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

本项目的目的是(1)检测胡萝卜植株、体细胞胚胎和合子胚中的赤霉素3-氧化酶-mRNAs(GA3ox-mRNAs)，(2)对两个胚胎中的DcGA3ox-mRNAs进行定量(或半定量)分析，(3)从这些基因中分离出5‘-上端区域用于启动子分析，(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.cv.美国-Harumakigosun)植株和体细胞胚胎作为植物材料进行了试验。我们从胡萝卜体细胞胚胎中分离到三个GA3ox基因，分别命名为DcGA3ox1、2、3。如果用半定量RT-PCR方法分析这些转录本，则这些基因在诱导体细胞胚胎中的表达显著增加。QRT-PCR分析表明，这些基因分别在幼苗的根、子叶和根中表达。其中以DcGA3ox3-mRNAs含量最高。然后，DcGA3ox2-mRNA量为DR…在体细胞胚中表达水平较高，至少在心期体细胞胚胎中高于DcGA3ox3。用原位杂交的方法，在鱼雷体细胞胚胎中，至少在茎前区域的表皮细胞和一些内胚层细胞中检测到其中的两个转录子。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发生在不同的胚胎中，我们引入了启动子：：GUS分析。首先，用基因组步移法分离了这些基因5‘-上端的2000B片段，并与GUS基因融合，构建了农杆菌侵染的重组表达载体。在体细胞胚胎诱导前，转基因细胞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。然而，这些信号在每个胚胎中的位置有时是不同的。因此，单个细胞团已经单独孵化，以建立每个细胞系。现在，下一代Three系列正在试管中生长。较少

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