共查询到19条相似文献,搜索用时 250 毫秒
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昆虫转座子及在家蚕中的应用 总被引:2,自引:1,他引:1
概述了近年来广泛应用于昆虫的若干转座子及其基本构造和应用;着重讨论了piggyBac转座子的构造和转座机制及所作成的转基因昆虫种类及标记基因:同时列举了在家蚕转基因中的实例. 相似文献
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通过PCR克隆,从家蚕基因组中获得大小为975bp的DNA片段(登录号:EU352872),Blast搜寻结果显示,该片段DNA序列的52-449nt区域与野蚕mariner-like元件DNA(登录号:AB237562)的同源性达90%,推测为家蚕类mariner转座子相关序列,在乙酰胆碱酯酶基因的第5内含子、微卫星S1104-R序列、BmBRC-NZ3 mRNA终止码的下游序列、ErB·1基因的内含子均检测到家蚕类mariner元件相关序列的同源区,暗示家蚕类mariner元件在家蚕基因组中发生了多次转座,并在家蚕基因组的不同区域随着进化发生了歧化。 相似文献
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为了实现利用非转座子载体介导的转基因家蚕整体表达人胰岛素样生长因子Ⅰ(hIGF-Ⅰ),将hIGF-Ⅰ基因克隆进非转座子类型的昆虫细胞表达载体pIZT/V5-His,构建转基因载体pIZT/V5-His-hIGF-Ⅰ。利用精子介导法将该转基因载体导入家蚕卵,通过绿色荧光筛选并结合PCR和Dot blotting检测鉴定,表明已成功获得hIGF-Ⅰ转基因家蚕。对培育至G2代的转基因家蚕5龄幼虫蛋白质样品进行Western blotting分析,结果显示hIGF-Ⅰ在转基因家蚕中获得表达,重组hIGF-Ⅰ的分子质量约12.5 kD;ELISA检测hIGF-Ⅰ在G2代转基因家蚕5龄幼虫全蚕以及后部丝腺、脂肪体冻干粉中的质量比分别为65、411、469 ng/g。试验结果再次证实通过非转座子载体pIZT/V5-His介导可以将外源基因导入家蚕基因组,并实现外源基因在转基因家蚕中整体表达。 相似文献
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人表皮生长因子(human epidermal growth factor,hEGF)具有刺激细胞增殖的强大能力,是伤口愈合的关键因子.利用转基因技术,在家蚕后部丝腺利用丝素轻链基因(fibroin light chain,FibL)启动子驱动FibL-hEGF融合蛋白的表达.借助piggyBac转座子将FibL-hEGF融合基因导入到家蚕基因组,利用荧光检测总共获得8个转基因阳性卵圈,转基因阳性率为36.36%.PCR和反向PCR检测证明外源融合基因FibL-hEGF已被成功整合到家蚕基因组中,且随机插入到家蚕染色体的5个位点.Western blotting检测表明FibL-hEGF融合蛋白在家蚕后部丝腺中表达且成功分泌到蚕茧中,进一步证实了转基因家蚕的产生.本研究中利用piggyBac转座子介导的转基因技术成功获得了转基因家蚕并得到重组hEGF蚕丝,该蚕丝可用于制造伤口敷料. 相似文献
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《中国蜂业》2021,(5)
转座因子(TEs)是真核生物基因组的主要序列成分,对真核生物基因组的结构、功能及进化具有重大影响。近年来,虽然有关昆虫转座子的研究在不断增加,并且科学家成功地应用转座子进行了功能基因的挖掘,但相关研究主要集中于双翅目的果蝇中。本研究利用生物信息学方法,基于de novo预测和结构预测两种策略,对地熊蜂参考基因组中的转座子进行了详细鉴定、分类和注释,并鉴定出潜在活跃的转座子。结果显示:虽然转座子序列仅占地熊蜂基因组全部序列的3.74%,但其超家族种类繁多,鉴定出的33282个转座子分属于22个超家族。本研究鉴定出2种具有潜在活性的MITE转座子,它们很可能正在地熊蜂基因组中发生转座。本研究为利用活跃的转座子创制地熊蜂的突变体库进而挖掘熊蜂的功能基因奠定了重要基础。 相似文献
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家蚕转座子表达载体pSVHK1.4的构建 总被引:2,自引:0,他引:2
研究利用PCR方法扩增黑腹果蝇 (D .melanogaster)hsp70启动子 ,然后插入到质粒pcDNA3上水母绿色荧光蛋白基因gfp的上游 ,与之顺向拼接构建成含hsp70和gfp的重组质粒pCGH。将家蚕K1 4转座子序列从pUK1 .4亚克隆到真核表达载体pSVL中PvuⅡ位点 ,获得中间载体pSK1 .4。最后将hsp70启动子和gfp报告基因的融合片段从pCGH中亚克隆到质粒pSK1 .4中K1 .4片段上的BglⅡ位点处 ,从而完成对转座子表达载体pSVHK1 .4的构建。 相似文献
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Nuttapone SANGKANJANAVANICH Tsutomu KAKUDA Yasunori SUZUKI Yukako SASAKI Shinji TAKAI 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2021,83(8):1182
Rhodococcus equi is a Gram-positive facultative intracellular bacterium that causes pyogranulomatous pneumonia in foals and immunocompromised people. In the present study, signature-tagged transposon mutagenesis was applied for the negative selection of R. equi mutants that cannot survive in vivo. Twenty-five distinguishable plasmid-transposon (plasposon) vectors by polymerase chain reaction (PCR), each containing a unique oligonucleotide tag, were constructed and used to select the transposon mutants that have in vivo fitness defects using a mouse systemic infection model. Of the 4,560 transposon mutants, 102 mutants were isolated via a real-time PCR-based screening as the mutants were unable to survive in the mouse model. Finally, 50 single transposon insertion sites were determined via the self-cloning strategy. The insertion of the transposon was seen on the virulence plasmid in 15 of the 50 mutants, whereas the remaining 35 mutants had the