共查询到19条相似文献，搜索用时 139 毫秒
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以蝴蝶兰"大辣椒"为试材,采用RACE技术克隆蝴蝶兰APETALA1(AP1)基因的cDNA全长,分析基本生物学信息,并初步探索其在花芽分化过程中的作用.结果表明:AP1基因的cDNA全长为1 155 bp,开放阅读框(open reading frame)752 bp,编码250个氨基酸,含有MADS盒与K盒等保守功能结构域.同源性比对结果显示,蝴蝶兰"大辣椒"与朵丽蝶兰、金蝶兰、文心兰、石斛兰和马兜铃等植物的MADS蛋白有较高的相似性,同源性均在80％以上;系统进化树分析显示,蝴蝶兰"大辣椒"AP1基因与朵丽蝶兰聚类关系最近;实时荧光定量PCR检测发现,AP1基因在叶片、根和花葶中均有表达,但表达量有差异.同一器官不同发育时期比较显示,叶片的AP1基因表达量没有明显的时空差异;根AP1基因的表达量随着花芽分化的进程呈递减趋势,其中始花期的表达量最低,盛花期最高;而花葶中AP1基因表达量随着花芽分化呈增加趋势,在盛花期表达量达到峰值.同一时期不同器官比较,花葶中AP1的表达量显著高于同一时期的叶片和根(P＜0.05).由此,推测AP1基因在调控蝴蝶兰花芽分化过程中发挥重要作用. 相似文献
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以CMV(黄瓜花叶病毒)外壳蛋白基因为靶标基因,针对CMV 3个常见株系(普通株系、黄化株系和坏死株系)外壳蛋白基因间高度同源序列设计引物,经RT-PCR获得目的序列,构建RNA干涉双元载体(其中目的序列反向重复插入)。以含双元载体的根癌农杆菌介导转化无菌番茄苗,培育转基因番茄。人工接种CMV验证转基因番茄抗病毒能力,荧光定量PCR分析转基因番茄中外源基因的表达效果。结果表明:RNA干涉转基因番茄呈现不同程度的抗病毒能力;对表现抗病毒能力的转基因番茄进行荧光定量PCR分析显示,转基因番茄中靶标基因的转录mRNA存在一定程度的降解,且抗病毒能力与降解程度成正相关。 相似文献
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用RT-PCR方法从‘大辣椒’蝴蝶兰叶片中克隆得到4个AP2/ERF家族基因，命名为PhAP2/ERF1 ~ PhAP2/ERF4。蛋白结构域和序列比对发现PhAP2/ERF1 ~ PhAP2/ERF4均含有1个AP2结构域， PhAP2/ERF2还含有1个B3结构域。进化树分析表明4个蛋白与兰科AP2/ERF家族成员亲缘关系最近，PhAP2/ERF1属于AP2/ERF家族DREB亚家族中的A1类，PhAP2/ERF2属于RAV亚家族，PhAP2/ERF3属于ERF亚家族的B4类，PhAP2/ERF4属于DREB亚家族中的A2类。用实时荧光定量PCR方法检测低温驯化和低温胁迫条件下PhAP2/ERF1 ~ PhAP2/ERF4在‘大辣椒’和‘富乐夕阳’蝴蝶兰叶片中的表达特性，结果表明：4个基因在‘大辣椒’叶片中的表达趋势一致，表达量在低温胁迫早期达到最高，在胁迫中晚期有所下降；而4个基因在‘富乐夕阳’叶片中的表达趋势有较大差异，PhAP2/ERF1和PhAP2/ERF2的表达在低温处理整个过程中没有被诱导或仅有较小幅度增加，PhAP2/ERF3对低温的响应时间显著晚于‘大辣椒’，PhAP2/ERF4的表达趋势与‘大辣椒’差异较小。蝴蝶兰AP2/ERF在抗冷品种‘大辣椒’和不抗冷品种‘富乐夕阳’叶片中的表达差异暗示AP2/ERF基因家族在蝴蝶兰低温胁迫响应中起重要调控作用。 相似文献
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辣椒N 基因介导抗根结线虫作用早期表达基因的抑制性消减杂交SSH分析 总被引：4，自引：0，他引：4
以含N 基因的辣椒(Capsicum annuum L. ) 为试验材料, 接种南方根结线虫(Meloidogyne incognita) 12、24、36 h的根尖材料作为测验方( tester) , 相应的未接种根尖材料作为驱动方( driver) , 构建一个南方根结线虫诱导N 基因表达早期的正向抑制消减杂交cDNA文库, 并结合文库高密度点阵膜杂交差异筛选, 获得了237条表达序列标签( EST) 。在GenBank上进行BLASTn与BLASTx分析, 得到148条功能已知的EST序列, 获得已知的上调抗性相关EST 68个。分离出了具有NBS2LRR结构的抗线虫蛋白和类LRR抗性蛋白的基因, 防御作用相关的类萌芽素(GLP) 、HSR203J 蛋白、蜜腺蛋白、蛇毒素肽等基因,抗性相关的WRKY、ERFBP等转录因子基因, 以及G蛋白、142323蛋白等多种信号蛋白基因。通过GeneOntology分析, N 基因介导的早期表达抗病基因涉及病原物的识别、抗性信号传导、过敏性坏死、系统获得性抗性以及植物细胞保护机制等多个方面, 并有许多功能未知的基因有待于进一步的研究。 相似文献
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以黑木耳DL202为试材，采用RT-PCR技术克隆了Mn-SOD基因全长，并对其进行生物信息学分析和表达分析，以期为进一步研究Mn-SOD基因在黑木耳抗逆过程中的功能奠定基础。结果表明：Mn-SOD基因cDNA全长为609 bp,编码202个氨基酸，命名为Ah-MnSOD;蛋白具有锰超氧化物歧化酶(Mn-SOD)家族的保守结构域，不具有跨膜结构和信号肽；亚细胞定位主要位于过氧化物酶体中；系统进化分析表明Ah-MnSOD与Auricularia subglabra亲缘关系最近。此外，构建了Ah-MnSOD基因的原核表达载体并成功诱导出目的蛋白。荧光定量PCR结果显示Ah-MnSOD基因在不同发育阶段均有表达，且在原基中表达最高。 相似文献
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绿色荧光蛋白基因转化柑橘胚性愈伤组织与高效再生 总被引：6，自引：0，他引：6
采用根癌农杆菌介导法对13份柑橘胚性愈伤组织进行绿色荧光蛋白基因(gfp)的转化,短时间内获得了温州蜜柑、橘橙杂种GWZ等11份柑橘材料的82个转化细胞系,其中佛罗斯特脐橙和日辉橘最易转化,分别获得28个和25个转化细胞系。选取3份材料的不同转化细胞系进行gfp的PCR分析,均获得了预期的gfp片段。研究表明,GFP标记可以在转化早期快速检测并分离转化子,及时剔除逃逸体,获得纯合的转化系;高水平的gfp表达不影响转化细胞增殖、生长和分化。这些带有GFP标记的转化细胞系为柑橘的有关基础研究提供了良好的试材,目前正用于柑橘体细胞杂交和不同倍性愈伤组织的社会化控制现象与机理等研究。 相似文献
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根癌农杆菌介导甘蓝枯萎病菌的遗传转化 总被引：2，自引：0，他引：2
