共查询到18条相似文献，搜索用时 109 毫秒
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[Objective] The aim was to isolate the CBL-interacting protein kinases(CIPK)from maize(Zea mays L.)and construct the fusion gene expression vector which consisted the ZmCIPK8 and GFP.[Method] The ZmCIPK8 cDNA was successfully cloned by using RT-PCR method.And then,it was connected to the pBlueScript SK(pSK)plasmid,which contained the GFP gene.So that the fusion gene vector pSK-CIPK-GFP was obtained.Then,the fusion gene was connected into the efficient plant expression vector PBI121 to construct the fusion gene expression vector PBI-CIPK-GFP.At last,the recombined expression vector was transformed to Agrobacterium tumefaciems LBA4404 to produce the engineering strain LBA4404-PBI-CIPK-GFP.[Result] The fusion gene expression vector which consisted of GFP and ZmCIPK8 gene and engineering strain LBA4404-PBI-CIPK-GFP were successfully constructed.[Conclusion] The results lays a foundation for further study of subcellular localization of ZmCIPK8,which can help to clarify the molecular mechanism of regulation serious stresses,and also provides an important basis for the research on resistance stress engineering of maize. 相似文献
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牛瑟氏泰勒虫P23基因的克隆与生物信息学分析(英文) 总被引：3，自引：0，他引：3
[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its genomic DNA by PCR amplification, and cloned into the pGEM-Easy vector; then the sequencing result was analyzed with bioinformatics methods. [Result] Whole length of the P23 gene from Theileria sergenti is 684 bp containing a 672 bp open reading frame. The deduced amino acid sequence (223 amino acid residues) contains a signal peptide of 19 amino acid residues and two fragments of transmembrane domains, with relative molecular weight of the 25.886 kD and with the pI of 9.22. The homology between the yielded sequence and Chitose of Theileria sergenti P23 gene(TS-Chitose type, D84446), Ikeda of Theileria sergenti P23 gene(TS-Ikeda type, D84447) reached 99% and 90%, respectively. The sequence has been accessed in GenBank(EU573168). [Conclusion] The protein encoded by the P23 gene has better stability and immunogenicity, thus can be used as the antigen candidate for preparing genetic engineering vaccine for Theileria sergenti. 相似文献
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[目的]研究牛瑟氏泰勒虫P23表面蛋白基因的克隆及原核表达。[方法]采用PCR方法扩增牛瑟氏泰勒虫中国延边株P23基因片段,将扩增产物克隆入pMD18-T载体构建重组质粒pMD18-P23,经PCR、双酶切鉴定后测序;将目的基因片段亚克隆入表达载体pGEX-4T-1构建重组表达质粒pGEX-4T-P23,转化宿主菌BL21获得重组菌。通过对诱导条件的优化,根据SDS-PAGE确定表达蛋白的最佳表达条件;Western-blotting检测表达蛋白的反应原性。[结果]所克隆的牛瑟氏泰勒虫P23基因片段长507 bp,与牛瑟氏泰勒虫日本株P23基因的核苷酸同源性达99.4%,表达的融合蛋白大小约为46 ku;诱导时机以接种培养后2 h为最佳,诱导时间以6 h为最佳,诱导温度以34℃为最佳,0.008-1.000 mmol/L的IPTG对表达量的影响不大。Western blotting检测表明该蛋白具有较好的抗原性。[结论]为牛瑟氏泰勒虫病的免疫学诊断和预防等研究奠定了基础。 相似文献
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Li Xue-lin Liu Xiao-fei Gao Xue-jun Qiao Bin Pan Hong-bao Ao Jin-xia 《东北农业大学学报(英文版)》2013,20(1):43-48
