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

本项目的目的是：(1)在胡萝卜植株、体细胞胚和合子胚中检测赤霉素3-氧化酶mrna (GA3ox-mRNA),(2)对两种胚中DcGA3ox-mRNA进行定量（或半定量）分析，(3)从这些基因中分离出5′-上部区域进行启动子分析，(4)生产引入每个启动子的转基因胡萝卜：GUS融合基因，(5)观察每个启动子的活性。胡萝卜（Daucus carota L.）实验材料选用US-Harumakigosun植物和体细胞胚。从胡萝卜体细胞胚中分离到3个ga3ox基因，分别命名为dcga3ox1、2、3。通过半定量RT-PCR分析，这些基因在诱导体细胞胚胎中的表达显著增加。qRT-PCR分析显示，这些基因分别在幼苗根、子叶和根上表达。其中以dcga3ox3 mRNA含量最高。DcGA3ox2-mRNA的表达量在体胚中明显增加，至少在心脏期体胚中高于DcGA3ox3。利用原位杂交技术，至少在鱼雷期体细胞胚胎的干前区表皮细胞和一些内胚层细胞中检测到其中两个转录本。而在鱼雷期合子胚胎的同一区域只检测到一个转录本。为了弄清楚为什么这些差异会在不同的胚胎中发生，我们引入了启动子：：GUS分析。首先，利用基因组行走法分离出这些基因的5′-upper区域的2000b以上，并与GUS基因融合制备农杆菌感染的质粒构建物。在体胚诱导前，在液体培养基中培养转基因细胞。在愈伤组织细胞和再生体细胞胚中观察到一些阳性信号。然而，这些信号在每个胚胎中的位置有时是不同的。因此，单个细胞群被单独培养以建立每个细胞系。现在，下一代的线正在试管中生长。少

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