCR analysis showed that the expression of GuPIP1 was up-regulated in response to drought, ABA, and salt stress.

Geophysical and Petro-Structural Characteristics of the Tarkwaïan and Associated Formations of the Gontougo Region (Northeastern Côte d’Ivoire)

Studies carried out in the Gontougo region aimed to describe the physical and petro-structural properties of the Tarkwaian formations of northeastern Côte d’Ivoire. The methodology developed is focused on the one hand on the gravimetric geophysical method and on the other hand, on petro-structural studies. The geophysical results highlighted two gravimetric facies characterized respectively by high density (ΔBg > 121 mGal) and low density (ΔBg < 114 mGal) anomalies. From a lithological point of view, the denser domains are made up of intrusive rocks dominated by granodiorites and tonalites cutting low density facies composed of Tarkwaian formations (polygenic conglomerates and arkosic sandstones) and volcanics (tuffs and cinerites). Structurally, these different lithological groups are affected by brittle (fractures, faults, strike-slip) and ductile (folds, schistosities and mineral lineations) deformations. These structures are mainly oriented NNW-SSW, WNW-ESE and NE-SW. The description of the sulphide minerals reveals a style of gold mineralization of the Tarkwaian formations.

Extracellular domain of kinase domain region mediated by adeno-associated virus inhibits growth and angiogenesis of bladder cancer in Balb-c mice

OBJECTIVE: To verify whether the extracellular domain of kinase domain region (KDR) has anti-angiogenesis activity in vivo. METHODS: cDNA was cloned into adeno-associated virus (AAV) vector pSNAV and transfected to baby hamster kidney (BHK) cells. Recombinant AAV was obtained from the cell culture supernatant after adding helper virus. Recombinant AAV-infected human bladder cancer EJ cell line (EJ cells) were injected subcutaneously into Balb-c nude mice. Tumor specimens were removed from the mice, paraffin-embedded and sliced, then stained by immunohistochemistry. Microvessel density (MVD) was determined under a microscope. RESULTS: The tumor volume developed by EJ cells transfected with the extracellular domain of KDR was significantly smaller (1.70 +/- 0.18 cm(3)) compared with that in the control (5.62 +/- 0.67 cm(3)) (P

Construction of Controllable Expression Vector of Mus musculus Growth Hormone (GH) shRNA and Its Gene Si- lencing Efficiency Detection

Growth hormone （GH） is a polypeptide hormone secreted by the pituitary, which promotes animal growth, muscle development, metabolism regulation and other important physiological functions. In this study, a pair of mGH short hairpin RNA （shRNA） was designed according to mouse （ Mus musculus） GH mRNA sequence; pSingle-tTS-mGH shRNA-RFP, an integrated controllable expression vector of mGH shRNA, was constructed successfully. The recombinant vector was transfected into mouse pituitary tumor cell line AtT-20. After addition of doxycyelin （ DOX）, the expression of red fluorescent protein （RFP） was observed un- der a fluorescent microscope. The expression level of mGH in cells was detected by quantitative Realtime PCR （qRT-PCR） and Western blot. After DOX induction, the relative expression level of GH mRNA in cells transfected with GH shRNA was reduced by about 70% compared with that in DOX-free group and other control groups, exhibiting extremely significant differences （P 〈 0.01 ） ; moreover, the relative expression level of GH protein was reduced by about 90% ; the expression level of GH mRNA and GH protein exhibited no significant difference among other groups （P 〉 0.05）. In this study, a controllable expression vector of GH shRNA with high gene silencing efficiency was constructed successfully, which could be used to reveal GH autocfine / paracrine interactions and analyze functions of GH gene in growth, development and disease occurrence of animals by regulating GH expression levels.

