FUNCTIONAL ANALYSIS OF APOMIXIS CANDIDATE GENE BY MEANS OF GENE TRANSFORMATION

本研究的主要结果如下：1)进行遗传转化首先要建立高效的植株再生体系。本研究利用可在任何季节、任何时间采集的各种材料，通过体细胞胚胎发生的方法，建立了有性和无融合生殖豚草的小叶、种子和子房以及有性水稻的植株再生体系。2)在建立了豚草和水稻愈伤组织再生体系的基础上，利用3种含Peta-GUS基因的双元载体，以农杆菌为介导剂，对其中几种愈伤组织进行了基因转化。将感染农杆菌的愈伤组织移到含有抗生素的选择培养基上进行筛选，并将存活的愈伤组织移到再生培养基上进行植株再生。完整的植物是…最终在不含激素的培养基上再生较多。对再生植株进行了Peta-GUS检测，表明GUS荧光可见。3)、构建了3种双元载体和无融合生殖候选基因asg-1。它们是pIG121Hm35S：：ASG1、pSMA35H2：：ASG1和pActnos/HM2：：ASG1。其中含有抗生素，但Peta-GUS基因被ASG1基因取代。而农杆菌GV3101/PMP90用于pSMA35H2：：ASG1，而EHA101用于其他载体。4)在构建3种双元载体和ASG1的基础上，用不同的农杆菌介导进行遗传转化。在取代Peta-Gus指示剂的情况下，采用农杆菌共培养、筛选培养基、愈伤组织增殖、再生培养基和无激素培养等步骤进行植株再生。最后获得完整的植株，并在P2级的培养箱中进行培养。5)为了检测ASG1基因的表达，分别从愈伤组织和水稻叶片中提取总DNA。根据ASG1基因序列设计引物，以总DNA为模板，用日立I-CHIP聚合酶链式反应系统进行扩增。从供试材料中均检测到一条约900bp的条带。这一结果表明无融合生殖候选基因ASG1已转入再生植株。现在，实验正在进行中，以观察植物的繁殖模式是否改变。较少

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