采用根癌农杆菌介导的方法,以农杆菌介导丝状真菌遗传转化的载体PCH-sGFP对尖孢镰刀菌甘蓝专化型两个生理小种进行转化,该载体含有报告基因-绿色荧光蛋白基因（gfp）和筛选基因-潮霉素磷酸转移酶基因（hph）。获得转化菌株后,经检测表明：T-DNA目的片段成功整合到枯萎病菌基因组中,并且单孢继代培养6代后荧光蛋白仍能稳定遗传。对转化菌株的生长速度和致病力进行分析,结果表明：野生菌株和转化菌株在生长速度和致病力上没有显著差异。因此,转化菌株可用于甘蓝枯萎病菌的组织病理学研究。 相似文献
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对在固体培养基上培养3 年的黄瓜转gfp 基因毛状根进行gfp 和rol 位点系列基因的PCR 扩增、gfp 的荧光定量PCR、Western blot 杂交以及荧光观察分析。结果显示,由组成型启动子35S 驱动的gfp 在转录水平上能正常表达,而且能够翻译出编码蛋白;此外,培养3 年后的毛状根,能扩增出与毛状根形态构成有关的rol 系列基因。本研究结果表明长期培养的毛状根能保持其遗传稳定性,这为利用毛状根长期工厂化生产外源基因表达的蛋白产物和药用植物的活性成分提供了理论依据。 相似文献
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YE Chuan-zhong LIN Zhen CHEN Shi-ping ZHANG Fang-lin PEI Xue-tao LI Liang FENG Kai 《园艺学报》2001,17(9):825-829
AIM: To facilitate the suicide gene delivery into neoplasm, a chimeric gene of HSV-tk and green fluorescent protein (gfp) was constructed. METHODS: Molecular cloning technique was used to construct this kind of eukaryotic vector. The internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV), which could coordinate expression of two genes in a single vector, was optioned. By using liposome-mediated transfection, eukaryotic expression vector tgCMV/hytk-IRES-gfp was transfected into human bladder carcinoma cells EJ. RESULTS: A bicistronic eukaryotic vector carrying gfp and hygromycin phosphotransferase-thymidine kinase fusion (hytk) gene was constructed. The results of PCR and microscopy detection show that the hytk-IRES-gfp gene was successfully transferred into EJ cells. There were no differences in the growth pattern or the morphology between EJ and EJ/hytk-GFP cells. In vitro experiments demonstrated dose- and time-dependent cell killing by transduction of the hytk-IRES-gfp gene followed by GCV treatment. The IC50 (the concentration required to elicit 50% growth inhibition) was 2.16 mg/L in treatment with GCV for 72 hours. CONCLUSION: These results suggest that this new kind of eukaryotic vector could serves as a new tool and method for neoplasm gene therapy. 相似文献
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GAO Guo-hui WANG Jin-dan HUANG Qi-di YANG Ji-feng YANG Shui-bing BAO Bing-bing ZHANG Yu HU Xiao-qu 《园艺学报》2011,27(5):1034-1040
AIM: To express a recombinant fusion protein anti-human epidermal growth factor receptor-2 single-chain variable fragment with green fluorescent protein (anti-HER2-ScFv-GFP) using the insect cells-Bac-to-Bac baculovirus expression system and to analyze the binding function of this fusion protein with HER2 on the surface of the breast cancer cells. METHODS: Human anti-HER2-ScFv gene from mice was fused with GFP gene. To obtain the recombinant plasmid pFAST Bac-to-Bac HT A/anti-HER2-ScFv-GFP, we inserted it into Bac-to-Bac baculovirus expression plasmid pFAST Bac-to-Bac HT A. The identified recombinant plasmid was transferred into Escherichia coli DH10Bac to allow the generation of a recombinant bacmid. After transfected the recombinant virus bacmid into the insect cells Tn-5B1-4, the recombinant virus was collected to infect Tn-5B1-4. SDS-PAGE and Western blotting analysis were used to verify the expression product in Tn-5B1-4. The fusion