Lysine-rich protein gene (lys) was cloned from winged bean (Psophocarpus tetragonolobus (L.) DC), and cloned into prokaryotic expression vector pHT43, the recombinant plasmid pHT43/lys were constructed and then transferred into Bacillus subtilis168, upon IPTG induction, the recombinant protein was expressed, and the content of lysine was detected by HPLC. The result showed that lysine content increased by 9.85%. It was suggested that introducing lys gene into Bacillus subtilis 168 was an effective way to improve its nutrition quality. 相似文献
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黑白花奶牛白细胞介素-2基因的克隆和表达(英文) 总被引：4，自引：2，他引：2
[Objective] The aim of this study is to clone bovine interleukin-2 gene(IL-2)and observe its expression in prokaryotic cells.[Method]Bovine IL-2 gene was amplified from total RNA of peripheral blood lymphocytes of holstein-friesian cows by RT-PCR.Subsequently,the gene was cloned into pGEX-2T prokaryotic expression plasmid to construct recombinant,which was then transformed into Escherichia Coli BL21.After IPTG induction,SDS-PAGE analysis was conducted.[Result]A 500 bp target fragment corresponding with expectation was obtained by RT-PCR.The cloned gene successfully expressed fusion protein of about 43 kD in prokaryotic cells.[Conclusion]This study provided a theoretical and material basis for further researches on IL-2 gene. 相似文献
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The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction （PCR） from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E. coli strain TG Ⅰ. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KG- Nanog, was constructed successfully. The pGEX-KG-Nanog plasmid was extracted from E. coli strain TG Ⅰ and was transformed into BL21（DE3） for expression. After induction by isopropyl-β-D-thiogalactoside （IPTG） at 37℃, the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and the expression condition was optimized. Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L^-1 IPTG for 5 h. The mouse Nanog gene was successfully expressed in E. coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody. 相似文献
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The small envelope protein （E） gene of avian infectious bronchitis virus （IBV） M41 strain was cloned, and then it was subcloned into prokaryotic expressing vector pGEX-6P-1. The recombinant plasmid was transformed into E.coli. BL21 and induced by IPTG. SDS-PAGE result showed that when objective protein fused with GST （about 20 ku）, the relative molecular mass of fusion protein was 38 ku. It indicated that objective protein was about 12.4 ku. The result showed that E protein was expressed successfully, it was useful to the subsequent E protein research. 相似文献
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ZHAO Pan TANG Shun-ming LIU Tingx LIU Ting QIN Guang-xing GUO Xi-j ie 《中国农业科学(英文版)》2010,9(12):1821-1828
