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Cloning and expression of swine myostatin gene and its application in animal immunization trial

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Science China(Life Sciences) 2005年第4期48卷 368-374页

作者： MA Xianyong CAO Yongchang SHU Dingming BI Yingzuo Genetic Engineering Laboratory College of Animal Science South China Agricultural University Guangzhou 510642 China Genetic Engineering Laboratory College of Animal Science South China Agricultural University Guangzhou 510642 China Genetic Engineering Laboratory College of Animal Science South China Agricultural University Guangzhou 510642 China Genetic Engineering Laboratory College of Animal Science South China Agricultural University Guangzhou 510642 China

We have amplified swine myostatin (MSTN) gene by reverse transcription poly-merase chain reaction (RT-PCR) and cloned it into pGEM-T Easy vector. The cloned swine MSTN gene consists of 1128 nucleotides, which has been submitted to GenBank (acquired reg-istered code- AY448008). The cloned swine MSTN gene was successfully expressed in E. coli without the first 25 amino acids. Crude extraction of the expressed recombinant MSTN protein was used to immunize mice to investigate the effects on their bodyweights. We show here that the body weights of the immunized mice were higher than that of the controls, even though the difference was not significant. Surprisingly, the progenies of the immunized mice also were heavier than the controls. Especially at day 3, the average body weight of the immunized mice was 10.5% higher than that of the controls , which is significant (p 0.05).

关键词： myostatin, gene cloning, sequencing, protein expression, detection, immunization.

Gene cloning and expression of cadherin in midgut of Helicoverpa armigera and its Cry1A binding region

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Science China(Life Sciences) 2005年第4期48卷 346-356页

作者： WANG Guirong WU Kongming LIANG Gemei GUO Yuyuan State Key Laboratory of Plant Disease and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences Beijing 100094 China State Key Laboratory of Plant Disease and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences Beijing 100094 China State Key Laboratory of Plant Disease and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences Beijing 100094 China State Key Laboratory of Plant Disease and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences Beijing 100094 China

Cadherins belong to one of the families of animal glycoproteins responsible for cal-cium-dependent cell-cell adhesion. Recent literatures showed that the cadherin-like in midgut of several insects served as the receptor of Bt toxin Cry1A and the variation of cadherin-like is re-lated to insect’s resistance to Cry1A. The full-length cDNA encoding cadherin-like of Helicoverpa armigera is cloned by degenerate PCR and RACE techniques and the gene was designated as BtR-harm, which is 5581 bp in full-length, encoding 1730 amino acid residues (BtR-harm was deposited in GenBank and the accession number is AF519180). Its predicted molecular weight and isoelectric point were 195.39 kDa and 4.23, respectively. The inferred amino acid sequence includes a signal sequence, 11 cadherin repeats, a membrane-proximal region, a transmem-brane region and a cytoplasmic region. Sequence analysis indicated that the deduced protein sequence was most similar to the cadherin-like from Heliothis virescens with 84.2% identity and highly similar to three other lepidopteran cadherin from Bombyx mori, Manduca sexta and Pectinophora gossypiella, with the sequence identities of 60.3.6%, 57.5% and 51.0%, respec-tively. The cDNA encoding cadherin gene was expressed successfully in E. coli and the recom-binant proteins can bind with Cry1Ac. Truncation analysis and binding experiment of BtR-harm revealed that the Cry1A binding region was a contiguous 244-amino acid sequence, which lo-cated between amino acid 1217 and 1461. Semi-quantitative RT-PCR analysis showed that BtR-harm was highly expressed in midgut of H. armigera, very low expressed in foregut and hindgut and was not expressed in other tissues. After H. armigera producing resistance to Cry1Ac, the expression quantity of BtR-harm significantly decreased in midgut of H. armigera. It is the first confirmation that BtR-harm can function as receptor of Cry1Ac in H. armigera and the binding region was located on a contiguous 244 amino acid sequence,

关键词： BtR-harm, Bt receptor, gene cloning and expression, Helicoverpa armigera.

