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
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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 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.
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.
引文
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[11]刘士平,王璐,薛艳红,寿惠霞.ARF-GEF基因家族的研究进展.遗传,2009,31:982–992Liu S P,Wang L,Xue Y H,Shou H X.Research progress in ARF-GEF gene family.Hereditas(Beijing),2009,31:982–992(in Chinese with English abstract)
[12]Wang L,Liao F L,Zhu L,Peng X B,Sun M X.NtGNL1 is involved in embryonic cell division patterning,root elongation,and pollen tube growth in tobacco.New Phytol,2008,179:81–93
[13]Liao F,Wang L,Yang L B,Peng X,Sun M.NtGNL1 plays an essential role in pollen tube tip growth and orientation likely via regulation of post-Golgi trafficking.PloS ONE,2010,5(10):e13401
[14]Liao F,Wang L,Yang L B,Zhang L,Peng X B,Sun M.Antisense oligodeoxynucleotide inhibition as an alternative and convenient method for gene function analysis in pollen tubes.PLoS ONE,2013,8(3):e59112
[15]He Y C,He Y Q,Qu L H,Sun M X,Yang H Y.Tobacco zygotic embryogenesis in vitro:the original cell wall of the zygote is essential for maintenance of cell polarity,the apical-basal axis and typical suspensor formation.Plant J,2007,49:515–527
[16]Sun M X,Moscatelli A,Yang H Y,Cresti M.In vitro double fertilization in Nicotiana tabacum(L.):fusion behavior and gamete interaction traced by video-enhanced microscopy.Sex Plant Reprod,2000,12:267–275
[17]Liu A W,Narayanan K K,AndréC P,Kaleikau E K,Walbot V.Co-transcription of orf25 and coxIII in rice mitochondria.Curr Genet,1992,21:507–513
[18]Sun C,Ridderstrle K,Hglund A S,Larsson L G,Jansson C.Sweet delivery-sugar translocators as ports of entry for antisense oligodeoxynucleotides in plant cells.Plant J,2007,52:1192–1198
[19]DinE,Tóth S Z,Schansker G,Ayaydin F,Kovács L,Dudits D,Bottka S.Synthetic antisense oligodeoxynucleotides to transiently suppress different nucleus-and chloroplast-encoded proteins of higher plant chloroplasts.Plant Physiol,2011,157:1628–1641
[20]Bolte S,Talbot C,Boutte Y,Catrice O,Read N D,SatiatJeunemaitre B.FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells.J Microscopy,2004,214:159–173
[21]Crooke S T,Bennett C F.Progress in antisense oligonucleotide therapeutics.Annu Rev Pharmacol Toxicol,1996,36:107–129
[22]Wen M,Li B,Ouyang Y,Luo Y,Li S.Preparation and quality test of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe:a preliminary study.Ann Biomed Eng,2009,37:1240–1250
[23]de Graaf B H,Cheung A Y,Andreyeva T,Levasseur K,Kieliszewski M,Wu H M.Rab11 GTPase-regulated membrane trafficking is crucial for tip-focused pollen tube growth in tobacco.Plant Cell,2005,17:2564–2579
[24]Voigt B,Timmers A C,amaj J,Hlavacka A,Ueda T,Preuss M,Menzel D.Actin-based motility of endosomes is linked to the polar tip growth of root hairs.Eur J Cell Biol,2005,84:609–621
[25]许珊,钱洁,宋馨,祝建.拟南芥愈伤组织细胞类58K蛋白定位及多泡体的分泌途径.中国科学(C辑:生命科学),2008,38:836–840Xu S,Qian J,Song Q,Zhu J.58K protein analogues localization in Arabidopsis callus cells and the secretory pathway of multivesicular bodies.Sci China:Ser C,2008,38:836–840(in Chinese)
[26]Vidali L,McKenna S T,Hepler P K.Actin polymerization is essential for pollen tube growth.Mol Biol Cell,2001,12:2534–2545
[27]陈琼,黄善金,于荣.植物微丝骨架动态变化的调节.植物生理学报,2011,47:18–26Chen Q,Huang S J,Yu R.Regulation of plant actin dynamics.Plant Physiol J,2011,47:18–26(in Chinese with English abstract)
[28]Crum C,Johnson J D,Nelson A,Roth D.Complementary oligodeoxynucleotide mediated inhibition of tobacco mosaic virus RNA translation in vitro.Nucl Acids Res,1988,16:4569–4581
[2]Zamecnik 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
[3]Gewirtz A M,Sokol D L,Ratajczak M Z.Nucleic acid therapeutics:state of the art and future prospects.Blood,1998,92:712–736