The gene of the non-structure protein 2 （NS2） was cloned by PCR from the genome ofBombyx mori densovirus Zhenjiang strain （BmDNV-Z）, inserted into prokaryotic expression vector pET28a to construct recombinant plasmid pET28a-NS2 and then expressed in bacteria Escherichia coli BL21 （DE3）. The expressed recombinant protein was identified by SDS-PAGE and Western blot analysis. Then, the recombinant protein was purified by Ni-NTA column, renatured and tested for enzyme activities. The purified NS2 protein exhibited a helicase activity unwinding double-stranded DNA substrates into single-strand primers, and higher unwinding activity to polarity substrate. Similarly, the purified NS2 protein possessed an ATPase activity and its enzyme activity was 0.276 μmol gg^-1 h^-1 in this study. The results indicated that the non- structure protein which encoded by the gene of BmDNV-Z NS2 possesses the biological activities of helicase and ATPase, and the helicase prefers to polarity substrates. Based on these results, it is speculated that the gene of BmDNV-Z NS2 plays an important role in the viral DNA replication. 相似文献
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铜绿假单胞菌脂肪酶Lipase基因的原核表达(英文) 总被引：3，自引：1，他引：3
[Objective] The aim of this study was to investigate the prokaryotic expression of pseudomonas aeruginosa Lipase gene.[Method]Lipase gene was amplified by PCR from the genome DNA of pseudomonas aeruginosa,and its nucleotide sequence was determined.The prokaryotic expression vector of Lipase gene was constructed by the gene recombination technique.The protein expression was induced for 4 hours by IPTG with the final concentration of 1.0 mmol/L,and then SDS-PAGE electrophoresis was analyzed.[Result]The sequence of mature peptides in Lipase gene cloned from pseudomonas aeruginosa had a 99.36% homology with that of pseudomonas aeruginosa lipase submitted in NCBI,so the prokaryotic expression vector of Lipase gene pET32a-Lip was successfully constructed.Furthermore,the results of SDS-PAGE electrophoresis showed that the target gene was expressed highly and effectively.[Conclusion]The cloned pseudomonas aeruginosa lipase with its signal peptide could be normally expressed in E.coli and also used for further study. 相似文献
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为构建牛瑟氏泰勒虫双拷贝p23表面蛋白基因真核表达质粒,根据GenBank牛瑟氏泰勒虫p23表面蛋白基因序列（D84447）,分别设计2对特异性引物,利用全血基因组DNA提取试剂盒提取牛瑟氏泰勒虫基因组DNA,采用SOE—PCR技术构建双拷贝p23基因,克隆到pMD-18-T载体上,经过PCR、酶切鉴定及测序后,亚克隆到pVAX-Ⅰ真核表达载体上,经过鉴定后采用脂质体法将重组质粒pVAXI-2p23转染到BHK-21细胞,用IFA和RT—PCR来鉴定目的基因的表达情况．结果表明,成功构建了牛瑟氏泰勒虫双拷贝p23表面蛋白基因真核表达质粒,并在BHK-21细胞中获得表达． 相似文献
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[目的]为研制牛瑟氏泰勒虫病基因工程苗和诊断试剂盒提供依据。[方法]通过PCR方法从牛瑟氏泰勒虫基因组DNA中克隆了1个P23基因,连接到pGEM-T-Easy载体中。运用生物信息学方法对该基因进行分析。[结果]P23基因全长为684 bp,包含1个长672 bp的开放阅读框,编码223个氨基酸,相对分子量是25.886 kD,等电点pI为9.22,包括1段19个氨基酸组成的信号肽和2段跨膜区。该序列在GenBank上的注册号为EU573168,与瑟氏泰勒虫Chitose型(D84446)和Ikeda型(D84447)的同源性分别为99%、90%。[结论]P23基因编码的蛋白有较好稳定性和免疫原性,可作为制备牛瑟氏泰勒虫基因疫苗的候补抗原。 相似文献
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小麦秸秆碳氮比调控施用对烟叶氮磷钾吸收的影响 总被引：1，自引：0，他引：1
[目的]研究小麦秸秆碳氮比调控施用对烟叶氮磷钾吸收的影响.[方法]2008～2009夏种季节于云南省曲靖市板桥镇进行田间小区试验,探讨小麦秸秆及其碳氮比调控后施用对烤烟产量、产值及上、中、下部烟叶对氮、磷、钾含量及累积吸收量的影响.[结果]小麦秸秆无论单独施用,还是碳氮比调控后施用均能促进烟叶产量、产值显著提高,同时,烟叶钾含量、钾和氮的累积吸收量也均有显著增加,而且小麦秸秆进行碳氮比调控后施用更有利于烟叶钾的吸收.与不施用秸秆相比,小麦秸秆单独施用、小麦秸秆+苕子、小麦秸秆+油枯、小麦秸秆+尿素进行碳氮比调控后施用,烟叶产量分别提高6.59%、3.58%、5.98%、8.80%,烟叶钾含量分别提高3.85%、7.76%、8.82%、11.21%,烟叶钾的总累积量分别提高10.71%、11.62%,15.32%、21.01%,烟叶氮的总累积量分别提高9.76%、1.22%、8.14%、14.00%;而不同处理之间烟叶氮含量无显著差异,烟叶对磷的吸收降低.[结论]在烤烟生产中施用高碳氮比小麦秸秆,即要注意补充氮素调节碳氮比,也要考虑磷素的补充.
