A dwarf mutant strain of Pharbitis nil, Uzukobito (kobito), has defective brassinosteroid biosynthesis.
A dwarf mutant strain of Pharbitis nil, Uzukobito (kobito), has defective brassinosteroid biosynthesis.
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DOI：
10.1046/j.1365-313x.2003.01887.x
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发表时间：
2003-11
期刊：
影响因子：
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通讯作者：
Yoshihito Suzuki;K. Saso;S. Fujioka;S. Yoshida;E. Nitasaka;S. Nagata;H. Nagasawa;S. Takatsuto;I. Yamaguchi
中科院分区：
文献类型：
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作者：
Yoshihito Suzuki;K. Saso;S. Fujioka;S. Yoshida;E. Nitasaka;S. Nagata;H. Nagasawa;S. Takatsuto;I. Yamaguchi
日本牵牛花（Pharbitisnil）是一种模式植物，其特征在于具有大量的自发突变体。隐性突变体Uzukobito表现出强烈的矮化性，叶片深绿色，具皱纹。通过应用油菜素内酯（一种具有生物活性的油菜素类固醇（BR））拯救了表型，表明Uzukobito是BR缺陷型突变体。对Uzukobito及其亲本野生型植物中内源BR水平的详细分析表明，Uzukobito在（24 R）-24-甲基-5 α-胆甾烷-3-酮下游具有较低水平的BR，（22 S，24 R）-22-羟基-24-甲基-5 α-胆甾烷-3-酮的活性高于野生型植物，而它们的直接前体（24 R）-24-甲基胆甾-4-烯-3-酮和（22 S，24 R）-22-羟基-24-甲基胆甾-4-烯-3-酮在Uzukobito中积累相对较多。这些结果表明Uzukobito在（24 R）-24-甲基胆甾-4-烯-3-酮和（22 S，24 R）-22-羟基-24-甲基胆甾-4-烯-3-酮向其5 α-还原形式的转化中具有缺陷，该转化由拟南芥中的去黄化2（DET 2）催化。克隆了水稻疫霉DET 2基因的直向同源物（PnDET 2），并显示在拟南芥中所有推定的基因中与DET 2具有最大的相似性。Uzukobito在推导的PnDET 2的氨基酸序列中有一个氨基酸由Glu 62替换为Val 62。在COS-7细胞中表达的重组PnDET 2被发现是功能性类固醇5 α-还原酶（S5 α R），其将（24 R）-24-甲基胆甾-4-烯-3-酮转化为（24 R）-24-甲基-5 α-胆甾烷-3-酮，而具有突变的PnDET 2没有显示任何催化活性。这表明植物S5 alphaR可以转化内在底物。所有这些结果清楚地表明，Uzukobito表型是由PnDET 2上的突变引起的，并且在大量的P. nil突变体中已经在分子水平上表征了形态突变体。
Japanese morning glory (Pharbitis nil) is a model plant characterized by a large stock of spontaneous mutants. The recessive mutant Uzukobito shows strong dwarfism with dark-green rugose leaves. The phenotype was rescued by the application of brassinolide, a bioactive brassinosteroid (BR), indicating that Uzukobito was a BR-deficient mutant. A detailed analysis of the endogenous BR levels in Uzukobito and its parental wild-type plant showed that Uzukobito had a lower level of BRs downstream of (24R)-24-methyl-5alpha-cholestan-3-one and (22S, 24R)-22-hydroxy-24-methyl-5alpha-cholestan-3-one than those in wild-type plants, while their immediate precursors (24R)-24-methylcholest-4-en-3-one and (22S, 24R)-22-hydroxy-24-methylcholest-4-en-3-one accumulated relatively more in Uzukobito. These results indicate that Uzukobito had a defect in the conversion of (24R)-24-methylcholest-4-en-3-one and (22S, 24R)-22-hydroxy-24-methylcholest-4-en-3-one to their 5alpha-reduced forms, which is catalyzed by de-etiolated2 (DET2) in Arabidopsis. The P. nil ortholog of the DET2 gene (PnDET2) was cloned and shown to have the greatest similarity to DET2 among all the putative genes in Arabidopsis. Uzukobito had one amino acid substitution from Glu62 to Val62 in the deduced amino acid sequence of PnDET2. Recombinant PnDET2 expressed in COS-7 cells was found to be a functional steroid 5alpha-reductase (S5alphaR) converting (24R)-24-methylcholest-4-en-3-one to (24R)-24-methyl-5alpha-cholestan-3-one, while PnDET2 with the mutation did not show any catalytic activity. This shows that a plant S5alphaR can convert an intrinsic substrate. All these results clearly demonstrate that the Uzukobito phenotype resulted from a mutation on PnDET2, and a morphological mutant has been characterized at the molecular level among a large stock of P. nil mutants.