共查询到19条相似文献，搜索用时 203 毫秒
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抗真菌蛋白Rs—AFPs基因克隆与序列分析 总被引：1，自引：0，他引：1
从开花后５０－９０ｄ的萝卜种子中提取总ＲＮＡ,逆转录合成ｃＤＮＡ第一链,通过ＰＣＲ反应,得到Ｒｓ－ＡＦＰ１和Ｒｓ－ＡＦＰ２基因扩增产物。采用Ｔ－载体克隆技术,将二分别插入ｐＢｌｕｅｓｃｒｉｐｔ／ＥｃｏＲＶ－Ｔ克隆载体,得到重组质粒ｐＧＲ－１和ｐＧＲ－２用其转化宿主菌Ｅ．ｃｏｌｉ ＤＨ５α,从阳性菌落制备测序用质粒ＤＮＡ样品,在ＡＢ１３７０Ａ ＤＮＡ测序仪上测序。 相似文献
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PEG介导BT基因转化大豆原生质体获转基因植株 总被引：5，自引：1，他引：4
通过ＰＥＧ法净Ｂ．Ｔ（Ｂａｃｉｌｌａｓ ｔｈａｒｉｎｇｉｅｎｓｉｓ ＣｒｙＩＡｃ）毒蛋白基因导入到大豆主栽品种黑农３５,黑农３７,合丰２５和合丰３５的原生质体中,经３０ｍｌ／Ｌ潮霉素筛选,选择有抗性的愈伤组织进行分化,获得了三棵再生植株,移栽后全部成活,对移栽后植株的总ＤＮＡ进行ＰＣＲ分析,均显阳性。对ＰＣＲ阳性植株进行Ｓｏｕｔｈｅｒｎ杂交分析,证明Ｂ．Ｔ毒蛋白基因已整合到大豆细菌基因组中。 相似文献
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从成熟番茄果实中分离ｍＲＮＡ,以Ｏｌｉｇｏ（ｄＴ）为引物在ＡＭＶ逆转录酶作用下合成ＡＣＣ２ｃＤＮＡ第一链,以ｃＤＮＡ为模板通过ＰＣＲ扩增ＡＣＣ２基因,获得１．４５ｋｂ的扩增片段,将此片段克隆到ＢＳ质粒的ＥｃｏＲＶ位点上,对插入片段两端进行部分序列分析。 相似文献
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一些含有１ＢＬ．１ＲＳ小黑麦易位的品种被认为耐Ａ１毒害，但这种耐性基因位于１ＲＳ还是位于小麦染色体上尚不清楚，１９８８年至１９９２年间的ＣｏｌｕｍｂｉａＭＯ用５５中软红粒冬小麦遗传背景材料回交获得含有Ｋａｖｋａｚ衍生的１ＢＬ．１ＲＳ和Ａｍｉｇｏ衍生的１ＡＬ．１ＲＳ易位的６个Ｆ４近等基因系，在液培条件下裂区设计法测定了１ＲＳ耐Ａ１性的表达。所有小区采用含１ｍｇ／ＬＡｌ的ＡｌＣｌ３进行处理，对照不含Ａ 相似文献
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天蚕抗菌肽B基因在广霍香抗病育种中的应用 总被引：9，自引：0，他引：9
用天蚕抗菌肽Ｂ基因转化广霍香,以期产生抗广霍香青枯病的转基因株系。通过农杆菌介导的叶盘法将抗菌肽Ｂ基因转入了广霍香的组织细胞,在Ｋａｎ的筛选培养基上经过丛芽途径获得转基因植株;采用ＤＮＡ Ｄｏｔ ｂｌｏｔ,Ｓｏｕｔｈｅｒｎ ｂｌｏｔ,ＲＮＡ Ｄｏｔ ｂｌｏｔ和大田攻毒试验证实的抗菌肽Ｂ基因已整合到再生植株的核基因组中,获得了高水平的表达,产生较强的抗病效果。 相似文献
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从解淀粉芽孢杆菌中提取染色体ＤＮＡ，经过ＰＣＲ扩增得到Ｂａｍａｓｅ（核酸酶ＢＮ）基因，然后克隆到质粒Ｐ＾ＧＥＭ－７２ｆ（＋）的Ｓｍａｌ位点上并进行序列分析。结果表明，核酸酶ＢＮ基因的核苷酸序列与已报道的序列相比具有９９．７％的同源性，长度为３３６ｂｐ，根据核苷酸序列推断的氨基酸序列则完全一致。该工作为以后利用核酸酶的特异表达来获得雄性不育植物打下了基础。 相似文献
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人α—干扰素2b基因转化番茄,番木瓜的研究（I）：人α—干扰素2b基因？… 总被引：2，自引：0，他引：2
以ＰＢＶ８８９（α－ＩＦＮ－２ｂ）为基因供体,利用ＥｃｏＥ Ｉ／Ｐｓｔ Ｉ双酶切分离出基因α－ＩＦＮ－２ｂ,再利用现有的植物表达质粒ＲＯＫ Ⅱ（带ＰＲＶ－ＣＰ基因,３５Ｓｐｒｏｍｏｔｅｒ／ＮＰＴ Ⅱ）,用ＸｂａＩ／ＳａｃＩ双酶切分离出大片段（Ｅ６）作为基因受体,采用定向连接法将α－ＩＦＮ－２ｂ拼接在Ｅ６上,完成植物表达载体构建。采用ＤＡＮ ｄｏｔ ｂｌｏｔ、ＲＮＡ ｄｏｔ ｂｌｏｔ对重组子进行筛选 相似文献
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Sadigh ZA Shahrabadi MS Shafyi A Bambai B 《Pakistan journal of biological sciences: PJBS》2008,11(9):1220-1226