Abstract：
[Objective] The aim was to study the effects of regulation C/N ratio wheat straw application on tobacco nitrogen,phosphorus,and potassium uptake.[Method]Effects of regulation C/N ratio wheat straw application on the flue-cured tobacco yield,output value,nitrogen,phosphorus and potassium content and cumulative uptake of the upper,middle and bottom leaf were studied by using the field plot experiments at Banqiao town,Qujing City,Yunnan Province during the 2008-2009 summer growing seasons, [Result] The results showed that the application of wheat straw alone or after C/N regulation,could significantly increase tobacco production,potassium content,the potassium and nitrogen accumulation amount of leaf,and was more conducive to the potassium uptake of tobacco leaf with wheat straW application after C/N regulation.Compared with non-straw application,the yield of tobacco increased by6.59%,3.58%,5.98%,8.80%with application of wheat straw alone,wheat straw and vetch,wheat straw and oilseed cake,wheat straw and urea nitrogen,the potassium content in tobacco leaf increasod by 3.85%,7.76%,8.82%,11.21%,respectively,the total potassium cumulative amount of leat increased by 10.71%,11.62%,15.32%,21.01%and the total nitrogen cumulative amount increased by9.76%,1.22%,8.14%,14.00%.However,the differences of tobacco leaf nitrogen content between the different treatments were not significant,the phosphorus uptake of tobacco leaf decreased. [Conclusion] The application of high C/N ratio wheat straw in flue-cured tobacco production,which should be concerned not only to adjust C/N ratio by adding nitrogen,but also considering additional phosphorus application. 相似文献
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牛瑟氏泰勒虫P23表面蛋白基因表达条件的优化 总被引：1，自引：0，他引：1
将已构建并测序正确的含有牛瑟氏泰勒虫P23表面蛋白基因的pGEX-4T—P23转化菌用IPTG进行诱导表达,经SDS—PAGE电泳可检测到相对分子量为46．0ku的融合蛋白．根据SDS—PAGE确定融合蛋白的最佳表达条件,结果显示诱导时机和时间是影响表达的主要因素,诱导温度和IPTG浓度次之;确定最佳诱导时机为转接种后2．0h,最佳诱导温度34℃,最佳诱导时间6．0h,最适IPTG浓度0．08mmol／L．表达产物主要以包涵体存在,在优化条件下融合蛋白的表达量经Bandscan5．0软件分析约占菌体总蛋白的31．7％． 相似文献
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牛体外发育胚胎特定阶段差异表达基因的研究 总被引：1，自引：0，他引：1
[目的]研究不同发育时期牛体外受精胚胎在基因表达模式上的差异.[方法]利用单个胚胎mRNA差异显示技术,对单个8细胞期胚胎与囊胚进行mRNA差异显示,获得1条特异表达条带,对其进行克隆、测序,并与GenBank进行对比.[结果]该序列与牛核糖体蛋白131基因(ribosomal protein 131,RPL3])具有99%的同源性.采用实时定量PCR技术检测8细胞期和囊胚期胚胎RPL31的mRNA表达量,结果表明,RPL31在8细胞期胚胎的相对表达量为囊胚期胚胎的3.2倍.[结论]为揭示和阐明控制牛早期胚胎发育的相关机理提供依据.
Abstract：
The cattle different stage embryos obtained from in vitro was studied using the technology of single preimplantation embryo mRNA different display:single 8-cell and blastocyst stage embryos were studied using technology of mRNA different display and one different fragment was found.The result suggested that this fragment displayed high homology (99%) to cattle mRNA for ribosomal protein L31.Then to detect the expression of RPL31 mRNA in 8 cell and blastocyst stage embryos by real-time quantitative PCR,the result showed the relative amount of 8 cells was 3.2 times of blastocyst's. 相似文献
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底栖鱼类对水田上覆水中磷素动态的扰动效应 总被引：2，自引：0，他引：2
[目的]研究底栖鱼类泥鳅对水田上覆水中磷素动态的扰动效应,探讨生物扰动机制.[方法]基于模拟试验,使用离子色谱法和分光光度法,对比分析上覆水中磷素含量在有/无泥鳅活动时的差异.[结果]扰动组的TP、DTP和PP浓度在试验开始阶段与对照组无显著差异,在试验中、后期显著高于对照(P<0.05).扰动组要的PP/TP高于对照组,扰动组中TP浓度的增加主要是由于PP的增加,扰动组的DIP/DT在试验中、后期显著高于对照(P<0.05).[结论]底栖鱼类对水田上覆水中的磷素产生了扰动作用,增加了水稻生长可利用的的磷素养分.