Effects of clinorotation on COL1A1- EGFP gene expression

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Science China(Life Sciences) 2004年第3期47卷 203-210页

作者： DAI Zhongquan1, LI Yinghui1, DING Bai1, ZHANG Yuguo2, LIU Weiquan2 & LIU Pengpeng2 1. Laboratory of Space Cellular and Molecular Biology, Institute of Space Medico-Engineering, Beijing 100094, China 2. College of Biological Science, Chinese Agriculture University, Beijing 100094, China Laboratory of Space Cellular and Molecular Biology Institute of Space Medico-Engineering Beijing 100094 China College of Biological Science Chinese Agriculture University Beijing 100094 China

Bone-formation related gene plays a critical role in bone loss induced by space mi-crogravity, however the exact mechanism is unclear. In this study, we aim to investigate the effect of microgravity on the activity of ?1(I) collagen (COL1A1) gene promoter and the expression of osteoblast-related genes. COL1A1 promoter was digested by restriction enzymes resulting in three DNA fragments. The fragments were ligated with the enhanced green fluorescent protein report gene, and subcloned into expression vectors. ROS17/2.8 cells transfected by these vec-tors were screened by G418, and enhanced green fluorescent protein (EGFP) positive colonies were isolated and cultured under clinostat condition. EGFP and Collagen type I expression level were detected by fluorescence intensity analysis and immunocytochemistry methods respectively. The results showed that the expression of EGFP and collagen type I was increased 24 h, 48 h after the cells were cultured under stimulated microgravity, illustrating that the activity of COL1A1 promoter might be increased. In conclusion, osteoblasts can compensatively increase the ex-pression of type I collagen by enhancing the activity of COL1A1 promoter under short-term simulated microgravity conditions.

关键词： simulated microgravity, osteoblast, gene expression, COL1A1 promoter.

Gene expression and molecular characterization of a thermostable trehalose phosphorylase from Thermoanaerobacter tengcongensis

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Science China(Life Sciences) 2005年第3期48卷 221-227页

作者： REN Yuanyuan 1,2 , DAI Xiuyu 1 , ZHOU Jian 1 , LIU Jingfang 1 , PEI Huadong 1,2 & XIANG Hua 1 1. State Key Laboratory of Microbial Resources and Center for Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China 2. Graduate School of Chinese Academy of Sciences, Beijing 100037, China State Key Laboratory of Microbial Resources and Center for Molecular Microbiology Institute of Microbiology Chinese Academy of Sciences Beijing100080 China Graduate School of Chinese Academy of Sciences Beijing100037 China State Key Laboratory of Microbial Resources and Center for Molecular Microbiology Institute of Microbiology Chinese Academy of Sciences Beijing100080 China

A gene encoding the trehalose phosphorylase (TreP), which reversibly catalyzes trehalose degradation and synthesis from α-glucose-1-phosphate (α-Glc-1-P) and glucose, was cloned from Thermoanaerobacter tengcongensis and successfully expressed in Escherichia coli. The overexpressed TreP, with a molecular mass of approximately 90 kDa, was determined by SDS-PAGE. It catalyzes trehalose synthesis and degradation optimally at 70℃ (for 30 min), with the optimum pHs at 6.0 and 7.0, respectively. It is highly thermostable, with a 77% residual ac- tivity after incubation at 50℃ for 7 h. Under the optimum reaction conditions, 50 μg crude en- zyme of the TreP is able to catalyze the synthesis of trehalose up to 11.6 mmol/L from 25 mmol/L α-Glc-1-P and 125 mmol/L glucose within 30 min, while only 1.5 mmol/L out of 250 mmol/L tre- halose is degraded within the same time period. Dot blotting revealed that the treP gene in T. tengcongensis was upregulated in response to salt stress but downregulated when trehalose was supplied. Both results indicate that the dominant function of the T. tengcongensis TreP is catalyzing trehalose synthesis but not degradation. Thus it might provide a novel route for indus- trial production of trehalose.

