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Construction of the subtracted cdna library of Striatal Neurons Treated with Long-term Morphine

Chinese Medical Sciences Journal 2011年第1期26卷 54-59页

作者： Bo Bai Hai-qing Liu Jing Chen Ya-lin Li Hui Du Hai Lu Peng-li Yu Department of Neurobiology Jining Medical College Jining 272067 China Department of Physiology Taishan Medical College Taian 271000 China

Objective To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization （SSH）. Methods Sbtracted cdna libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons （10^-5 mol/L for 72 hours）. To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein （CREB） mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons （62.85± 1.98） compared with normal striatal neurons （28.43 ± 1.46, P〈0.01）. Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis. And the 36 clones showed homology with 19 known genes and 2 novel genes. The expression of 2 novel genes, mitochondrial carrier homolog 1 （Mtchl ; 96.81±2.04 vs. 44.20±1.31, P〈0.01） and thyrnoma viral proto-oncogene 1 （Akt1 ; 122.10±2.17 vs 50.11±2.01, P〈0.01）, showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons. Conclusions A reliable differential cdna library of striatal neurons treated with long-term morphine is constructed. Mtchl and Aktl might be the candidate genes for the development of morphine tolerance.

关键词： neuron morphine tolerance suppression subtractive hybridization subtracted cdna library differential gene expression

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Construction and analysis of a subtracted cdna library of Betula platyphylla female inflorescence

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Journal of Forestry Research 2005年第2期16卷 97-100页

作者： WEIJi-cheng YANGChuan-ping WANGChao JIANGJing ForestryCollege NortheastForestryUniversityHarbin150040P.R.China ForestryCollege NortheastForestryUniversityHarbin150040P.R.China／／MudanjiangTeachersCollegeMudanjiang157012PR.China

Female inflorescence of Betula platyphylla was sampled at an interval of eachtwo days to analyze the background of gene expression in floral phase. On the basis of SMARTstrategy, the driver cdna was obtained from total RNA of the last sample and the tester cdna wasfrom that of the others by RT-PCR which were subsequently used to construct a subtracted cdnalibrary. The result of the ESTs (expression sequence tags) blastX showed that the genes in thesubtracted cdna library could be mainly clustered into 5 groups related to metabolism,transportation and signal transduction, cell cycle, stress response, and regulation. Therelationship between gene expression and development was also discussed.

关键词： betula platyphylla subtracted cdna library SMART

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Cloning and characterization of a novel gene, LRRP1, related with liver regeneration

Cloning and characterization of a novel gene, LRRP1, related...

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中国细胞生物学学会第八届会员代表大会暨学术大会

作者：徐存拴袁金云杨柯金张安世沈国民邢雪琨李玉昌张会勇林俊堂章静波 Sahman Rahman College of Life Sciences Henan Normal University Xinxiang 453002 China Key Laboratory for Cell Differentiation Regulation built by Province and Ministry Xinxiang 453002 China Homophilita Research Center London UniversityLondon SE17EHUnited Kingdom

The mammal liver has a very strong regeneration capacity after partial hepatectomy (PH) (Michalopoulos & Defrance, 1997;Fausto, 2000). It was confirmed that lots of genes participate in liver regeneration (Fukuhara et al, 2003;Yang et al, 1999). To further learn the genes related with liver regeneration (LR), we cloned a novel cdna full-length from a subtracted cdna library of regenerating rat liver following short interval successive partial hepatectomy (SISPH), whose accession number was AF509819 from GenBank, named gene related with liver regeneration 1 (LRRP1). Sequence analysis showed that the cdna held 2337 bases containing an open reading frame (ORF) encoding a protein of 500 amino acids (ca.55 kD a ). A comparison of base sequence of the cdna and amino acid sequence of the protein with GenBank Database to perform homologous analysis showed that its base sequence and amino acid sequence were highly homologous to the 3’-terminal 1000 bases and N-terminal 300 amino acids of human T-cell lymphotropic/leukemia binding protein (TxBp151). But they are not the same gene and protein, because their gene structures are different. The in situ hybridization analysis found that 3 peaks of its mRNA content appeared respectively at 2h, 36h and 72h after PH and 2 peaks did at 36-40h and 72h in SISPH. The microarray analysis showed that a high peak of its mRNA content appeared at 2h and 72h after PH and 72h in SISPH, respectively. The expression product of the gene was generated by prokaryotic fusion system and the corresponded antiserum was prepared by immunizing rabbit with the fusion antigen. The inmmunohistochemistry analysis revealed that this protein was located in cell nuclear and increased 3-5 folds in the regenerating rat liver of 4h and 44-48h in SISPH and after PH. It was existed in the cell nuclears of the rat embryo of morula phase, in that of the embryophoric membrane of 5-7 day rat embryo, in that of cerebrum and vertebra bone of 8-15 day rat embryo and in that

关键词： subtracted cdna library Complementary DNA microarray Gene related with liver regenration 1 (LRRP1) Partial hepatectomy (PH) Short interval successive partial hepatectomy (SISPH) Liver regeneration (LR)

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