【作者】 王天云；

【导师】 薛乐勋；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2005， 博士

【摘要】 核基质结合区(matrix attachment regions, MARs)又叫核骨架附着区(scaffold attachment regions, SARs)是染色质被限制酶消化后仍与核基质或核骨架结合的DNA序列。MAR长度由300bp至2,000bp不等,通常富含AT碱基对,常含有一些特征性基序(motifs),如A-box(AATAAAAA/CAA)、T-box(TTTTATTTTT)、酵母自主复制序列(autonomously replicating DNA sequence, ARS)、果蝇拓朴异构酶Ⅱ(Topoisomerase Ⅱ)识别位点和能形成蛋白质识别位点的松散DNA(unwinding DNA)、弯曲DNA(curved DNA)等。MARs与核基质结合受AT区的位置及结构影响,其二级结构表现为狭窄的DNA小沟,易于弯曲和解链。MAR一般位于功能转录单位的侧翼,作为一种边界元件(boundary elements),但也有一些MAR位于某些基因的内含子中。虽然MAR具有一些序列特征,但比较不同MAR的碱基序列,发现MAR在碱基组成上并不具保守性。近年来研究发现,将MAR构建表达载体能提高外源基因的表达水平,增强外源基因表达的稳定性,克服外源基因沉默等。MAR还能使染色质形成环状结构,还可以作为DNA复制的起始点或调控基因的转录。但关于MAR的研究尚处于早期阶段,其分子调控机制目前仍不清楚。 迄今已有一些MAR从酵母、动物、植物及人类中分离出来,并进行了其功能的相关研究,但目前所分离的MAR绝大多数来源于高等动植物,单细胞的真核生物研究的较少,真核藻类的MAR研究未见相关报道。高等动植物存在器官组织分化问题,不同的分化组织细胞基因表达的方式及其种类不同,MAR序列也可能存在差别。因此以单细胞的真核生物研究MAR的调控机制更具有特点及优势。杜氏盐藻(Dunaliella salina)是一种无细胞壁的单细胞绿藻,含有一个大的杯状叶绿体进行光合作用,具鞭毛,能游动,可在0.05～5M NaCl培养液中更多还原

【Abstract】 Matrix attachment regions (MARs) or scaffold attachment regions (SARs) are DNA fragments that still bind to the nuclear matrices or scaffold after chromatin digested with restriction endonuclease. MARs, usually 300~2000bp long with AT-rich, contain some sequence motifs, such as A-box(AATAAAAA/CAA),T-box (TTTTATTTTT), autonomously replicating DNA sequence(ARS), topoisomerase, unwinding DNA, curved DNA consensus sequence recognizing sites, etc. The binding of MARs to the nuclear matrices is affected by the position and structure of AT bases, and their second structures are narrow DNA grooves, which easily curve or unwind. MARs are commonly located in the flanking sites of functional transcription units as boundary elements, some in the introns. MARs have some typical sequence features, but the sequences are not conserved. It has been demonstrated that MARs increase transgene expression, enhance transgene stability and prevent transgene silencing when they are used to construct the expression vectors, and also make chromatin form such loop structure as DNA replication origin site or regulate gene transcription. However, the studies on MARs are still in the early phase, and the regulation mechanisms are unclear.So far, a few MARs have been isolated from yeast, plants, animals and human, but not from unicellular algae. Because the gene expression and regulation matter of the differential organs and tissues are diverse in higher plants and animals, the MARs may be different So it is very interesting to study the MARs of single cell eukaryotic organisms. Dunaliella salina is a kind of single green alga without cell wall, contains a big cup chloroplast, can grow between the concentration of 0.05M~5M NaCl. D. salina has more advantages than other plant organisms as bioreactor producingpharmaceutical proteins. The aim of this study is to isolation MARs from D.salina and study their effects on transgene, provide some experimental bases for the study the effect and mechanism of MARs in theoretic, and do some exploring for the establishing the steady and effective D. salina expression systems.In the present study, the MARs were isolated from D. salina and their functions on transgene expression were also studied. Firstly, we prepared D.salina nuclear matrices through diiodosalicylic acid lithium salt (LIS) extraction of D.salina cell nuclei, restriction enzymes digestion. Secondly the nuclear matrices DNAs were extracted by SDS-proteinase K, and then digested with restriction enzymes and inserted into the pUC18 vector cut with the corresponding restriction enzymes to construct the D.salina randomly MAR libraries. The libraries were screened through in vitro binding assay, three DNA fragments were found to bind specifically to the nuclear matrices in vitro. Sequence analysis showed they all had the typical MAR features, such as AT-rich, A-box, T-box sequence motifs etc. The MARs were cloned into the both flanking regions of D.salina expression vector pRNC, which constructed by our laboratory containing chloraphericol acetyltransferase (CAT) reporter gene, to generate the D.salina