关键词： gene expression, Thermoanaerobacter tengcongensis, trehalose phosphorylase, dot blot.

expression and function of leptin and its receptor in mouse mammary gland

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Science China(Life Sciences) 2007年第5期50卷 669-675页

作者： LIN Ye LI QingZhang Key Laboratory of Dairy Science of Ministry of Education Northeast Agricultural University Harbin 150030 China Key Laboratory of Dairy Science of Ministry of Education Northeast Agricultural University Harbin 150030 China

Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lacta-tion-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway.

关键词： mammary gland, leptin, OB-Rb, expression, signal transduction

Comparative analysis of gene expression at early seedling stage between a rice hybrid and its parents using a cDNA microarray of 9198 uni-sequences

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Science China(Life Sciences) 2006年第6期49卷 519-529页

作者： HUANG Yi LI Lihua CHEN Ying LI Xianghua XU Caiguo WANG Shiping ZHANG Qifa National Key Laboratory of Crop Genetic Improvement National Center of Plant Gene Research Wuhan Huazhong Agricultural University Wuhan 430070 China National Key Laboratory of Crop Genetic Improvement National Center of Plant Gene Research Wuhan Huazhong Agricultural University Wuhan 430070 China National Key Laboratory of Crop Genetic Improvement National Center of Plant Gene Research Wuhan Huazhong Agricultural University Wuhan 430070 China National Key Laboratory of Crop Genetic Improvement National Center of Plant Gene Research Wuhan Huazhong Agricultural University Wuhan 430070 China National Key Laboratory of Crop Genetic Improvement National Center of Plant Gene Research Wuhan Huazhong Agricultural University Wuhan 430070 China National Key Laboratory of Crop Genetic Improvement National Center of Plant Gene Research Wuhan Huazhong Agricultural University Wuhan 430070 China National Key Laboratory of Crop Genetic Improvement National Center of Plant Gene Research Wuhan Huazhong Agricultural University Wuhan 430070 China

Using a cDNA microarray consisting of 9198 expressed sequence tags,we surveyed the gene expression profiles in shoots and roots of a rice hybrid,Liangyoupei 9 and its parents Peiai 64s and 93-11 at 72 h after germination. A total of 8587 sequences had detectable signals in both shoots and roots of the three genotypes. A total of 1571 sequences exhibited significant (Pexpression differences in shoots or roots among the three genotypes,of which 121 showed expression poly-morphisms in both shoots and roots,and 870 revealed significant expression differences between the hybrid and one of the parents. The expression polymorphism of the sequences was associated with the functional categories of the sequences. They occurred more frequently in categories of carbohy-drate,energy and lipid metabolisms and stress response than expected,while less frequently in categories of amino acid metabolism,transcription and translation regulation,and signal transduction. A total of 214 sequences exhibited significant (Pexpression,of which 117 had homology to genes with known functions,assigned in the categories of basic metabolism,genetic information processing,cell growth and death,signal transduction,transportation and stress response. The results may provide useful information for exploring the relationship between gene expression polymorphism and phenotypic variation,and for characterizing the molecular mechanism of seedling development and heterosis in rice.

关键词： Oryza sativa shoots, roots, gene expression, microarray, heterosis.

Characterization and developmental expression of AmphiMef2 gene in amphioxus

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Science China(Life Sciences) 2007年第5期50卷 637-641页

作者： ZHANG Ying WANG LiFeng SHAO Ming ZHANG HongWei Institute of Developmental Biology Life Science College Key Lab of Experimental Teratology of Ministry of Education Shandong University Jinan 250100 China Institute of Developmental Biology Life Science College Key Lab of Experimental Teratology of Ministry of Education Shandong University Jinan 250100 China Institute of Developmental Biology Life Science College Key Lab of Experimental Teratology of Ministry of Education Shandong University Jinan 250100 China Institute of Developmental Biology Life Science College Key Lab of Experimental Teratology of Ministry of Education Shandong University Jinan 250100 China

