拟南芥花粉和胚胎两个突变体的筛选和分子遗传研究
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摘要
通过卡那霉素抗性(由突变体中标记基因编码)非孟德尔分离,从Ds插入的拟南芥突变体库中筛选到一个雄配子体发育突变体:ovp1(over vacuolatedpollen)和一个早期胚胎发生突变体eed1(early embryo defect),我们对这两个突变体的表型和相关基因进行了研究。
光学和电子显微镜观察及细胞学分析结果表明ovp1突变体有约50%的花粉粒在二细胞时期已经明显空泡化(over vacuolated),随后发生皱缩并最终败育。四分体分析和正反交实验进一步证明ovp1杂合体是一个雄配子体致死突变体。反向PCR_和序列分析结果表明Ds插入在At2G01560基因的第一个外显子内。等位突变体分析和分子互补证明ovp1的表型是由Ds插入该基因引起的。GFP∷OVP1融合蛋白显示OVP1定位于细胞核上。启动子-GUS染色、RNA原位杂交实验表明At2G01560在成熟花粉粒中特异表达。这些结果说明OVP1是拟南芥中一个特异性调控雄配子体发育的必须因子。
观察eed1果荚内的弯曲子叶胚时期的种子,发现约20%的种子变黄退化。整体透明观察发现部分eed1胚胎早期细胞表现为不正常的细胞分裂模式,部分胚胎细胞膨大、细胞之间联系松散,使胚胎形态不正常。异常胚胎在心形胚前退化,导致种子皱缩变黄。正反交结果证明eed1是纯合胚胎致死的突变体。反向PCR和序列分析结果表明Ds插入在At2G26120基因的终止密码子后361bp处。分子互补实验表明At2G26120编码EED1。启动子-GUS染色、RNA原位杂交和半定量RT实验表明At2G26120在各个器官都有表达,尤其是根尖、叶片的周缘、花粉粒,和Dr-5-GUS很相似,暗示该基因对早期胚胎发育模式的作用可能涉及生长素信号转导途径。
To further understand mechanisms that control male gametogenesis and embryo development,we performed an genetic screen for mutations that caused distorted Mendelian segregation in Ac/Ds transposon system in Arabidopsis thaliana.One mutant(ovp1)that displayed defective pollen development and another mutant(eed1) that displayed defective embryo development were focused on.The mutant phenotypes and disrupted gene molecular genetic research were studied.
Phenotypic analyses revealed that ovp1 heterozygotes have about 50%aborted mature pollen.Reciprocal crosses and pollen tetrad directly demonstrate that ovp1 is a male gametophytic lethal mutant in the Landsberg erecta ecotype.Ds insertion resulted in At2G01560.Situ hybridization and OVP1_(promoter)∷GUS analysis revealed that OVP is mature pollen-specific expression.Subcellular localization study demonstrated that the OVP1 is localized to cell nucleus.
eed1 displayed 20%embryo defects,and whole stain-clearing technique revealed that the embryo development was arrested before heart embryo stage in eed1. Reciprocal crosses demonstrate that eed1 homozygote is lethal in the Landsberg erecta ecotype.Ds insertion resulted in 3'-end of At2G26120,and we can't obtain eed1 homozygote plant.RT-PCR and promoter-GUS analysis revealed that EED1 is expressed ubiquitously in Arabidopsis plants and strongly expressed in root tip, mature pollen,leaf edge.
光学和电子显微镜观察及细胞学分析结果表明ovp1突变体有约50%的花粉粒在二细胞时期已经明显空泡化(over vacuolated),随后发生皱缩并最终败育。四分体分析和正反交实验进一步证明ovp1杂合体是一个雄配子体致死突变体。反向PCR_和序列分析结果表明Ds插入在At2G01560基因的第一个外显子内。等位突变体分析和分子互补证明ovp1的表型是由Ds插入该基因引起的。GFP∷OVP1融合蛋白显示OVP1定位于细胞核上。启动子-GUS染色、RNA原位杂交实验表明At2G01560在成熟花粉粒中特异表达。这些结果说明OVP1是拟南芥中一个特异性调控雄配子体发育的必须因子。
观察eed1果荚内的弯曲子叶胚时期的种子,发现约20%的种子变黄退化。整体透明观察发现部分eed1胚胎早期细胞表现为不正常的细胞分裂模式,部分胚胎细胞膨大、细胞之间联系松散,使胚胎形态不正常。异常胚胎在心形胚前退化,导致种子皱缩变黄。正反交结果证明eed1是纯合胚胎致死的突变体。反向PCR和序列分析结果表明Ds插入在At2G26120基因的终止密码子后361bp处。分子互补实验表明At2G26120编码EED1。启动子-GUS染色、RNA原位杂交和半定量RT实验表明At2G26120在各个器官都有表达,尤其是根尖、叶片的周缘、花粉粒,和Dr-5-GUS很相似,暗示该基因对早期胚胎发育模式的作用可能涉及生长素信号转导途径。
To further understand mechanisms that control male gametogenesis and embryo development,we performed an genetic screen for mutations that caused distorted Mendelian segregation in Ac/Ds transposon system in Arabidopsis thaliana.One mutant(ovp1)that displayed defective pollen development and another mutant(eed1) that displayed defective embryo development were focused on.The mutant phenotypes and disrupted gene molecular genetic research were studied.
Phenotypic analyses revealed that ovp1 heterozygotes have about 50%aborted mature pollen.Reciprocal crosses and pollen tetrad directly demonstrate that ovp1 is a male gametophytic lethal mutant in the Landsberg erecta ecotype.Ds insertion resulted in At2G01560.Situ hybridization and OVP1_(promoter)∷GUS analysis revealed that OVP is mature pollen-specific expression.Subcellular localization study demonstrated that the OVP1 is localized to cell nucleus.
eed1 displayed 20%embryo defects,and whole stain-clearing technique revealed that the embryo development was arrested before heart embryo stage in eed1. Reciprocal crosses demonstrate that eed1 homozygote is lethal in the Landsberg erecta ecotype.Ds insertion resulted in 3'-end of At2G26120,and we can't obtain eed1 homozygote plant.RT-PCR and promoter-GUS analysis revealed that EED1 is expressed ubiquitously in Arabidopsis plants and strongly expressed in root tip, mature pollen,leaf edge.
引文
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Cannon MC,Terneus K,Hall Q,Tan L,Wang Y,Wegenhart BL,Chen L,Lamport DT,Chen Y,Kieliszewski MJ(2008)Self-assembly of the plant cell wall requires an extensin scaffold.Proc Natl Acad Sci USA 105:2226-2231
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Ciapa B,Chiri S(2000)Egg activation:upstream of the fertilization calcium signal.Biol Cell 92:215-233
Clough SJ and Bent AF(1996)Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J 16:735-743
Colcombet J,Boisson-Dernier A,Ros-Palau R,Vera CE,Schroeder JI(2005)Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 are essential for tapetum development and microspore maturation.Plant Cell 17:3350-3361
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Durbarry A,Vizir I,Twell D(2005).Male germ line development in Arabidopsis.duo pollen mutants reveal gametophytic regulators of generative cell cycle progression.Plant Physiol 137:297-307
Friml J,Vieten A,Sauer M,Weijers D,Schwarz H,Hamann T,Offringa R,J(u|¨)rgens G(2003)Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis.Nature 426:147-53
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