共查询到18条相似文献，搜索用时 109 毫秒
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牡丹ACC氧化酶基因的克隆与反义载体的构建 总被引：1，自引：0，他引：1
根据报道的牡丹ACC氧化酶基因(DQ337251)cDNA序列,设计一对特异引物,以牡丹品种"洛阳红"基因组DNA为模板,用PCR扩增方法克隆出牡丹ACC氧化酶基因的部分片段,并将其连接到pMD18-T载体上进行测序.结果表明,克隆的序列全长为467 bp,其中包括一个长度为157 bp的序列,推测它可能是一个内含子,其它序列与已报道序列同源性为98.2%;用SacⅠ和XbaⅠ对重组质粒和载体pBI 121酶切、连接,构建牡丹ACC氧化酶基因的反义表达载体. 相似文献
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甜瓜蔗糖磷酸合成酶基因全克隆及工程载体的构建 总被引：1，自引：0，他引：1
蔗糖磷酸合成酶在甜瓜果实蔗糖合成途径中起关键性的调节作用,克隆该基因并导入甜瓜低糖自交系,可改良甜瓜果实品质创新优良种质。从甜瓜幼苗叶片提取总RNA,利用RT-PCR与Southern blot方法相结合,重组子质粒经限制性内切酶酶切和PCR验证以及测序同源性比较,克隆到基因的5’(2859bp)和3’(852bp)。应用高保真Taq聚合酶PCR拼接蔗糖磷酸合成酶完整cDNA序列3692bp,该片段与GenBank中其它植物基因序列具有97%￣99%的同源性,登录号DQ364058。应用BamHⅠ、KpnⅠ、BglⅡ限制性酶切获得植物双元表达载体pROK2及基因的线性片段,通过T4连接酶反应,构建了以CaMV35S为启动子,以Tnos为终止子的工程载体pROK-SPS,该载体含有Npt-II选择标记基因。 相似文献
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甜瓜果实蔗糖磷酸合成酶基因cDNA片段的克隆及表达分析 总被引：2，自引：0，他引：2
根据在GenBank中登录的番茄、马铃薯等蔗糖磷酸合成酶基因的保守序列设计引物, 采用RT-PCR方法从甜瓜花后25 d的果实总RNA中扩增出目标cDNA片段, 克隆到pMD18-T载体中。序列分析表明, 该片段与番茄蔗糖磷酸合成酶氨基酸序列同源性为98.9% , GenBank中登记号为DQ355797。通过Northern blot检测其在甜瓜果实不同发育时期的表达变化, 结果表明该基因在甜瓜果实花后25 d开始表达,随着果实的成熟, 表达量升高。 相似文献
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应用RNA反义技术抑制甜瓜果实发育过程中酸性转化酶活性,促进蔗糖积累,从而为培育优质品种提供了可行的新方法。将已克隆到pMD18-T载体上的甜瓜酸性转化酶基因用BamHⅠ和HincⅡ双酶切,得到该基因编码区1038bp的cDNA片段,将其定向插入到植物表达载体pROK2的BamHⅠ/SmaⅠ克隆位点,构建了甜瓜酸性转化酶cDNA反义表达载体(Anti-MAI1)。采用冻融法将其转入根癌农杆菌LBA4404,得到了完整的Ti质粒表达载体系统。利用叶盘法转化烟草,经PCR和PCR-Southern杂交检测,证明此基因已整合入烟草的核基因组中。 相似文献
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柿果ACC合成酶基因的克隆与植物表达载体的构建 总被引：3，自引：0，他引：3
以柿果组织中分离的总RNA为模板,经RT-PCR扩增到约1.1 kb的富有柿果ACC合成酶(Fuyu-ACS)的 基因片段,将此片段克隆到pGEMR-T easy vector上,经测序分析与Genbank中平核无柿果的DK-ACS1基因核苷酸 序列的同源性为99％。设计了2对带限制性内切酶位点的特异性引物,以测序质粒为模板,PCR扩增到2个ACS- Fuyu片段。2个片段经双酶切消化后,分别以正反2个方向插入到植物表达载体pBI121的35 S启动子和NOS终止 子之间,构建成Fuyu-ACS基因的植物表达载体。 相似文献
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以新疆哈密瓜(Cucumis melo L. ) 幼叶为材料, 根据已报道的甜瓜ACC合成酶3(1-aminocyclopropane-1-carboxylic acid synthase, ACS) 基因片段设计特异引物, 采用RT-PCR和RACE (Rapid Amplification cDNA Ends, cDNA末端快速扩增) 等技术, 克隆得到编码哈密瓜CM e2ACS3基因的全长cDNA 序列,长1 895 bp, 编码482个氨基酸, 分子量约为54.12 kD, 等电点8.45, GenBank登录号为FJ383171。利用软件MEGA4构建进化树, 发现哈密瓜ACS3基因与黄瓜中的同类基因亲缘关系最近。哈密瓜CMe-ACS3基因的克隆及序列分析为认识此基因在甜瓜中的功能研究奠定基础。 相似文献
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为探讨甜瓜N-乙酰氨基葡萄糖基转移酶基因(CmGnT)的功能,选取甜瓜CmGnT基因保守序列,利用特异性引物PCR扩增得到一段238 bp的干扰片段。利用Swa I单酶切将正向片段亚克隆到质粒载体p FGC1008的35S启动子与GUS之间,利用Spe I单酶切将反向片段亚克隆到质粒载体p FGC1008的GUS与终止子之间,构建了RNAi表达载体p FGC1008-CmGnT。通过冻融法将其转入根癌农杆菌C58中,菌液PCR验证表明RNAi表达载体p FGC1008-CmGnT构建成功并成功转入农杆菌。为进一步研究甜瓜CmGnT基因功能及甜瓜分子育种奠定重要基础。 相似文献
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荔枝败育胚S-腺苷甲硫氨酸合成酶基因的全长扩增和序列分析 总被引：3，自引：1，他引：3
从荔枝‘桂味’的败育胚中提取总RNA，利用GeneRacerTM Kit将RNA经去磷酸化反应、去mRNA的帽子结构及RNA Oligo连接，反转录合成cDNA，以cDNA为模板，分别用SSH分离得到的胚败育相关差异表达s一腺苷甲硫氨酸合成酶基因的部分cDNA序列(599 bp)设计出3’和5’端基因特异引物，进行PCR末端扩增。荔枝败育胚的s一腺苷甲硫氨酸合成酶基因序列全长为1515 bp，编码393个氨基酸，与其它物种的S一腺苷甲硫氨酸合成酶基因的核苷酸序列同源性在79%~82%之间，氨基酸序列同源性在88%～91%之间。结果表明所获得的基因为与荔枝胚败育相关的S一腺苷甲硫氨酸合成酶基因。关键词： 相似文献