[4]Dagle J M,Weeks D L.Oligonucleotide-based strategies to reduce gene expression.Differentiation,2001,69:75–82
[5]Zheng 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
[6]Cho-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
[7]Potts 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
[8]Estruch 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
[9]Moutinho A,Camacho L,Haley A,Pais M S,Trewavas A,MalhóR.Antisense perturbation of protein function in living pollen tubes.Sexual Plant Reproduct,2001,14:101–104
[10]Sun C,Hglund 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
[11]刘士平,王璐,薛艳红,寿惠霞.ARF-GEF基因家族的研究进展.遗传,2009,31:982–992Liu S P,Wang L,Xue Y H,Shou H X.Research progress in ARF-GEF gene family.Hereditas(Beijing),2009,31:982–992(in Chinese with English abstract)
[12]Wang L,Liao F L,Zhu L,Peng X B,Sun M X.NtGNL1 is involved in embryonic cell division patterning,root elongation,and pollen tube growth in tobacco.New Phytol,2008,179:81–93
[13]Liao F,Wang L,Yang L B,Peng X,Sun M.NtGNL1 plays an essential role in pollen tube tip growth and orientation likely via regulation of post-Golgi trafficking.PloS ONE,2010,5(10):e13401
[14]Liao F,Wang L,Yang L B,Zhang L,Peng X B,Sun M.Antisense oligodeoxynucleotide inhibition as an alternative and convenient method for gene function analysis in pollen tubes.PLoS ONE,2013,8(3):e59112
[15]He Y C,He Y Q,Qu L H,Sun M X,Yang H Y.Tobacco zygotic embryogenesis in vitro:the original cell wall of the zygote is essential for maintenance of cell polarity,the apical-basal axis and typical suspensor formation.Plant J,2007,49:515–527
[16]Sun M X,Moscatelli A,Yang H Y,Cresti M.In vitro double fertilization in Nicotiana tabacum(L.):fusion behavior and gamete interaction traced by video-enhanced microscopy.Sex Plant Reprod,2000,12:267–275
[17]Liu A W,Narayanan K K,AndréC P,Kaleikau E K,Walbot V.Co-transcription of orf25 and coxIII in rice mitochondria.Curr Genet,1992,21:507–513
[18]Sun C,Ridderstrle K,Hglund A S,Larsson L G,Jansson C.Sweet delivery-sugar translocators as ports of entry for antisense oligodeoxynucleotides in plant cells.Plant J,2007,52:1192–1198
[19]DinE,Tóth S Z,Schansker G,Ayaydin F,Kovács L,Dudits D,Bottka S.Synthetic antisense oligodeoxynucleotides to transiently suppress different nucleus-and chloroplast-encoded proteins of higher plant chloroplasts.Plant Physiol,2011,157:1628–1641
[20]Bolte S,Talbot C,Boutte Y,Catrice O,Read N D,SatiatJeunemaitre B.FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells.J Microscopy,2004,214:159–173
[21]Crooke S T,Bennett C F.Progress in antisense oligonucleotide therapeutics.Annu Rev Pharmacol Toxicol,1996,36:107–129
[22]Wen M,Li B,Ouyang Y,Luo Y,Li S.Preparation and quality test of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe:a preliminary study.Ann Biomed Eng,2009,37:1240–1250
[23]de Graaf B H,Cheung A Y,Andreyeva T,Levasseur K,Kieliszewski M,Wu H M.Rab11 GTPase-regulated membrane trafficking is crucial for tip-focused pollen tube growth in tobacco.Plant Cell,2005,17:2564–2579
[24]Voigt B,Timmers A C,amaj J,Hlavacka A,Ueda T,Preuss M,Menzel D.Actin-based motility of endosomes is linked to the polar tip growth of root hairs.Eur J Cell Biol,2005,84:609–621
[25]许珊,钱洁,宋馨,祝建.拟南芥愈伤组织细胞类58K蛋白定位及多泡体的分泌途径.中国科学(C辑:生命科学),2008,38:836–840Xu S,Qian J,Song Q,Zhu J.58K protein analogues localization in Arabidopsis callus cells and the secretory pathway of multivesicular bodies.Sci China:Ser C,2008,38:836–840(in Chinese)
[26]Vidali L,McKenna S T,Hepler P K.Actin polymerization is essential for pollen tube growth.Mol Biol Cell,2001,12:2534–2545
[27]陈琼,黄善金,于荣.植物微丝骨架动态变化的调节.植物生理学报,2011,47:18–26Chen Q,Huang S J,Yu R.Regulation of plant actin dynamics.Plant Physiol J,2011,47:18–26(in Chinese with English abstract)
[28]Crum C,Johnson J D,Nelson A,Roth D.Complementary oligodeoxynucleotide mediated inhibition of tobacco mosaic virus RNA translation in vitro.Nucl Acids Res,1988,16:4569–4581