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Acta Pharmaceutica Sinica B 2014年第2期4卷 161-166页

作者： Ying Liu Xiaoqing Zhu Wendong Li Hao Wen Ya Gao Yong Liu Chunsheng Liu School of Chinese Pharmacy Beijing University of Chinese MedicineBeijing 100102China Department of Antibiotics Beijing Institute for Drug ControlBeijing 100102China

The rate-limiting enzyme in the mevalonic acid(MVA)pathway which can lead to triterpenoid saponin glycyrrhizic acid(GA)is 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR).In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway,the HMGR gene from Glycyrrhiza uralensis Fisch.(GuHMGR)was cloned and over-expressed in Pichia pastoris *** recombinant *** strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by *** results showed that all the recombinant *** strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other ***,as the copy number increased,the content of ergosterol showed an increasing–decreasing–increasing *** study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of ***.

关键词： Glycyrrhiza uralensis Fisch. 3-Hydroxy-3-methylglu-taryl-CoA reductase gene over-expression Pichia pastoris Copy number variation

over-expression of VEGF165 in the adipose tissue-derived stem cells via the lentiviral vector

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Chinese Medical Journal 2011年第19期124卷 3093-3097页

作者： SUN Xiang-zhou LIU Gui-hua WANG Zhuo-qing ZHENG Fu-fu BIAN Jun HUANG Yan-ping GAO Yong ZHANG Ya-dong DENG Chun-hua Department of Urology First Affiliated Hospital of Sun Yet-sen University Guangzhou Guangdong 510080 China Department of Scientific Research First Affiliated Hospital of Sun Yet-sen University Guangzhou Guangdong 510080 China

Background Many researchers studied the possibility of using stem cells as gene therapeutic vector. But few related reports on the adipose tissue-derived stem cells （ADSCs） are available. Therefore we intended to construct a lentiviral VEGF165 expression vector and then infect the ADSCs to produce therapeutic seed *** EHS1001-68950485313912 clone was mutated by PCR method to produce consensus fragment of VEGF165 transcript （NM_001025368）. Lentivirus was enveloped with pGC-FU, pHelper 1.0 and pHelper 2.0 plasmids in 293T *** then the ADSCs （multiplicity of infection=20） were transfected with the vectors after titer determination. Stable expression of VEGF165 in ADSCs was confirmed by immunofluorescence staining, enzyme-linked immunosorbent assay （ELISA） and Western blotting *** DNA sequencing and 293T transfection verified VEGF165 was linked to the GFP fused vector. The virus titer is up to 2x10a determined by quantitative PCR. VEGF165 transduced cells could show green fluorescence confirmed by immunofluorescence staining （almost 95%）. ELISA analyses could detect out the density of VEGF was 850.86-1202.13pg/ml （mean （923.00±31.22） pg/ml） in the supernatant of VEGF16s-transduced cells but not detected in the GFP-transduced cells （P 〈0.001） and the Western blotting analyses also confirmed VEGF165 expression in VEGF165-transduced *** The VEGF165 over-expression ADSCs were obtained and may be used as a cell therapeutic tool and may be applied for vascular regeneration, especially in the treatment of erectile dysfunction.

关键词： adipose tissue-derived stem cells VEGF16s lentivirus over-expression

over-expression of Brassica napus MPK4 Enhances Resistance to Sclerotinia sclerotiorum

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Over-expression of Brassica napus MPK4 Enhances Resistance t...

over-expression of Ultrabithorax alters embryonic body plan and wing patterns in the butterfly Bicyclus anynana

中国植物病理学会2008年学术年会

作者： WANG Zheng MAO Han JI Rui-qing CAI Li LIU Sheng-yi

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus) resulting in significant economic losses. Studies on Arabidopsis MPK4 loss-of-function mutant have implicated that AtMPK4 is involved in plant defense regulation and its effect on disease resistance is various in different plant-pathogen interactions. Here, we isolated an oilseed rape MAP kinase, B. napus MPK4 (BnMPK4). BnMPK4 along with PDF1.2 are promptly inducible in a resistant line Zhangshuang 9, but suppressed in a susceptible line 84039 after inoculation with Sclerotinia sclerotiorum. Transgenic oilseed rape over-expressing BnMPK4 (BnMPK4ox) markedly enhanced resistance to S. sclerotiorum. BnMPK4ox inhibited growth of the pathogen and constitutively activated PDF1.2, but decreased H202 production and suppressed PR-1 expression constitutively. Our results supported the idea that MPK4 positively regulate jasmonic acid (JA)-mediated defense response, which plays an important role in resistance to S. sclerotiorum, and achieved the attempt that through altered regulation of plant defense mechanisms we efficiently improved resistance to S. sclerotiorum in oilseed rape, an important oil crops resource worldwide.

关键词： Brassica jasmonic acid MAP kinase over-expression Sclerotinia

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Over-expression of Ultrabithorax alters embryonic body plan ...