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HOU Wei-hong YUAN Bao-mei WANG Tian-yun CHAI Yu-rong HOU Gui-qin WANG Jian-min XUE Le-xun 《园艺学报》2004,20(6):998-1002
AIM: To clone and express mouse canstatin (m canstatin) cDNA and provide a basis for the further research on its anti-angiogenic activity and potential application for cancer therapy. METHODS: Total RNA was extracted from mouse liver tissue by Trizol Reagent, and mouse canstatin cDNA was amplified by RT- PCR, then cloned into vector pMD18-T for sequencing. pET30a(+)-m canstatin recombinant plasmid was constructed and expressed in E.coli BL21 with induction of IPTG. RESULTS: Mouse canstatin cDNA is 684 bp coding 227 amino acids. The sequences of both cDNA and amino acid share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. In the present study, pET30a(+)-m canstatin recombinant plasmid was expressed in E.coli BL21. CONCLUSION: Mouse canstatin cDNA has been cloned for the first time. Constructed pET30a(+)-m canstatin recombinant plasmid is highly expressed in E.coli BL21. 相似文献
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AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5α was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5α was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1. 相似文献
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马铃薯S病毒外壳蛋白基因的克隆及其在大肠杆菌中的表达 总被引：2，自引：1，他引：2
以田间采集的马铃薯病叶中提取的马铃薯病毒s(PVS)总RNA为模板，通过RT—PCR获取长度为890 bp的P 一cp的cDNA，克隆至pGEM—T载体上。酶切回收该基因片段，并构建了该基因的原核表达质粒pBV—pvs。SDS—PAGE凝胶电泳和Western印迹分析表明：P 一印基因在大肠杆菌JM109中可特异地高效表达分子量约33kD的蛋白，且表达蛋白具有良好的抗原活性。利用该表达产物免疫动物家兔，获得的抗血清可用于大田马铃薯s病毒的快速检测。 相似文献
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桃果实ACC合酶cDNA的克隆 总被引：12，自引：0，他引：12
根据其它植物ＡＣＣ合酶氨基酸保守区设计两组简并引物，用套式ＰＣＲ技术从桃成熟果实ｃＤＮＡ中扩增出１．１ｋｂ大小特异性片段，将其克隆至ｐＧＥＭ－Ｔ载体上，对重组克隆ｐＰＡＣＳＩ进行全序列测定表明，插入片段全长１１００个碱基，编码３６６个氨基酸，该基因与番茄、笋瓜、小西葫芦、康乃馨、苹果ＡＣＣ合酶ｃＤＮＡ氨基酸序列同源性分别为７２．７％，７１．３％，７１．１％，６９．１％，６５．４％。ＲＮＡ点杂交表明ｐＰＡＣＳＩ基因随着桃果实成熟表达活性增强，乙烯处理能诱导桃果实该基因的表达。 相似文献
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AIM: To construct a eukaryotic expression plasmid for enhanced green fluorescent protein (EGFP) and ZNF580 fusion protein, and study its subcellular localization in the transfected MGC803 cells. METHODS: The primers were designed according to the cDNA encoding sequence of ZNF580 full-length open reading frame (1-172aa), ZNF580 amino terminus (1-93aa) and ZNF580 carboxyl terminus (94-172aa). The three cDNA segments of PCR were cloned into pGEM-T vector. Then they were subcloned respectively into plasmid pEGFP-C1 (enhanced green fluorescent protein). The subcellular localization of the fusion protein in MGC803 cells transfected with the vector was monitored by autofluorescence microscopy. RESULTS: Restricted enzymes analysis and DNA sequencing showed that the sequences of the pEGFP-ZNF580 (1-172), pEGFP-ZNF580 (1-93) and pEGFP-ZNF580 (94-172) transgenic plasmid were correct. The fusion proteins of EGFP-ZNF580 (1-172) and pEGFP-ZNF580 (94-172) were localized in the nuclei. CONCLUSION: The recombinant eukaryotic expression vector pEGFP-ZNF580 has been successfully constructed. The nuclear localization signal is within amino acid residues 94 and 172 of ZNF580 carboxyl terminus (C2H2 zinc finger domain). 