Myocyte enhancer factor 2 proteins are members of MADS family of transcription factors, which can control the expression of muscle-specific genes in vertebrates. However, not all Mef2 genes are es-sential for muscle development in invertebrates. Here we have isolated a full-length cDNA from am-phioxus, designated AmphiMef2. The predicted amino acid sequence has highly conserved MADS and MEF2 domains, showing higher identity with the corresponding regions of its homologues in verte-brates than those in invertebrates. Results from whole-mount in situ hybridization show that the ex-pression of AmphiMef2 initially appears in the presomitic mesoderm at early neurula stage, then the transcripts are detected in both the somites and the unsegmented presomitic mesoderm. At 36 h larval stage, the expression is only detected in the posterior somites. By 48 h larval stage, the expression is shifted to the preoral pit (a homologous organ to the vertebrate adenohypophysis) and persists until at least 72 h larval stage. The results suggest that AmphiMef2 may be not only involved in the myogenesis but also the development or function of the preoral pit in amphioxus.

关键词： AmphiMef2, amphioxus, developmental expression, myogenesis, preoral pit

Polynomial analysis of canopy spectra and biochemical component content inversion

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Science China Earth Sciences 2005年第10期48卷 1780-1791页

作者： YAN Chunyan1, LIU Qiang1, NIU Zheng1, WANG Jihua2, HUANG Wenjiang2 & LIU Liangyun 2 1. State Key Laboratory of Remote Sensing Science, Jointly Sponsored by the Institute of Remote Sensing Applications of Chinese Academy of Sciences and Beijing Normal University, Beijing 100101, China 2. National Engineering Research Center for Information Technology in Agriculture, Beijing 100089, China Jointly Sponsored by the Institute of Remote Sensing Applications of Chinese Academy of Sciences and Beijing Normal University National Engineering Research Center for Information Technology in Agriculture

A polynomial expression model was developed in this paper to describe directional canopy spectra, and the decomposition of the polynomial expression was used as a tool for re-trieving biochemical component content from canopy multi-angle spectra. First, the basic formula of the polynomial expression was introduced and the physical meaning of its terms and coeffi-cients was discussed. Based on this analysis, a complete polynomial expression model and its decomposition method were given. By decomposing the canopy spectra simulated with SAILH model, it shows that the polynomial expression can not only fit well the canopy spectra, but also show the contribution of every order scattering to the whole reflectance. Taking the first scattering coefficients a10 and a01 for example, the test results show that the polynomial coefficients reflect very well the hot spot phenomenon and the effects of viewing angles, LAI and leaf inclination angle on canopy spectra. By coupling the polynomial expression with leaf model PROSPECT, a canopy biochemical component content inversion model was given. In the simulated test, the canopy multi-angle spectra were simulated by two different models, SAILH and 4-SCALE re-spectively, then the biochemical component content was retrieved by inverting the coupled polynomial expression + PROSPECT model. Results of the simulated test are promising, and when applying the algorithm to measured corn canopy multi-angle spectra, we also get relatively accurate chlorophyll content. It shows that the polynomial analysis provides a new method to get biochemical component content independent of any specific canopy model.

关键词： polynomial expression, canopy spectra, decomposition, biochemical component content, inversion.

Internal signal stochastic resonance of a synthetic gene network

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Science China Chemistry 2005年第3期48卷 189-194页

作者： WANG Zhiwei HOU Zhonghuai XIN Houwen Department of Chemical Physics University of Science and Technology of China Hefei 230026 China Department of Chemical Physics University of Science and Technology of China Hefei 230026 China Department of Chemical Physics University of Science and Technology of China Hefei 230026 China

The dynamics behavior of a synthetic gene network controlled by random noise is investigated using a model proposed recently. The phenomena of noise induced oscillation (NIO) of the protein concentrations and internal signal stochastic resonance (SR) are studied by com-puter simulation. We also find that there exists an optimal noise intensity that can most favor the occurrence of effective oscillation (EO). Finally we discuss the potential constructive roles of SR on gene expression systems.

关键词： noise, noise-induced oscillation, stochastic resonance, gene expression, synthetic gene network.