Protocol for Artificial MicroRNA Mediated over-expression of miR820 in Indica Rice

第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Xiaoling Tong Steven Hrycaj Ondrej Podlaha Aleksandar Popadic Antónia Monteiro Department of Ecology and Evolutionary Biology Yale University State Key Laboratory of Silkworm Genome Biology Southwest University Department of Biological Sciences Wayne State University Department of Biological Sciences National University of Singapore

In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A variety of RNAi experiments across insect species have shown that the hox gene Ultrabithorax（Ubx） is necessary to promote hindwing identity. However, it remains unclear whether Ubx is sufficient to confer hindwing fate to forewings across insects. Here, we address this question by over-expressing Ubx in the butterfly Bicyclus anynana using a heat-shock promoter. Ubx whole-body over-expression during embryonic and larvae development led to body plan changes in larvae but to mere quantitative changes to adult morphology, respectively. Embryonic heat-shocks led to fused segments, loss of thoracic and abdominal limbs, and transformation of head limbs to larger appendages. Larval heat-shocks led to reduced eyespot size in the expected homeotic direction, but neither additional eyespots nor wing shape changes were observed in forewings as expected of a homeotic transformation. Interestingly, Ubx was found to be expressed in a novel, non-characteristic domain – in the hindwing eyespot centers. Furthermore, ectopic expression of Ubx on the pupal wing activated the eyespot-associated genes spalt and Distal-less, known to be directly repressed by Ubx in the fly’s haltere and leg primordia, respectively, and led to the differentiation of black wing scales. These results suggest that Ubx has been co-opted into a novel eyespot gene regulatory network, and that it is capable of activating black pigmentation in butterflies.

关键词： Ultrabithorax over-expression Homeotic transformation Wing Eyespot Bicyclus anynana

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American Journal of Plant Sciences 2015年第12期6卷 1951-1961页

作者： Neha Sharma Sandeep Panchal Neeti Sanan-Mishra Plant Molecular Biology Group International Center for Genetic Engineering and Biotechnology New Delhi India

In the present study, we illustrate the strategy and protocol required to generate rice transgenics over-expressing the 21-nt form of Osa-miR820. The miR exists in two size variants of 21-nt and 24-nt so the natural precursor cannot be employed for the purpose of miR over-expression as the cellular machinery can process both size variants thereby masking the role of PTGS regulation. Hence, we adopted the artificial miR technology to specifically over-express the 21-nt species in the transgenics. During the course of experiments it was observed that the amiR constructs probably interfered with the regeneration of the transformed callus, necessitating protocol modifications. The results indicate the successful over-expression of the 21-nt miR species. These plants can serve as a useful source for the functional dissection of the role played by the 21-nt Osa-miR820 species. They will also be valuable in highlighting the importance for the existence of a dual mode of miR mediated target regulation.

关键词： Osa-miR820 over-expression Agrobacterium Artificial microRNA (amiR)

LAC2 down-regulated by miR-397a over-expression leads to hyper sensitization and less tolerance to salt in safflower

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LAC2 down-regulated by miR-397a over-expression leads to hyp...

LAC2 down-regulation by miR-397a over-expression leads to hyper sensitization and less tolerance to salt in safflower

2013全国植物生物学大会

作者： DONG Yuanyuan LIU Weican ZHOU Ying ZHOU Yonggang ZHAO Lidan YANG Shulin School of Environmental and Biological Engineering Nanjing University of Science and Technology Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development Jilin Agricultural University

miR-397a has been known to play a key role in plant development and in stress response by regulating mRNAs that code for the LAC2 in *** this study,we had verified that miR-397a was highly expressed in leaf of safflower by small RNA sequencing technology. LAC2 targeted by miR-397a was down-regulated when miR-397a plasmid was transferred and over-expressed in the protoplast of safflower by *** phenotypes illustrated LAC2 was negative regulated by miR-397a in *** observing functions of safflower miR-397a,miR-397a over-expressing Arabidopsis plants were *** new phenotypes of decreased percents of seeds germination,early flowering and reduced tolerance to salt were observed in the safflower miR-397a over-expressed Arabidopsis were *** data indicated that the phenotype of reduced tolerance to salt may be caused through hyper sensitization to the NaCl signal by reduced expression of LAC2 *** expression of LAC2 was down-regulated by over-expression of safflower miR-397a,which suggested that less salt tolerance of miR-397a over-expression Arabidopsis may be from down-regulation of LAC2, which may affect the transportation of *** phenotypes of reduced tolerance to salt could present a new phenomenon for molecular breeding.

关键词： microRNA tolerance over-expression salt sensitive

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LAC2 down-regulation by miR-397a over-expression leads to hy...

