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双抗转基因烟草纯合系的选育及田间试验   总被引:12,自引:0,他引:12  
为了将双抗转基因烟草推向实用,进行了双抗转基因烟草纯合系的选育。在试验田中利用人工攻毒进行了T1代植物的抗病毒特性的分离试验;在抗性良好的T1植株的后代T2代植物上进行了田间的病毒接种试验和转基因表达的分析,从中选育出双抗转基因烟草纯合系。又于试验田中,在这些纯合系后代T3代上验证了抗病毒特性的遗传稳定性。在转基因表达分析中,发现CMV外壳蛋白基因的表达在翻译水平上受到抑制。  相似文献   
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The genomie DNA of CaMV (Xinjiang isolate) was mapped on the virion DNA with anumber of restriction endonueleases by double digestions and partial digestions of one-end~(32)P-labelled fragments. It contained the unique sites of BglI, SalI and XhoI. BamHI andHhaI each cut it at two sites, EeoRI at five sites (three of which were located), Hpa Ⅱ atseven sites, BglⅡ at eight sites, and HaeⅢ at ten sites. The sites of all the enzymes usedabove, as well as HindⅢ which cut at ton sites were also mapped on the cloned CaMVDNA. Virion DNA and cloned viral DNA gave the same results in the number and locationof cleavage sites of the restriction enzymes tested on both DNAs. Like most of the otherisolates, CaMV-Xinjiang isolate has three single-stranded discontinuities. From the diges-tion patterns of SalI, XhoI, EcoRI and HhaI, it is concluded that the restriction map ofCaMV-XJ genome is closely related to that of BKMV isolate from East Germany.  相似文献   
3.
本文用混合酶解和一端以~(32)P标记的DNA的部分酶解测定了花椰菜花叶病毒新疆分离物(CaMV-XJ)基因组DNA的限制性内切酶图.它含有BglⅠ,SalⅠ,XhoⅠ的单一切点.BamHⅠ和HhaⅠ各有2个切点,EcoRⅠ有5个切点,HpaⅡ有7个切点,BglⅡ有8个切点,HaeⅢ有10个切点,HindⅢ有10个切点.在病毒粒体DNA和克隆的病毒DNA上这些酶的切点数目和位置是一样的.同其他大多数CaMV分离物一样,CaMV-XJ DNA上也有3个单链不连续区.从SalⅠ,XhoⅠ,EcoRⅠ,HhaⅠ的酶切情况看,CaMⅤ-XJ基因组的内切酶图谱与东德的CaMⅤ分离物BkMⅤ比较接近。  相似文献   
4.
从感病的萝卜叶片中得到芜菁花叶病毒(TuMV)的分离物,以oligo(dT)为引物,合成了其基因组RNA的互补DNA,并克隆到载体λ-ZAPⅡ中。通过单链cDNA探针杂交和DNA末端序列分析找出含有poly-A尾巴的阳性克隆。我们对一个含有1429个碱基插入片断的克隆进行了序列分析,根据芜菁花叶病毒外壳蛋白分子量的大小和马铃薯Y病毒组蛋白质加工位点氨基酸序列的保守性,确定了外壳蛋白基因的5′末端。利用PCR方法对其5′末端进行改造,加进起始密码ATG和两个限制性内切酶位点。通过基因重组操作,将该基因插入到双元载体pBI121的衍生物pBIN437中,得到芜菁花叶病毒外壳蛋白基因的植物表达载体。  相似文献   
5.
An intermediate expressing vector carrying the tobacco mosaic virus (TMV, Chinese common strain) coat protein (CP) gene was constructed by recombinant DNA techniques. The TMV-CP gene was transferred into the tobacco genome via Ti plasmid and a large number of regenerated plants, including both systemic and local lesion hosts for TMV, were obtained. Southern blot analysis revealed that 1-5 copies of the CP gene were integrated into the tobacco genome. RNA and protein analysis demonstrated that the TMV-CP gene was correctly expressed in the transgenic plants. The abundance of TMV-CP mRNA in total leaf RNA accounted for 0.005-0.01%, while the amount of coat proteins reached 0.05-0.2% of the total leaf soluble proteins. Virus challenge experiments showed that the symptom development of virus infection was markedly delayed and the replication as well as the spread of the virus was significantly inhibited in the transgenic plants expressing the TMV-CP gene. Three of these plants were completely protected afte  相似文献   
6.
