Study on mechanisms of specific expression of gibberellin 3-oxidase genes in carrot somatic embryogenesis.

胡萝卜体细胞胚胎发生中赤霉素3-氧化酶基因特异性表达机制研究

• 批准号: 16580084
• 负责人: MITSUHASHI Wataru
• 金额: $ 2.43万
• 依托单位: Yamagata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16580084/
• 关键词: Carrot; Somatic embryo; Gibberellin

Aims of this project were (1)detection of gibberellin 3-oxidase-mRNAs (GA3ox-mRNA) in carrot plant, somatic embryo, and zygotic embryo, (2)quantitative (or semi-quantitative) analysis of DcGA3ox-mRNA in both embryos, (3)isolation of 5'-upper regions from those genes for promoter analysis, (4)production of transgenic carrot which was introduced each promoter :: GUS fusion gene, (5)observation of each promoter activity. Carrot (Daucus carota L. cv. US-Harumakigosun) plants and somatic embryos were used as plant materials for these experiments. We isolated three GA3ox-genes named as DcGA3oxl, 2, 3 from carrot somatic embryos. Expression of these genes was dramatically increased in induced somatic embryos if these transcripts were analyzes by semi-quantitative RT-PCR method. qRT-PCR analysis showed these genes were expressed at root, cotyledon and root, respectively in young seedlings. Especially, DcGA3ox3-mRNAs were most abundant mRNA among them in it. Then, amount of DcGA3ox2-mRNA was dr … More astically increased in somatic embryo, and it became higher than that of DcGA3ox3, at least, at heart stage-somatic embryos. Two of those transcripts were detected in epidermal and some endodermis cells at pre-stem region, at least, in torpedo stage-somatic embryos by using in situ hybridization. While, only one transcript was detected at the same region in torpedo stage-zygotic embryos. To be clear why these differences were happened in different embryos, we had introduced promoter :: GUS analysis. At first, more than 2000b of 5'-upper region in these genes were isolated by using genome-walking, and those were fused with GUS gene to produce plasmid-constracts for agrobacterium infection. Transgenic cells were grown up in liquid medium before induction of somatic embryo. Some of positive signals were observed in callus cells and regenerated somatic embryo. However, position of those signals were sometimes different in each embryo. So individual cell cluster has incubated separately to establish each cell-line. Now, next generation of there line are growing in tube. Less

该项目的目的是 (1) 检测胡萝卜植物、体细胞胚和合子胚中的赤霉素 3-氧化酶-mRNA (GA3ox-mRNA)，(2) 对两个胚胎中的 DcGA3ox-mRNA 进行定量（或半定量）分析, (3) 从这些基因中分离 5'-上部区域用于启动子分析, (4) 导入每个启动子的转基因胡萝卜的生产 :: GUS (5)各启动子活性的观察以胡萝卜(Daucus carota L. cv. US-Harumakigosun)植物和体细胞胚为植物材料进行这些实验，我们分离了三个GA3ox-基因，命名为DcGA3oxl、2、3。如果这些转录物通过半定量 RT-PCR 方法配对，则这些基因的表达在诱导体细胞胚胎中显着增加。 qRT-PCR分析显示，这些基因分别在幼苗的根、子叶和根中表达，其中DcGA3ox3-mRNA的含量最高，并且在体细胞中DcGA3ox2-mRNA的量明显增加。至少在心脏阶段体细胞胚胎中，它的转录水平高于 DcGA3ox3。在表皮和一些内皮细胞中检测到了其中两个转录本。通过使用原位杂交，至少在鱼雷期体细胞胚胎的前茎区域中检测到了一个转录本，而在鱼雷期合子胚胎中的同一区域仅检测到了一个转录本，以明确为什么这些差异发生在不同的胚胎中。 ，我们引入了启动子::GUS分析，首先，通过基因组行走分离出这些基因中超过2000b的5'-上部区域，并将它们与GUS基因融合以产生。转基因细胞在诱导体细胞胚之前在液体培养基中生长，在愈伤组织细胞和再生体细胞胚中观察到一些阳性信号，但是这些信号在每个胚胎中的位置有时不同。细胞簇已分别培养以建立每个细胞系，现在，该细胞系的下一代正在试管中生长。