黄瓜果实维生素C合成关键基因克隆与分析

【目的】鉴定调控黄瓜果实中L-半乳糖途径维生素C(Vc)合成相关基因的位置、数量及表达特征,同时对关键基因进行克隆分析,旨在为黄瓜果实中Vc合成调控研究奠定基础。【方法】根据已报道的拟南芥中L-半乳糖途径合成Vc相关基因,利用蛋白编码的氨基酸序列在黄瓜9930_V2参考基因组数据库中进行BLAST比对,确定黄瓜中的同源基因,借助TBtools软件绘制基因在染色体上的位置。通过qRT-PCR分析上述基因在果实Vc含量差异显著的两份黄瓜材料中的表达量。利用PCR扩增对限速酶GDP-L-半乳糖磷酸化酶(GGP)及GDP-甘露糖-3′5′-差向酶(GME)同源基因进行克隆,测序分析这些基因在高Vc含量与低Vc含量黄瓜果实中的序列差异。构建系统进化树,分析黄瓜果实GME、GGP与其他物种中同源基因的亲缘关系。【结果】在黄瓜基因组中比对到21个参与L-半乳糖途径合成Vc相关酶PMI、PMM、GMPase、GME、GGP、GPP、GalDH、GalLDH的同源基因,7条染色体均有分布,在5号染色体和1号染色体上分布最多。通过对21个基因在两份果实Vc含量高低差异显著的两份材料CG45(高Vc含量)和R48(低Vc含量)的表达量分析,发现调控PMI、PMM、GMPase、GME、GalLDH这5个酶的基因在CG45和R48中有极显著的表达差异。对Vc合成限速酶GGP和GME相关基因在CG45和R48两份材料中进行克隆发现,CsGME2在R48中基因全长为3537 bp,在CG45中基因全长为3541 bp,该基因在两份材料存在多个SNP位点差异和Indel差异,有一个突变位点位于CDS区,且导致了氨基酸序列的改变。通过对调控Vc合成限速酶GME、GGP蛋白性质分析,发现限速酶GME、GGP在不同物种的蛋白性质差异不大,均为亲水性蛋白,功能相对保守。进化树分析发现不同物种亲缘关系较近的聚类在一起,进化过程高度保守。【结论】鉴定出21个分布于7条染色体上的黄瓜果实Vc合成的L-半乳糖途径相关基因,推测关键酶PMI、PMM、GMPase、GME、GalLDH、GGP可能影响黄瓜果实中Vc含量变化,调控Vc合成限速步骤关键酶GME、GGP功能相对保守,Vc合成限速步骤关键酶GME基因CsGME2在高Vc和低Vc两份材料中的一个SNP位点变异导致氨基酸序列的变化。

Balb/c小鼠甘露聚糖结合凝集素-C基因的克隆与鉴定

目的获得小鼠甘露聚糖结合凝集素-C(MBL-C)基因的全长编码区cDNA。方法利用RT-PCR方法,从Balb/c小鼠的肝细胞中分离出MBL-C基因cDNA片段,将其克隆入pUC-T载体并测序,对基因结构及其与人MBL基因的同源性进行比较分析。结果扩增得到的Balb/c小鼠MBL-C基因cDNA全长735 bp,编码245个氨基酸残基,包含了完整的编码区基因序列。经核苷酸序列比较分析发现,扩增得到的Balb/c小鼠MBL-C基因与Genbank中发表的序列具有100%的同源性,与人的MBL基因的同源性为71.4%。结论成功地克隆到完整的Balb/c小鼠MBL-C编码区基因,为进一步在整体水平研究MBL分子的天然免疫功能提供了必要的条件。

翘嘴鳜早期基因c-fos的分子特征与表达

【目的】分析翘嘴鳜(Siniperca chuatsi)早期基因c-fos的两个亚型fosaa和fosab的分子特征,探究二者学习记忆功能。【方法】根据翘嘴鳜c-fos编码区设计引物,克隆翘嘴鳜fosab基因,通过翘嘴鳜全基因组数据库获取fosaa基因,用生物信息学分析翘嘴鳜fosaa、fosab的结构特征,用荧光定量PCR检测fosaa、fosab在翘嘴鳜健康组织以及投喂饲料7、56 d后脑组织中的表达量。【结果】fosab全长2498 bp,开放阅读框(ORF)1152 bp,与fosaa基因相同,有4段外显子;二者基因序列差异较大,DNA序列及氨基酸序列相似性分别为40.4%、46.0%。进化树分析显示,翘嘴鳜FOSAB与其他鱼类以及人(Homo sapiens)、褐家鼠(Rattus norvegic)、小鼠(Mus musculus)和野猪(Sus scrofa)等哺乳动物的C-FOS聚为一支,所有动物FOSAA聚为一支,翘嘴鳜FOSAB蛋白与哺乳类动物C-FOS亲缘关系比与FOSAA的关系更近。同线性分析显示,c-fos基因进化较为保守,处于tmed10和jdp基因之间。荧光定量分析表明,翘嘴鳜fosaa和fosab基因在脑和皮肤中均有较高的表达量,fosaa基因在脑中表达量最高,而fosab基因在皮肤中表达量最高。经饲料驯食后,驯化56 d时fosaa基因表达量显著高于驯化7 d(P<0.05),而驯化7、56 d的fosab基因相对表达量无显著差异(P>0.05)。【结论】翘嘴鳜fosaa和fosab基因分子特征差异较大,发挥的功能不尽相同,学习记忆相关基因fosaa在56 d饲料驯食中有重要作用。