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作物学报 ›› 2014, Vol. 40 ›› Issue (02): 355-361.doi: 10.3724/SP.J.1006.2014.00355

• 研究简报 • 上一篇    下一篇

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

廖芳蕾1,王鲁2,辛可行1,陈文荣1,郭卫东1,*   

  1. 1 浙江师范大学化学与生命科学学院, 浙江金华 321004; 2 中国科学院武汉植物园, 湖北武汉 430074
  • 收稿日期:2013-03-30 修回日期:2013-09-08 出版日期:2014-02-12 网络出版日期:2013-11-14
  • 基金资助:

    本研究由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)和浙江师范大学博士启动基金(ZC304010042)资助。

Comparison and Analysis of Effects of Antisense Oligodeoxynucleotide Inhibition of NtGNL1 in Three Culture Systems of Tobacco

LIAO Fang-Lei1,WANG Lu2,XIN Ke-Xing1,CHEN Wen-Rong1,GUO Wei-Dong1,*   

  1. 1 College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China; 2 Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China
  • Received:2013-03-30 Revised:2013-09-08 Published:2014-02-12 Published online:2013-11-14

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。显微缩时对FM4-64染色的花粉管观察发现, 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。

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

Abstract:

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 antisense 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 staining 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.

Key words: Antisense oligonucleotide inhibition, NtGNL1, in vitro culture systems, Pollen tube, Endosome trafficking, Membrane trafficking3

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