In this study, one of the measles virus membrane proteins, named hemagglutinin (H) which has a key role in tropism, receptor binding, hemagglutinating activity and also induction of protective immunity against viral infection, was expressed by the baculovirus expression system using specific plasmid (pDONR221) to produce entry clone. Measles Virus (AIK-C strain) genome was extracted from infected Vero cells. H gene was amplified by specific primers during RT-PCR reaction and inserted into the specific plasmid (pDONR221) using BP recombination reaction. Recombinant baculovirus harboring H gene was consequently constructed by LR reaction. Insect cells (Sf9) were infected with recombinant baculovirus. In order to increase viral titer, recombinant baculoviruses were passaged four times in Sf9 cells. Synthesis of H protein was verified by SDS-PAGE, western-blot and indirect immunoflourescene using goat polyclonal antibody against Measles Virus. The results showed that H protein was partially glycosylated, but it appeared to be active in hemagglutination assay. 相似文献
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为了验证dhbC基因的功能,以质粒pEGFP-NI为模板,通过PCR扩增获得新霉素抗性基因(neo~r)DNA片段,构建重组质粒pMD18-neo,电击转化法将pMD18-neo导入枯草芽孢杆菌Bacillus subtilis CAS15感受态细胞中,使neo'基因片段与dhbC基因片段发生置换,获得dhbC基因缺失突变株.再通过电击转化将dhbC 基因全长编码序列导入dhbC基因缺失突变株CAS15 dhbC-del,获得dhbC基因回复株CAS15 dhbC-com.经CAS平板法检测表明,CAS15 dhbC-del不能产生橘黄色晕圈,而CAS15 dhbC-tom产生与CAS一致的橘黄色晕圈,证明了dhbC基因与嗜铁索的合成密切相关. 相似文献
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应用PCR方法从感染香蕉束顶病毒的香蕉植株幼嫩假茎和叶片总DNA中克隆了Rb(Rb-binding-like protein)基因的编码区,将其插入原核表达载体pET-28b中构建重组质粒pET28b-Rb。转化重组质粒的E.coli BL21(DE3)进行IPTG诱导表达,SDS-PAGE分析结果表明,表达产物大小为22.6ku,且主要以包涵体形式稳定表达。目的蛋白经Ni2+-NTA琼脂糖亲和层析纯化后免疫家兔并获得抗血清。Western blot检测结果表明,抗血清与诱导表达的BBTV编码Rb蛋白发生特异性反应。间接ELISA法测定的抗血清效价大于1:250000。用抗血清对感病香蕉和健康香蕉进行检测,结果表明,抗血清对感病香蕉有很高特异性,最佳工作浓度为1:1500。 相似文献
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利用Trizol法提取接种PRsv 4 d的番木瓜种苗总RNA,分离纯化mRNA,反转录合成双链cDNA,双链cDNA末端经Pfu-DNA聚合酶补平,与EcoR I接头连接,XhoI酶切消化产生粘端.用Sepharose CL-2B柱分离纯化去除小分子cDNA片段,再与pMyr酵母表达载体连接,转化受体菌XL10-Gold,构建了接种PRSV番木瓜种苗胞质酵母双杂交cDNA文库.所获得的原始文库的克隆总数为1.8×106cfu,重组率为100%,文库滴度为2.6×109cfu/mL.对随机选取的34个克隆进行PCR鉴定,结果表明插入片段长度均大于O.5 kb且集中在1 kb左右.文库质量鉴定结果表明,该文库具有较好的库容量、较高的重组率以及较大的插入片段. 相似文献
