兰州大学机构库 >生命科学学院
CKRC1在生长素合成中的作用以及与细胞分裂素作用关系的研究
Alternative TitleStudies on the function of CKRC1 in auxin biosynthesis and its relation to cytokinin action
周朝阳
Thesis Advisor郭光沁
2011-05-29
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Keyword拟南芥 CKRC1 细胞分裂素 生长素的合成 生长素的运输 根的生长
Abstract细胞分裂素影响植物生长和发育的很多方面。然而,细胞分裂素以及细胞分裂素与其它激素相互作用发挥功能的分子机制还是未知的。 实验室前期在含有细胞分裂素的培养基上分离筛选到了一株根卷曲生长的突变体(Cytokinin induced root curling_1),把它命名为ckrc1。ckrc1突变体表现出主根对细胞分裂素的不敏感性以及向地性(GR)缺陷的表型。通过反向PCR的方法克隆到了这一基因,T-DNA插入位置在At1g70560的第四个内含子区。ckrc1-1的等位突变体ckrc1-2和ckrc1-3表现出相似的表型,在含有细胞分裂素的培养基上根卷曲生长(张春广,2007)。 表型重复实验表明ckrc1突变体具有主根对细胞分裂素的不敏感性以及向地性(GR)缺陷的表型的表型,而且这一缺陷能够被外源的生长素(NAA, IAA, 2;4-D)或IPA所互补。 为了进一步确定ckrc1-1的表型是由At1g70560突变引起的,我们将35S::CKRC1转到ckrc1-1 突变体中,转基因植物在含有细胞分裂素的培养基上根不再卷曲生长可以将突变体的表型恢复到野生型的水平,证明突变体的表型是的确是由CKRC1突变引起的。 CKRC1编码一色氨酸氨基转移酶(TAA1),将生长素合成途径中的Trp转变成IPA,是生长素生物合成过程的重要成员之一。用GC-MS的方法测定植物内源生长素的含量结果显示在ckrc1-1突变体中生长素的含量明显降低。 为了进一步研究CKRC1组织表达的特异性。我们把长2.2kb的CKRC1启动子连上GUS报告基因转化野生型拟南芥。5个独立转基因株系的染色结果显示:CKRC1在茎顶端分生组织,子叶的维管组织,茎,萼片和雄蕊的花丝,初级和次级根都有较高的表达。 在整株和根尖水平上,用细胞分裂素和乙烯合成前体ACC处理野生型小苗和TAA1::TAA1:GFP转基因植物,Real-Time PCR和TAA1::TAA1:GFP转基因植物荧光信号分析表明细胞分裂素和乙烯能诱导CKRC1的表达。GC-MS分析表明细胞分裂素处理后生长素的含量在WT和ckrc1-1中分别增加41%和23%,说明细胞分裂素促进生长素的合成在很大程度上依赖于CKRC1。 进一步的研究分析表明,在细胞分裂素信号转导的突变体ahk3-1 和arr1-3/12-1中,细胞分裂素不能上调CKRC1的表达,而在乙烯信号转导突变体etr1-3中细胞分裂素却能上调CKRC1的表达。这些结果表明细胞分裂素可以通过AHK3 ARR1/12信号途径使CKRC1/TAA1表达上调进而增加IAA的含量,这个过程是不依赖于乙烯的。 我们进一步研究了细胞分裂素和乙烯对生长素极性运输的影响,3H-IAA极性运输的结果表明细胞分裂素能够抑制而乙烯能够促进生长素的向基和向顶运输。该结果进一步证实了细胞分裂素通过AHK3/ARR1/12信号途径而不是ETR1介导的乙烯信号途径来调节生长素运输的载体,进而达到对生长素运输的抑制。 综上所述,我们的结果表明细胞分裂素通过刺激生长素的合成抑制生长素的运输来调节根的生长和发育的。
Other AbstractCytokinin (CK) influences many aspects of plant growth and development, whose function often involves its intricate interactions with other phytohormones such as auxin and ethylene. However, the molecular mechanisms underlying the role of CK and its interactions with other growth regulators are still poorly understood. Here we describe the isolation and characterization of the Arabidopsis CK induced root curling1 (ckrc1) mutant. The ckrc1 mutant exhibits defective root gravitropic response (GR) and an increased resistance to CK in primary root growth. These defects can be rescued by exogenous auxin (IAA, NAA, 2, 4-D) and IPA. Inverse-PCR amplification of T-DNA flanking sequence identified a T-DNA insertion in the 4th intron of an annotated gene At1g70560. Allelic analysis and molecular complementation showed that At1g70560 was the CKRC1 gene. CKRC1 encodes a previously identified tryptophan aminotransferase (TAA1) involved in the indole-3-pyruvic acid (IPA) pathway of indole-3-acetic acid (IAA) biosynthesis. Consistent with a defect in auxin biosynthesis, GC-MS result showed that the endogenous IAA level in ckrc1-1 mutant was significantly reduced. To investigate the details of tissue-specific expression of CKRC1/TAA1, we introduced a CKRC1 promoter (2.2 kb) GUS reporter fusion (pCKRC::GUS) into Arabidopsis WT plants. Staining of the GUS activity in independent transgenic lines revealed that the CKRC1 promoter was highly active in the shoot apical meristem, vascular tissues of cotyledons, stems, sepals, stamen filaments, and primary and secondary roots. Furthermore, we show that CK up-regulates CKRC1/TAA1 expression in roots in AHK3/ARR1/12-dependent and ethylene-independent manner using chemical inhibitors and mutants. To more precisely determine the amount of the IAA increase upon the CK treatment, we conducted the GC-MS analysis of the endogenous IAA levels. The IAA level in 7-d old seedlings significantly increased by 41% in WT, but only 23% (not statistically significant) in ckrc1-1, indicating that a significant part of CK induced auxin biosynthesis is dependent on CKRC1. [3H]IAA transport assays showed that ZT significantly reduced the rate of both acropetal (a-, from shoot to root) and basipetal (b-, from root tip to the elongation zone) PAT in WT and ckrc1-1 roots, through its AHK3 ARR1/12 signalling pathway that is independent of ethylene. Our results suggest that CK regulates root growth and development not only by down-reg...
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/222035
Collection生命科学学院
Recommended Citation
GB/T 7714
周朝阳. CKRC1在生长素合成中的作用以及与细胞分裂素作用关系的研究[D]. 兰州. 兰州大学,2011.
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