Shoot differentiation and gene-transfer in Periploca sepium Bunge

杠柳芽分化和基因转移

• 批准号: 13660148
• 负责人: GYOKUSEN Koichiro
• 金额: $ 2.3万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660148/
• 关键词: Periploca; Tree; Shoot differentiation; Polyisoplene; Gene-transfer; Agrobacterium method; GFP; Ferritin; ゴム産生植物; Periploca sepium; 遺伝子組換え; クロバナカズラ

Cotyledon and hypocotyl of Periploca sepium Bunge, which is a native liana tree species and contents rubber element in china, were used for shoot differentiation. MS-medium was used and both of BAP and NAA are used as hormones for shoot differentiation. Shoot differentiation was recognized in both cotyledon and hypocotyls, but differentiation rate was higher in hypocotyl. Root differentiation was recognized by using 1/2MS with 6μ mol/l IAA.Stem and leaf of Periploca were used for shoot differentiation. MS-medium was used and both of BAP and NAA were added as hormones for shoot differentiation. Differentiation rate in stem was higher in the combination of BAP 1μM〜3μM and NAA 0.1μM and the combination of BAP 3μM〜10μM and NAA 0.1〜1μM in leaf. High differentiation rate was recognized and that was in the range from 90% to 100%.Agrobacterium-mediated gene-transfer method was introduced for Periploca. GFP gene was used as a model-gene and stem from micropropagated shoot was used for explant. A plantlet that had green fluorescent under microscope was regenerated from a stem. PCR analysis and Western Analysis were used to investigate the DNA and GFP protein. From the result of both analysis, gene transfer of GPF gene had succeeded in Periploca.Agrobacterium-mediated gene -transfer method was introduced to Ferritin gene. Two plantlets had been regenerated from shoot stem, and DNA segment was detected in these two plantlets by PCR analysis.

以我国特有的藤本植物杠柳（PeriplocasepiumBunge）的子叶和下胚轴为外植体，进行芽分化研究。以MS为基本培养基，以BAP和NAA为激素诱导不定芽分化。子叶和下胚轴均能分化芽，但下胚轴分化率较高。根的分化用1/2 MS +6μ mol/lIAA进行鉴定，不定芽的分化用杠柳的茎和叶。以MS为基本培养基，附加BAP和NAA作为芽分化的激素。茎段分化以BAP 1μM × 3μM + NAA 0.1μM组合最高，叶片分化以BAP 3μM × 10μM + NAA 0.1 μM组合最高。采用农杆菌介导法对杠柳属植物进行了基因转移研究。以GFP基因为模型基因，以微繁殖的茎段为外植体。从茎再生出在显微镜下具有绿色荧光的植株。用PCR和Western分析检测DNA和GFP蛋白。从两种分析结果来看，GPF基因在杠柳属植物中的转基因已经成功。从茎段再生出2株再生植株，PCR检测到2株再生植株的DNA片段。

项目成果

宮柱明日香: "ペリプロカ(Periploca sepium Bunge)への外来遺伝子導入に関する研究"九州森林研究. 56. 178-179 (2003)

Asuka Miyabashira：“将外源基因引入Periploca sepium Bunge的研究”九州森林研究56。178-179（2003）。

Asuka,Miyabashira: "Shoot differentiation from stem and leaf of Periploca sepium Bunge"Kyushu Univ.For.Res.. 84. 42-50 (2003)

Asuka，Miyabashira：“从Periploca sepium Bunge的茎和叶的芽分化”九州大学研究. 84. 42-50 (2003)

宮柱明日香: "ゴム産生植物ペリプロカ(Periploca sepium Bunge)の葉茎からの再分化"九大農学部演習林報告. 84. 42-50 (2003)

Asuka Miyabashira：“橡胶生产植物Periploca sepium Bunge的叶和茎的再分化”九州大学农学部实验林报告书84. 42-50 (2003)。

Asuka,Miyahbashira: "Establishment of gene-transfer method in Periploca sepium Bunge"Kyushu For.Res.. 56. 178-178 (2003)

Asuka，Miyahbashira：“Periploca sepium Bunge 基因转移方法的建立”Kyushu For.Res.. 56. 178-178 (2003)