expression vectors pcDMM-1, pcDMM-2 and pcDMM-3. The plasmids were co-electroporated with selective pSP-Bl vector containing the bar gene into D.salina cells, and the stably transformed D.salina alga strains were obtained via phosphinothricin (PPT) and chloramphenicol screening. When the cells grew to the logarithmic growth phase, CAT reporter gene expressions were analyzed by ELISA methods, the result showed that mean CAT enzyme activities were increased 1.5, 4.5 and 2.2 fold as compared to that of transformants without MARs. It was demonstrated that the CAT gene was integrated into the transgenic D.salina genomic DNA via Southern blotting, and Northern blotting suggested CAT gene could transcribe and express at RNA levelsPart I: Isolation and characterization of matrix attachment region from Dunaliella salina1 Methods:1.1 Preparation of D.salina cell nucleiThe D.salina cells in mid-logarithmic phase were disrupted with 0.5% TritonX-100, purified by 15% Percoll, and screened by microscope observation.1.2 Preparation of D.salina cell nuclear matrices and characterization of nuclear matrices proteinD.salina cell nuclear matrices were prepared through 25mM LIS extraction, followed the nuclear matrices proteins were identified by 10% SDS-PAGE electrophoresis. The nuclear matrices were digested with EcoBl overnight, centrifuged and removed the DNA not binding the nuclear matrices closely. 13 Extraction of nuclear matrices DNA and construction of MAR libraryNuclear matrices DNA were extracted by SDS-proteinase K, digested by restriction enzymes EcoRI, HindUl, Taql and Rsal, ligated to the pUC18 vector cut with the corresponding enzymes, transformed into E.coli JM109 cells and picked up the positive colonies.1.4 In vitro binding assaysThe positive colonies were picked and cultured in liquid medium overnight, the resultant plasmids were extracted and the inserted DNA fragments and pUC18 vector were released through restriction enzymes digestion. Both the pUC18 and the inserts themselves were labeled by Horseradish Peroxidase (HRP), and then bound to the prepared nuclear matrices completely. The DNA bound to the nuclear matrices and existed in supernatant were all extracted again, analyzed by agarose gel electrophoresis and fixed by TCA, finally the gels were dried and exposed to X-ray film.1.5 Sequencing of DNA fragmentsDNA sequencing was performed by Shanghai Sangon Biological Engineering & Technology services Co., Ltd (China). Data were analyzed by the DNA sequencing analysis software primer5.0, BioEdit and MAR-wizfhttp://www.futursoft.ore/MAR-wiz).2 Results:2.1 Preparation of D.salina cell nuclei0.5 %Triton X-100 could efficiently lyse the membranes of D. salina cells, and over 90% of the cells were at the bottom of tube through 15% Percoll purification. Microscopic observation showed that the structures of isolation D.salina cell nuclei were conserved well, and the nucleolus could be seen clearly, indicating that the purified nuclei of D.salina were obtained through the isolation procedure.2.2 Preparation of D.salina cell nuclear matrices and characterization of nuclear matrices proteinNearly all histone and nonhistone chromosomal proteins from D. salina were removed at 25mM LIS concentration through analysis by 10 % SDS polyacrylamide gels electrophoresis. The histones were removed specifically thoroughly, indicating the nuclear matrices could be prepared through 25mM LIS extraction. 23 Extraction of nuclear matrices DNA and construction of MAR libraryThe nuclear matrices DNAs were extracted and digested by restriction enzymes then ligated to pUC18 vector to construct the D.salina MAR libraries, 56 positive colonies were obtained. 2.4 In vitro binding assaysThree DNA fragments were found to bind to nuclear matrices via in vitro binding assay. 