FUNCTIONAL ANALYSIS OF APOMIXIS CANDIDATE GENE BY MEANS OF GENE TRANSFORMATION

通过基因转化对无融合生殖候选基因进行功能分析

• 批准号: 14560005
• 负责人: CHEN Lanzhuang
• 金额: $ 2.37万
• 依托单位: UNIVERSITY OF MIYAZAKI
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14560005/
• 关键词: apomixis; apomixis-specific gene(ASG1); sexual; guineagrass; rice; genetic transformation; plant regeneration; binary vector; pAct-ASG1コンストラクト

The results of this study were summarized as following :1),to carry out genetic transformation, as a first step, efficient plant regeneration system should be established. In this study, we used various materials which could be collected in any season and any times, and have established plant regeneration systems by the way of somatic embryogenesis from leaflets, seeds and ovaries of sexual and apomicitic guineagrass, and from sexual rice. The somatic embryogenesis on calli of guineagrass was observed and confirmed with eletronic microscope.2),based on the establishments of plant regeneration systems on guineagrass and rice, several kinds of calli of them were used for gene transformation with 3 kinds of binary vectors containing peta-GUS gene and mediated with agro-bacterium. The calli infected with agro-bacterium were moved onto selection medium containing antibiotics for selection, and survived calli were removed onto regeneration medium for plant regeneration. Complete plants were … More regenerated on hormone free medium in final. The peta-GUS examination was carried out on regenerated plants, indicating the GUS fluorescence be visible. Therefore, the genetic transformation systems have been established in guineagrass and rice.3),the construction of 3 kinds of binary vectors and apomixis candidate gene ASG-1l were carried out. They were pIG121Hm35S :: ASG1, pSMA35H2 :: ASG1, and pActnos/Hm2 :: ASG1. In them, the antibiotics were contained but the peta-GUS was replaced with ASG1 gene. However, the agro-bacterium of GV3101/PMP90 was used for pSMA35H2 :: ASG1, and the EHA101 was for the others.4),based on the construction of 3 kinds of binary vectors and ASG1, they were mediated with different agro-bacterium for genetic transformation. As the peta-GUS indicator was replaced, the culture was continued in steps of co-culture with agro-bacterium, selection medium, callus propagation, regeneration medium and hormone free medium for plant regeneration. In final, complete plants were achieved and they are cultured in incubator of P2 level.5),for the examination and gene expression of ASG1,total DNA were extracted from calli and leaves of guineagrass and rice, respectively. When the primers were made according to the sequences of ASG1, the total DNAs were used as template and amplified with the primers using HITACHI i-chip PCR system. A band with 900bp in size was detected from all the materials provided from guineagrass and rice. This result indicated that apomixis candidate gene ASG1 has been transferred into the regenerated plants. Now, the experiments are in progress, in order to observe whether the reproductive mode of the plants is changed or not. Less

这项研究的结果总结如下：1），要进行遗传转化，作为第一步，应建立有效的植物再生系统。在这项研究中，我们使用了各种材料，这些材料可以在任何季节和任何时候收集，并通过从传单，种子和卵巢中的性和尖端的豚鼠以及性稻的叶子，种子和卵巢中建立了植物再生系统。观察到豚鼠书法上的体细胞胚发生，并用Eletronic显微镜证实。感染农业 - 细菌的书法被移至含有抗生素的选择培养基上，并将生存的书法移至再生培养基上以进行植物再生。完整的植物是……在最终的无荷兰培养基上更加重生。 PETA-GUS检查是在再生植物上进行的，表明可见GUS荧光。因此，已经在几内亚草和大米中建立了遗传转化系统。3），进行了3​​种二元载体和Apomixis候选基因ASG-1L的构建。它们是PIG121HM35S :: ASG1，PSMA35H2 :: ASG1和PACTNOS/HM2 :: ASG1。在其中，抗生素被含有，但PETA-GUS被ASG1基因代替。然而，GV3101/PMP90的农业细菌用于PSMA35H2 :: ASG1，而EHA101的农业 - 基于其他二进制载体和ASG1的eHA101。4），它们是用不同的农业杆菌介导的，用于遗传转化。当更换PETA-GUS指标时，培养物以与农业 - 细菌，选择培养基，愈伤组织繁殖，再生培养基和无刺激性培养基的共同培养的步骤继续进行。在最终，实现了完整的植物，并在P2水平的孵化器中进行培养。5），对于ASG1的检查和基因表达，分别从Calli和几内亚草和大米的叶子中提取了总DNA。当根据ASG1的序列进行引物时，将总DNA用作模板，并使用日立I-ChIP PCR系统用引物进行放大。从几内亚草和米饭提供的所有材料中检测到具有900bp的带。该结果表明，Apomixis候选基因ASG1已转移到再生植物中。现在，正在进行实验，以观察植物的生殖模式是否已更改。较少的

项目成果

陳 蘭荘: "茎頂・脇芽培養によるウイルスフリーかんしょ種苗の大量増殖法の確立"宮崎大学農学部研究報告. (In press). (2004)

陈兰庄：“通过茎尖和侧芽培养大规模繁殖无病毒甘松种子和幼苗的方法”，宫崎大学农学部研究报告（2004年出版）。

Chen, L.Z.: "Effect of harvest seasons on the efficiency of ovary culture in Panicum maximum"Plant Biotechnology. 19. 173-179 (2002)

陈L.Z.：“收获季节对大黍子房培养效率的影响”植物生物技术。

Asymmetric somatic hybrids between Lycopersicon esculentum and Lycopersicon hirsutum

番茄和陆地番茄之间的不对称体细胞杂种

• 发表时间: 2002
• 期刊: SABRAO Journal of Breeding and Genetice 34
• 作者: Chen;L.Z.
• 通讯作者: L.Z.

Characterization of the apomixis-specific gene in Panicum maximum.

大黍无融合生殖特异性基因的表征。

• 发表时间: 2003
• 期刊: Proceedings of The International Colloquium on Plant Biotechnology
• 作者: Chen;L.Z.
• 通讯作者: L.Z.

Somatic hybrids between Lycopersicon esculentum and Lycopersicon chmielewskii.

番茄和番茄之间的体细胞杂交。

• 发表时间: 2002
• 期刊: Plant Biotechnology 19
• 作者: Chen;L.Z.;K.Murai;M.Inoue;Y.Kaneko;Y.Sato;T.Adachi.
• 通讯作者: T.Adachi.