protein was purified with Ni2+-NTA affinity chromatography. The purified fusion protein was bound to the surface of HER2-positive breast cancer cells SKBR3 and HER2-negative breast cancer cells MCF7. The binding effects on the surface of breast cancer cells were observed under laser confocal microscope. RESULTS: The fusion gene anti-HER2-ScFv-GFP was successfully constructed with the length of 1 539 bp. The green fluorescence was also observed in Tn-5B1-4 cells infected with the recombinant virus under fluorescent microscope. A 60 kD protein was examined and confirmed by SDS-PAGE and Western blotting. Under laser confocal microscope, strong green fluorescence was observed on the surface of the HER2-positive breast cancer cells SKBR3. However, no green fluorescence was observed on the surface of HER2-negative breast cancer cell MCF7. Obvious green fluorescence on the surface of HER2-positive breast cancer cell SKBR3 was also found after the cells were eluted with 1×PBS. CONCLUSION: The fusion protein anti-HER2-ScFv-GFP was successfully expressed in insect cells Tn-5B1-4, and can firmly bind to the surface of breast cancer cells SKBR3 and emit the green fluorescent light. 相似文献
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AIM: To study the feasibility, security and validity of the intrapericardial injection with a trans-diaphragmatic approach in chronic heart failure rats and its value in the study of gene therapy for heart diseases, and further investigate whether adeno-associated virual gene transfer of sarcoplasmic reticulum Ca2+-ATPase （SERCA2a） gene can improve ventricular function in chronic heart failure (HF) rats. METHODS: An animal model of heart failure was obtained by creating descending aortic constriction in rats. Recombinant adeno-associated virus, carrying enhanced green fluorescent protein (EGFP) gene and recombinant adeno-associated virus carrying SERCA2a gene, were respectively injected into pericardium of heart failure rats in different groups (group HF+EGFP and group HF+SERCA2a) by intrapericardial injection with a trans-diaphragmatic approach. After 30 days, hemodynamic parameters were measured and analyzed. Cryosection was analyzed by fluorescence microscopy to examine the expression of green fluorescent protein, and Western blotting was performed to detect the expression of SERCA2a. RESULTS: Green fluorescence was detected in cryosection of the hearts in all rats in group HF+EGFP and the expression of green fluorescent protein was ubiquitously. The expression of SERCA2a in all rats in group HF+SERCA2a was more than those in group HF and group HF+EGFP. And overexpression of SERCA2a improved the systolic and diastolic function of heart failure rats significantly and the hemodynamic parameters were similar with those of the controls. CONCLUSION: The intrapericardial injection with a trans-diaphragmatic approach suggests a simple, safe, efficient and cheap technique for the gene therapy of chronic heart failure. Gene thransfer of SERCA2a may be a new approach for the treatment of chronic heart failure. 相似文献