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短短芽胞杆菌FJAT-0809-GLX具有抑菌、抗褐变和保鲜功能。以短短芽胞杆菌菌株FJAT-0809-GLX总DNA为模板,采用PCR扩增超氧化物歧化酶(Supseroxide dismutase,SOD)基因,将该片段纯化回收后与p MD18-T连接并转入大肠杆菌(Escherichia coli)DH5α,进行序列测定,结果显示SOD基因序列长度为609 bp(Gen Bank登录号:KM255665),编码202个氨基酸残基。将SOD基因片段与同样酶切的表达载体p ET-28a连接,构建重组表达载体p ET-28a-SOD,转入大肠杆菌BL-21,采用异丙基-β-D-硫代吡喃半乳糖苷(Isopropyl-β-D-thiogalactopyranoside,IPTG)进行诱导表达。SDS-PAGE结果表明,在菌体中存在约25 ku的蛋白表达产物,与该基因ORF预期大小接近。以上结果为进一步研究该酶的生理生化特性奠定了基础。 相似文献
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Background: Herpes simplex virus type 2 (HSV-2) is highly prevalent and major cause of genital herpes in humans. The life-long nature of infection and the increasing prevalence of genital herpes imply that vaccination is the best strategy for controlling the spread of infection and limiting HSV disease. HSV glycoprotein D (gD) is one of the most important viral immunogen which has an essential role in virus infectivity and induction of immune responses. Methods: HSV-2 DNA was extracted and used as template in polymerase chain reactions to amplify gD2 gene. The PCR product was confirmed by restriction enzyme analysis, cloned into a cloning vector and then sequenced. The Bac-to-Bac expression system was used to express HSV-2 gD in insect cells. The expressed protein was used as subunit vaccine to immunize guinea pigs after confirmation. Results: The expressed protein was confirmed with SDS-PAGE and Western-blot analysis. In Western-blot analysis, two major protein bands, with approximate molecular weights of 52-55 and 41-43 kDa corresponding to the glycosylated and non-glycosylated forms of gD2 protein, were observed, respectively. Immunization with the recombinant gD2 could elicit humoral responses in guinea pigs as measured by neutralization test and ELISA, and offered high protection against induced HSV-2 genital disease. Conclusion: The baculovirus expression of heterologous genes permits proper folding, post-translational modification and oligomerization in manners that are often identical to those that occur in mammalian cells. Expression of proteins under the control of the strong polyhedrin promoter, allowing high level protein production, can be used as subunit vaccine. 相似文献