2.5 Sequencing of DNA fragmentsThe sequences of three DNA fragments have the MAR typical features, 62.5% AT, and contain A-box, T-box, Topoisomerase II sites motifs etc.Part II: Studies of the Effects of D.salina MARs on TransgeneExpression 1 Methods:1.1 Construction of D.salina expression vector containing MARsThe D.salina MARs were amplified through PCR using primers designed according to the GenBank sequence, inserted into pMD18-T vector and sequenced. The pMD18 T-MAR plasmid were digested with BamHUSaR and kpnVSaR to release the inserted MARs, and then cloned into the D.salina expression vector pRNC to construct the D.salina expression vector pcDMM, which flanked by the MARs in the flanking regions of expression cassettes. The resultant pcDMM vectors were identified through restriction enzymes digestion and agarose gel electrophoresis.12 Transformed into D.salina and screened the stably transformed D.salina strainsD.salina cells were collected in the logarithmic growth phase, and the cells density was adjusted to 107 cells/ml. Plasmid pcDMM and pSP-Bl were added to the cell suspension and co-electroporated. The cells were immediately cultured in PKS liquid medium for 12 h darkness and 24 h with light after transformation, and then added PPT to the concentration of 1.5ug/ml, followed two days later the PPT concentration increased to 3ug/ml, and then after a week the D.salina cells were spread on solid selective medium containing 3|xg/ml PPT and 60ug/ml chloramphenicol. When single small green alga colonies appeared, they were picked into liquid medium containing 3 u g/ml PPT.13 Analysis of CAT reporter gene expressionWhen the D.salina cells cultured to the logarithmic growth phase in the selective liquid medium, the CAT reporter gene expression levels were analyzed by ELISA method. 1.4 PCR detection and Southern blottingThe transformed D.salina strains genomic DNAs were extracted, and CAT gene was amplified through PCR. The genomic DNA was digested with restriction enzymes, separated by agarose gel electrophoresis and transferred to a Hybond N+ nylon membrane, and hybridized with the CAT gene labeled by Dig, analyzed if itwas integrated into the transgenic D.salina genomic DNA. 1.5 Northern blottingThe total RNA of transformed D.salina strains was extracted, separated on 1.0 agarose gel containing 0.1% formaldehyde, then blotted onto Hybond N+ nylon membrane and labeled with CAT gene probe.2 Results:2.1 Construction of D.salina expression vector containing MARsThe D.salina MARs were amplified through PCR and sequenced. The D.salina expression vectors pcDMM-1, pcDMM-2 and pcDMM-3 containing MARs were successfully constructed, and they were identified to be right through restriction enzyme digestion and agarose electrophoresis.2.2 Transformed into D.salina and screened the stably transformed D.salina strainsThe expression vectors pcDMM-1, pcDMM-2 and pcDMM-3 and the selective vector pSP-Bl were co-transformed into D.salina by electroporation. The positive transformed D.salina alga strains were screened via PPT selection and the liquid cultured D.salina algae were obtained. 23 Analysis of CAT reporter gene expression levelCAT enzyme activities of transformed D.salina were assayed by ELISA methods, and the result showed the mean CAT enzyme activities of the vectors containing MARs were higher than the control vectors 1.5-fold, 4.5-fold and 2.2-fold.2.4 PCR detection and Southern blotting analysisPCR and Southern blotting demonstrated that CAT gene was integrated into the transformed D.salina genome DNA.2.5 Northern blotting analysisThe results of Northern blotting showed that the CAT gene could transcribe and express at RNAtranscriptional level.Conclusions:1 Three DNA fragments having the typical features of MARs have been first isolated from D.salina and they can bind to the nuclear matrices in vitro and have2 The CAT reporter gene can be expressed stably in transgenic D.salina.3 The three isolated three MARs can increase the CAT gene expression with 1.5-4.5-fold as compared that of without MARs.4 There is no relationship between the effect of MARs and the binding strength to nuclear matrices in vitro.5 The CAT gene has been integrated into the transgenic D.salina genomic DNA demonstrated by PCR and Southern blotting..6 Northern blotting has shown that CAT gene can be transcribed and expressed at RNA level.更多还原