Lentivirus-mediated Persephin overexpression in Parkinson's disease rats

第十四届全国植物基因组学大会

作者： Yuanyuan Dong Ying Zhou Yonggang Zhou Lidan Zhao Shulin Yang School of Environmental and Biological Engineering Nanjing University of Science and Technology

关键词： microRNA tolerance over-expression salt sensitive

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Neural Regeneration Research 2015年第11期10卷 1814-1818页

作者： Xiao-feng Yin Hua-min Xu Yun-xia Jiang Yun-lai Zhi Yu-xiu Liu Heng-wei Xiang Kai Liu Xiao-dong Ding Peng Sun Department of Neurosurgery the Second Affiliated Hospital of Shanxi Medical University Department of Physiology Qingdao University Nursing College of Qingdao University Department of Pediatric Surgery Affiliated Hospital of Qingdao University Department of Nursing Affiliated Hospital of Qingdao University Department of Neurosurgery Affiliated Hospital of Medical College Qingdao University

Persephin, together with glial cell line-derived neurotrophic factor and neurturin, has a neurotrophic effect and promotes the survival of motor neurons cultured in vitro. In this study, dopaminergic neurons in the substantia nigra of rats were transfected with the Persephin gene. One week later 6-hydroxydopamine was injected into the anterior medial bundle to establish a Parkinson＇s disease model in the rats. Results found that the number of dopaminergic neurons in the substantia nigra increased, tyrosine hydroxylase expression was upregulated and concentrations of dopamine and its metabolites in corpus striatum were increased after pretreatment with Persephin gene. In addition, the rotating effect of the induced Parkinson＇s disease rats was much less in the group pretreated with the Persephin gene. Persephin has a neuroprotective effect on the 6-hydroxydopamine-induced Parkinson＇s disease through protecting dopaminergic neurons.

关键词： nerve regeneration Persephin lentivirus Parkinson's disease dopaminergic neurons gene therapy over-expression transfection striatum neural regeneration

Bzw2 Promotes Proliferation and Lactation of Mammary Epithelial Cell in Dairy Goat

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Journal of Integrative Agriculture 2012年第11期11卷 1884-1891页

作者： SUN Rui-qiu LI Qing-zhang YAN Hong-bo ZHAO Jing GAO Xue-jun Key Laboratory of Dairy Science Ministry of Education/College of Life ScienceNortheast Agricultural UniversityHarbin 150030P.R.China

Mitosis of mammary epithelial cell is foundation of mammal lactation. We developed a strategy of combined application of generation of longer cDNA fragments from the serial analysis of gene expression （SAGE） tags for gene identification （GLGI） to screen and identify genes influencing lactating ability of mammary epithelial cell in dairy goat. GLGI as a new tag identification technique was brought about with SAGE. Bzw2 was found as a candidate gene related to lactation by screening Long-SAGE library of mammary gland in dairy goat. Bzw2 cDNA was synthesized by switching mechanism at 5＂-end of RNA transcript （SMART） technology. The mRNA level of Bzw2 was relatively higher in early lactation than in other development stages of mammary gland. The proliferation of mammary epithelial cell was inhibited by transfecting specific shRNA of Bzw2. The mRNA levels of Stat5, Csn2 and Prlr were also down-regulated, suggesting the lactating ability of mammary epithelial cell was attenuated after Bzw2 RNAi. The reduction of mammary epithelial cell growth and lactation by Bzw2 RNAi was rescued through over-expression of Bzw2. These results revealed that Bzw2 might play an important role in lactation though the molecular mechanism was still unclear.

关键词： Bzw2 mammary epithelial cells proliferation lactation RNAi over-expression

Enhancing ergosterol production in Pichia pastoris GS115 by overexpressing squalene synthase gene from Glycyrrhiza uralensis

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Chinese Journal of Natural Medicines 2015年第5期13卷 338-345页

作者： LIU Ying ZHU Xiao-Qing LI Wen-Dong WEN Hao LIU Chun-Sheng School of Chinese Pharmacy Beijing University of Chinese Medicine Beijing Institute for Drug Control

The present study was designed to determine the effects of copy number variations(CNVs) of squalene synthase 1(SQS1) gene on the mevalonate(MVA) ***1 gene from ***(Gu SQS1) was cloned and over-expressed in Pichia pastoris *** recombinant *** strains containing different copy number of Gu SQS1 were *** was used to assay the level of ergosterol in all transgenic *** strains containing Gu *** analysis showed that the contents of ergosterol in all of the transgenic *** containing Gu SQS1 were higher than that in the negative *** with the increase of copy number of Gu SQS1,the content of ergosterol showed an increasing-decreasing-increasing *** contents of ergosterol in 10-copy-Gu SQS1 *** and 47-copy-Gu SQS1 *** were significantly higher than that in the rest recombinant *** *** conclusion,the CNVs of Gu SQS1 influence the content of secondary metabolites in the MVA *** present study provides a basis for over-expressing Gu SQS1 and increasing the content of glycyrrhizin in *** cultivars.

关键词： Glycyrrhiza uralensis GuSQS1 over-expression Pichia pastoris Copy number variations