相似文献
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AIM:To construct a eukaryotic expression vector expressing outer membrane lipoprotein LipL41 of Leptospira lai and express it in mammalian cell. METHODS:LipL41 gene was amplified by PCR from genome of Leptospira lai 017 strain, and was subcloned into vector pGEX-4T-1. After sequencing, LipL41 gene digested by restriction endonuclease and cloned into vector pcDNA3. After confirming the correctness of the eukaryotic recombinant vector by restrication enzyme digestion, it was transfected into COS7 cells by liposome. Its expression was analyzed by RT-PCR. RESULTS:A fragment of 1 011 bp was amplified, and sequence analysis showed it had a 98% homology with Leptospira kirschneri. The analysis of restriction enzyme indicated that the eukaryotic recombinant vector was correctly constructed. A specific amplified fragment was showed in the cells transfected with recombinant plasmid by RT-PCR, but the cell transfected with blank plasmid did not show this band. CONCLUSIONS:The LipL41 gene of Leptospira lai was successfully inserted into eukaryotic expression plasmid and the recombinant plasmid expressed the LipL41 mRNA. 相似文献
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AIM: To construct prokaryotic expression vector of His-tagged human IP-10 for further study of its biological function in the inflammatory response. METHODS: The coding sequence of IP-10 lacking signal peptide was amplified from human lung cDNA library by polymerase chain reaction (PCR) and the fragment was cloned into pET-14b plasmid for the construction of His-tagged fusion protein expressing vector, pET-14b/IP-10. After being identified by enzyme digestion and sequencing, the recombinant vector was transformed into a strain of E. coli, BL21 (DE3). The expression of His-tagged fusion protein was induced with IPTG and purified with Ni+-NTA affinity chromatography. Then the chemotactic activity of IP-10 was determined by transwell migration assay on THP-1 cells. RESULTS: The construction of pET-14b/IP-10 recombinant vector was proved by enzyme digestion and sequencing. The fusion protein IP-10, which was purified by a routine Ni+ affinity method, had an activity on the induction of cell migration of THP-1. CONCLUSION: We successfully construct IP-10 fusion protein expressing vector and get the fusion protein with high bioactivity, which provides essential materials for the future studies on IP-10. V 相似文献