Abstract：
[Objective] The research aimed to investigate the bioturbation effects of benthic fish Misgurnus anguillicaudatus on phosphorus dynamic in overlying water of paddy field,as well as to explore the bioturbation mechanism.[Method]Based on simulation experiment,the phosphorus contents in overlying water were analyzed comparatively with and without Misgurnus anguillicaudatus by the using of ion chromatography and spectrophotometry. [Result] The concentrations of total phosphorus (TP),dissolved total phosphorus(DTP)and particular phosphorus(PP) in bioturbation group had no significant differences with those in control group in initial stage of experiment,and became significantly higher than control group in middle and late stages of experiment(P<0.05).The PP/TP ratios in bioturbation group were bigger than those in control group,the increase of TP concentration in bioturbation group was mainly due to the increase of PP.The ratios of dissolved inorganic phosphorus(DIP) to DTP (DIP/DTP) were significantly bigger than those in control group in middle and late stages of experiment (P<0.05). [Conclusion] The benthic fish had bioturbation effects on phosphorus in overlying water of paddy field,which increased the available phosphorus for rice growth. 相似文献
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[目的]构建抗黄瓜花叶病毒RNAi载体,并将载体转入烟草.[方法]采用RT-PCR方法,扩增黄瓜花叶病毒NS04加工番茄分离物的RNA2基因组的序列选取CMV RAN2基因组中的复制酶片段作为靶序列,构建pBi35SCR2真核表达载体,并对表达载体时行鉴定;通过农杆菌介导的方法将表达载体转入烟草,用PCR的方法检测载体是否转入.[结果]系统进化树分析结果表明,RNA2中编码CMV-2a的序列与中国浙江的DQ412731 分离物有较高核苷本乡酸及氨基酸同源性,分别达到98.0%和96.5%;RCR结果表明,试验成功构建了pBi35SCR2真核表达载体,并成功将表达载体转入烟草[结论]试验获得的转基因烟草可作为后期攻毒试验的材料,并为研究加工番茄抗黄瓜花叶病毒奠定了基础.
Abstract：
[Objective] Aimed to construct RNAi vector resistant to cucumber mosaic virus and transferred this vector into tobacco.[Method]RT-PCR method was used to amplify cucumber mosaic virus NSO4 and process RNA2 gene sequen of tomato isolates.The analysis results of phylogenetic tree demonstrated that the sequence in RNA2 encoded CMV-2a had 98.0% and 96.5% homology with nucleotide and amino acid of DQ412731 isolate of Zhejiang,China.The replicase fragment in CMV RAN2 gene was taken as target sequence to construct pBi35SCR2 eukaryotic expression vector,then the expression vector was identified.Through agrobacterium-mediated method,the expression vector was transferred into tabacco and PCR method was used to check the transfer.The PCR results demonstrated that the experiment had successfully construct eukaryotic expression vector of pBi35SCR2 and the expression vector was successfully transferred into tabacco. [Conclusion] The obtained transgenic tobacco could be used as challenge test material in following experiment and provided foundation for studying processing tomato resist cucumber mosaic virus. 相似文献
18.
一株产紫杉醇的曼地亚红豆杉内生真菌的分离及鉴定 总被引：1，自引：0，他引：1
[目的]分离并鉴定1株产紫杉醇的曼地亚红豆杉内的内生真菌.[方法]从曼地亚红豆杉树皮内表皮中分离得到32株内生真菌,并通过高效液相色谱法检测其发酵产物.[结果]筛选获得1株可以产紫杉醇的内生真菌M57,其紫杉醇产量为45～50μg/L,并通过对M57菌落的形态学观察以及18S rDNA序列分析初步将其鉴定为根霉属(Rhizopus)真菌.[结论]该菌株的发现为微生物发酵法生产紫杉醇提供了具有潜在应用价值新的菌种.
Abstract：
[Objective] The aim was to isolate and identify a taxol-producing endophytic fungus from Taxus media.[Method]32 strains of endophytic fungi were identified form the inner bark of T.media,and their fermentation products were detected by high performance liquid chromatography (HPLC). [Result] Through the screening,a strain of taxol-producing endophytic fungi M57 was obtained,which could produce 45-50 μg/L of taxol,and M57 was defined as Rhizopus sp.through morphological observation and 18S rDNA sequence analysis. [Conclusion] The finding of Rhizopus sp.M57 provided a promising strain for producing taxol with taxol-producing fungi fermentation process. 相似文献