期刊文献+

比较和分析NtGNL1的反义寡聚核苷酸抑制在烟草三种培养体系中的效果 被引量:2

Comparison and Analysis of Effects of Antisense Oligodeoxynucleotide Inhibition of NtGNL1 in Three Culture Systems of Tobacco
下载PDF
导出
摘要 为探讨反义寡聚核苷酸抑制(antisense oligodeoxynucleotides inhibition)技术在植物材料上的应用,以已知功能的NtGNL1(Nicotiana tabacum GNOM-Like1)为目标基因,寻找反义寡聚核苷酸抑制最适作用体系,并进一步分析NtGNL1的具体作用。根据目标基因mRNA序列设计反义寡聚核苷酸序列,商业合成后并将其添加到烟草胚珠、种子和花粉管离体培养的培养基中,以抑制NtGNL1的表达。结果表明,将反义寡聚核苷酸引入离体培养系统,短时间内抑制了目标基因mRNA的表达,但对胚胎发育过程的影响和种子萌发的抑制都不明显。在花粉离体萌发系统中,反义寡聚核苷酸抑制能够高效地引起目标基因mRNA的下调。对FM4-64染色的花粉管显微缩时观察发现,NtGNL1的下调能够引起囊泡分布和运输方向的改变。对花粉管膜流相关的5个基因的半定量分析也显示反义寡聚核苷酸进入花粉管后,导致3个基因的表达下调,暗示NtGNL1抑制表达会影响花粉管囊泡运输的多个节点。 To develop the antisense oligonucleotide inhibition technology in plant materials, we used the known-function-gene NtGNL1 as the target to establish the optimum application system and further analyze the specific role of NtGNL1. Based on the mRNA of target gene, we designed, commercially synthesized and then applied the antisense oligonucleotide sequences into the in vitro cultured ovules, seeds and pollen tubes to inhibit the expression of NtGNL1. The results revealed that the co-culture of an- tisense oligodeoxynucleotides hardly affected the pattern formation of the embryos, as well as the seeds germination, despite the expression of NtGNL1 decreasing during short periods. Significantly, the antisense oligodeoxynucleotides down-regulated the expression of target gene in the in vitro cultured pollen tubes. Microscopic time laps observation of pollen tubes by FM4-64 stain- ing indicated that the inhibition also changed the vesicles distribution model and the direction of vesicle trafficking. Furthermore, the mRNA expression of five genes related to membrane trafficking was also analyzed by semi-quantitative PCR, showing that three genes were down-regulated after the antisense oligodeoxynucleotides entered into the pollen tubes. All these results implied that the inhibition of NtGNL1 expression will affect several key points of vesicle trafficking in pollen tubes.
出处 《作物学报》 CAS CSCD 北大核心 2014年第2期355-361,共7页 Acta Agronomica Sinica
基金 国家自然科学基金项目(31100228) 浙江省自然科学基金项目(Y3110218) 浙江省"重中之重"学科"现代农业生物技术与作物病害防控"开放基金(ZC323012042) 浙江师范大学博士启动基金(ZC304010042)资助
关键词 反义寡聚核苷酸抑制 NtGNL1 离体培养 花粉管 囊泡运输 膜流 Antisense oligonucleotide inhibition NtGNL1 in vitro culture systems Pollen tube Endosome trafficking Mem-brane trafficking
  • 相关文献

参考文献28

  • 1Paterson B M, Roberts B E, Kuff E L. Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation. Proc Natl Acad Sci USA, 1977, 74: 4370-4374.
  • 2Zamecnik P C, Stephenson M L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci USA, 1978, 75: 280-284.
  • 3Gewirtz A M, Sokol D L, Ratajczak M Z. Nucleic acid therapeutics: state of the art and future prospects. Blood, 1998, 92: 712-736.
  • 4Dagle J M, Weeks D L. Oligonucleotide-based strategies to reduce gene expression. Differentiation, 2001, 69: 75-82.
  • 5Zheng H, Sahai B M, Kilgannon P, Fotedar A, Green D R. Specific inhibition of cell-surface T-cell receptor expression by antisense oligodeoxynucleotides and its effect on the production of an antigen-specific regulatory T-cell factor. Proc Natl Acad Sci USA, 1989, 86: 3758-3762.
  • 6Cho-Chung Y S, Nesterova M V, Severin E S, Vinogradov S V. Chemical modification enhances the inhibitory effect of regulatory subunit antisense oligodeoxynucleotide of cAMP-dependent protein kinase type I on cell proliferation. Biochem Int, 1991, 25: 767-73.
  • 7Potts J D, Dagle J M, Walder J A, Weeks D L, Runyan R B. Epithelial-mesenchymal transformation of embryonic cardiac endothelial cells is inhibited by a modified antisense oligodeoxynucleotide to transforming growth factor beta 3. Proc Natl Acad Sci USA, 1991, 88: 1516-1520.
  • 8Estruch J J, Kadwell S, Merlin E, Crossland L. Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase. Proc Natl Acad Sci USA, 1994, 91: 8837-8841.
  • 9Moutinho A, Camacho L, Haley A, Pais M S, Trewavas A, Malhó R. Antisense perturbation of protein function in living pollen tubes. Sexual Plant Reproduct, 2001, 14: 101-104.
  • 10Sun C, H?glund A S, Olsson H, Mangelsen E, Jansson C. Antisense oligodeoxynucleotide inhibition as a potent strategy in plant biology: identification of SUSIBA2 as a transcriptional activator in plant sugar signalling. Plant J, 2005, 44: 128-138.

二级参考文献142

  • 1王昕,种康.植物小G蛋白功能的研究进展[J].植物学通报,2005,22(1):1-10. 被引量:20
  • 2Tanchak M A, Fowkel C. The morphology of mulfivesicular bodies in soybean protoplasts and their role in endocytosis. Protoplasma, 1987, 138:173-182
  • 3Wileman T, Hxrdrno C, Stahl P. Receptor-mediated endocytosis. Biochem J, 1985, 232: 1-14
  • 4Segui-Simarro J M, Staehelin L A. Cell cycle-dependent changes in Golgi stacks, vacuoles, clathrin-coated vesicles and multivesicular bodies in meristematic cells ofArabidopsis thaliana: A quantitative and spatial analysis. Planta, 2006, 223:223-236
  • 5Pear J R, Kawagoe Y, Schrenckengost W E, et al. Higher plants contain homologs of the bacterial celt genes encoding the catalytic subunit of cellulose synthase. Proc Natl Acad Sci USA, 1996, 93:12637-12642
  • 6Perrin R M, DeRocher A E, Bar-Peled M, et al. Xyloglucan fucosyltransferase, an enzyme involved in plant cell wall biosynthesis. Science, 1999, 284:1976-1979
  • 7Gregory A C E, Smith C, Kerry M E, et al. Comparative subcellular immunolocation of polypeptides associated with xylan and canose synthases in French bean (Phaseolus valgaris) during secondary wall formation. Phytochemistry, 2002, 59:249-259
  • 8Bloom G S, Brashear T A. A novel 58-kDa protein associates with the Golgi apparatus and microtubules. J Biol Chem, 1989, 264: 16083-16092
  • 9Ktistakis N T, Roth M G, Bloom G S. PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A. Cell Biol, 1991, 113:1009-1023
  • 10Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacterriophage T4. Nature, 1970, 227:680-685

共引文献11

同被引文献11

引证文献2

二级引证文献2

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部