insertion of transposon on the chromosome. The chromosomal mutants contained transposon insertions in genes involved in cellular metabolism, DNA repair and recombination, gene regulation, non-ribosomal peptide synthesis, and unknown functions. Additionally, seven of the chromosomal mutants showed a reduced ability to multiply in the macrophages in vitro. In this study, we have identified several biosynthetic pathways as fitness factors associated with the growth within macrophages and survival in mice. 相似文献
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piggyBac转座子介导的家蚕细胞转基因研究初探 总被引:5,自引:4,他引:1
为探讨稳定转化家蚕细胞表达外源基因的新方法,以家蚕核型多角体病毒(BmNPV)极早期蛋白基因(ie-1)启动子元件驱动新霉素抗性基因(neor),并克隆至具有绿色荧光蛋白基因(gfp)标记的昆虫转座子载体piggyBac中,构建转基因载体pigA3-IE-Neo。以该载体转染家蚕BmN细胞,用终浓度800μg/mL的遗传霉素(geneticin,G418)筛选3个月,获得了稳定转化的细胞,呈现绿色荧光的细胞数达80%以上。通过PCR鉴定证实了细胞基因组DNA中neor基因和gfp基因的存在。 相似文献
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Actinobacillus pleuropneumoniae is a gram-negative bacterium and is the causative agent of swine pleuropneumonia, a highly contagious respiratory disease. Biofilm formation is an important ability possessed by numerous bacterial pathogens. The purpose of this study was to identify and characterize biofilm mutants of A. pleuropneumoniae serotype 1 strain S4074 created using a mini Tn-10 transposon. The transposon library was screened to identify mutants with a modified ability to form biofilms in polystyrene microtiter plates. A total of 1200 mutants were screened and the analysis identified 24 mutants that exhibited abnormal biofilm formation, at least 16 unique genes were identified. Most genes identified in the enhanced-biofilm mutants encoded proteins with unknown functions, whereas most genes identified in the biofilm-reduced mutants encoded proteins related to transport, protein synthesis and nucleic acid synthesis. Approximately 50% of genes, including hns, potD2, ptsI, tig and rpmF, identified in our screen have been previously associated with biofilm formation in A. pleuropneumoniae and other bacterial species, and thus validated the screening method. The rest of genes identified, such as APL_0049, APL_0637 and APL_1572, have not been previously associated with biofilm formation. Interestingly, gene APL_0049 was previously seen among the genes differentially expressed during a natural infection of pig lungs. Preliminary characterization of the mutants was also initiated by assessing their hydrophobicity, their biofilm matrix composition and their ability to adhere to a polystyrene surface or NPTr cells. Based on the preliminary characterization, some of the mutants identified appear to have deficiencies during the initial attachment or growth of the biofilm. In conclusion, transposon mutagenesis analysis allowed the identification of new genes associated with biofilm formation in A. pleuropneumoniae. 相似文献
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为了研究7种家养动物全基因组微卫星的分布是否存在差异,本试验利用软件MSDB搜索了猪、马、牛、山羊、绵羊、鸡和犬7种家养动物全基因组微卫星序列,并对其进行了分析研究。初步结果显示,7种家养动物全基因组微卫星在数量、丰度和密度上都存在差异,其中数量、丰度和密度最高的是犬,共1 436 242个位点,数量最少的是鸡,共276 564个位点,但丰度和密度最低的是马,共430 760个位点。对这些微卫星的分析表明,所有物种全基因组中单碱基重复微卫星最丰富,六碱基重复微卫星数量最少,且6种重复类型的优势微卫星都富含碱基A和T。除这些共同点之外,微卫星的分布规律也存在差异。总的来说,牛、山羊、绵羊微卫星的分布规律最为相似,鸡与其他物种微卫星的分布规律相差最远。分析还发现,7种动物微卫星长度大多集中在12~20 bp之间,这可能是受到趋同选择压力的结果。据以上可以推测,物种间微卫星的分布存在差异,但也存在一定的保守性,且物种间亲缘关系越近,微卫星的分布也越相似。以上结果为以后微卫星的功能研究提供依据。 相似文献