以马铃薯Y病毒(PVY,中国分离株)基因组RNA的cDNA为模板,我们用聚合酶连锁反应(PCR)合成了完整的外壳蛋白(CP)基因及部分3′末端非编码区序列。在5′-引物Y5中引入了限制性酶NcoI位点及起始密码AUG,3′-引物Y3中引入了Sall位点。PCR产物经NcoI及Sall双酶切后克隆入pGEM衍生质粒,并测定了其中一个克隆pPCY6的CP基因的序列,以此克隆的CP基因片段作探针,从cDNA库中钓出相关的几个克隆,并测定了序列,由此我们获得了包含部分NIb基因,全部的CP基因及3′-非编码区共1317个碱基。序列分析表明,该株系CP基因核苷酸序列与0株系同源性(94.2%)较与N株系(89.6%)同源性稍高,但氨基酸序列的同源性差别不大(分别为93.3%及91.8%)。目前我们已将该基因克隆入双元载体pBI121.1,并通过脓杆菌转化马铃薯,以期得到抗PVY的马铃薯工程株。  相似文献   
7.
表达烟草花叶病毒外壳蛋白的转基因烟草及其对TMV的抗性   总被引:14,自引:0,他引:14  
利用体外重组DNA技术构建携带烟草花叶病毒(TMV,国内流行的普通株)外壳蛋白基因(CP)的中间表达载体,通过土壤农杆菌(Agrobacterium tumefaciens)Ti质粒,重组TMV-CP基因被转移至烟草细胞,并获得大量再生的转基因烟草。工程烟草的基因组经Southern印迹法分析证明,CP基因在再生工程株染色体中有1—5个拷贝的插入。分子杂交分析确证TMV-CP基因在转基因株中获得正确表达,其mRNA和蛋白产物的丰度分别达0.005—0.01%及0.05—0.2%。攻毒实验表明90%以上的能表达TMV—CP基因的工程烟草能不同程度地抑制病毒的复制、扩散,并显著延缓,减轻系统病状的发生。有3株工程植株直至花期无病状表现,生长正常。这一抗性作用机制在于转化细胞中的外壳蛋白在病毒侵染早期有效地抑制了入侵病毒颗粒的脱壳,从而阻断病毒的复制。  相似文献   
8.
从感染TMV的普通烟叶中分离到含有TMV-RNA复制酶的无细胞提取物(20,000×g沉淀),它能在四种三磷酸核苷(其中三磷酸尿苷用氚标记)、镁离子和放线菌素D的存在下离体合成双链RNA,经酚-SDS提取、Serva纤维素柱层析获得的3H-双链RNA,用聚丙烯酰胺-琼脂糖凝胶电泳、核糖核酸酶处理、热变性、自退火等手段,证实3H-双链RNA中含有TMV-RNA的复制型(RF)和复制中间体(RI),分子量分别为4.0×106和5.0×106,分子杂交竞争试验表明3H-双链RNA中新生链的60-70%为TMV-RNA正链。  相似文献   
9.
The entire coat protein (CP) gene and part of the 3'-noncoding sequence of the potatovirus Y (PVY, the Chinese isolate) genome were synthesized with polymerase chain reaction(PCR) using cDNA of its genomic RNA as a template. A restriction endonuclease site Ncoland the initiation codon AUG were included in primer Y5 while the SalI site was includedin primer Y3. After being double digested with Ncol and SalI enzymes, the PCR product wascloned into a pGEM derivative plasmid, and the CP gene in one of the clones, pPCY6, wassequenced. Several clones were selected from the cDNA library by using the CP gene frag-ment of pPCY6 as a probe and the sequences of these clones were determined. These se-quences included part of the NIb gene, entire CP gene and 3'-noncoding region, 1317 bp alltogether.Sequence analysis indicated that the nucleotide sequence homology of the CP geneof this strain with that of the 0 strain (94.2%) was a little higher than with that of the Nstrain (89.6%), but the homology of amino acid se  相似文献   
10.
Complementary DNAs to turnip mosaic virus (a radish Raphanus stativus isolate) genomicRNA were synthesized using oligo (dT) as primer and cloned into the vector λ-ZAP Ⅱ. Afterhybridization with a single-stranded cDNA probe and the sequencing of inserted DNA,positive clones with poly--A tails were obtained. One clone containing 1429-base pair insertwas sequenced. The coat protein gene was identified based on the molecular weight of theTuMV coat protein and the consensus sequences of the polyprotein processing sites ofpotyviruses. The 5' end of the coat protein gene was modified by PCR to introduce aninitiation codon, ATG, and two restriction enzyme sites. The gene was then manipulatedinto a binary vector pBIN437 which was derived from pBI121, and the plant expressionvector is being used to transform Brassica napus.  相似文献   
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