摘要
将转座子标签法应用于水稻，构建水稻突变体库，是进行功能基因组学研究的重要手段。本研究将双元表达载体CamDs转入水稻中，发展了一个带有基因捕获器的Ac／Ds激活转座子系统作为插入标签来研究水稻基因组。主要研究结果分述如下：
1．利用农杆菌介导的转化方法，将CamDs载体转入水稻品种秀水11，获得了62个独立可育的转基因株系。
2．对转Ds因子T_1代株系及转Ac基因T_1代株系进行了PCR检测，PCR结果证明分别有92.8％和93.5％的转Ac株系和转Ds株系为阳性。32个样品的Southern杂交检测结果表明，Ac／Ds确已整合到水稻基因组中并可稳定遗传给子代，且拷贝数多为1～2个。
3．转Ds植株T_0代、T_2代以及转Ac植株T_0代、T_1代经过抗性筛选，移入大田。先后共构建了290个杂交群体，其中Ac×Ds杂交群体234个，Ds×Ac杂交群体56个。
4．本研究对12个杂交组合的108株水稻进行了Ds转座分析。杂交F_1代水稻苗经过抗性筛选，苗期进行Basta抗性检测及PCR检测，验证了杂交种中Ds转座事件的发生。检测结果表明，Ds因子的转座频率在0～50％的范围，平均转座频率约13％。
5．对经过抗性筛选的杂交种F_1代在不同发育阶段、不同器官取样进行GUS染色。结果表明，杂交植株A4-1在开花期时花和叶子均有gus基因的表达。说明本研究中的基因捕获器能够捕获到特异时期特异基因的表达。
6．转基因植株及其后代出现了一些可见的形态突变，包括Ds31-1的穗型突变、Ds147的黄叶子突变、Ds9的细高秆突变以及其他一些突变类型，如白化苗、斑纹状叶片、分蘖早、苗期生长快、叶角大等。这些形态突变是否与T-DNA插入有关还需进一步验证。
Transposon tagging is an important tool for the study of functional genomics. By constructing the binary vector CamDs and transformation mediated by Agrobacterium, we developed an Ac/Ds transposon system that could be used for both gene trapping and activation tagging. The following results are obtained :
1. Through Agrobacterium-mediated transformation, 62 independent fertile transgenic rice lines were produced.
2. In T1 generations, 92.8% Ac lines and 93.5% Ds lines were identified as transgenic plants by PCR analysis. Southern blotting analysis for 32 independent transgenic plants indicated that the maize transposable element Ac/Ds had been integrated into the rice genome and most integration was one or two insertion sites.
3. Resistance assay was applied to TO, T2 transgenic Ds rice and TO, T1 transgenic Ac rice. The resistant plants were planted in field. In order to induce transposition of the inserted Ds elements, the Ds-inserted transgenic plants were crossed with the transgenic plants carrying AcTPase. A population of about 290 hybrids consisting of Ac × Ds and Ds × Ac was constructed in this study.
4. For the F1 population of Ac × Ds crossing offspring, 108 single hybrid plants of 12 combinations were studied. First, the hybrids consisting of both Ac and Ds were screened by hygromycin and kanamycin. Second, we could estimate that Ds were excised based on the Basta-resistance analysis. Third, the excision frequency of Ds element was evaluated with PCR amplification. If Ds element did not excise, PCR amplification would fail because of the long distance (8.3kb) between the two primers. Otherwise, a 860bp DNA fragment would be amplified. The result indicated that the excision frequency of Ds element trans-activated by Ac transposase was from 0 ~ 50% and the average excision frequency was about 13%.
5. To assess the efficiency of gene trapping using the gus reporter gene, we
examined GUS expression patterns in various organs of F1 hybrids in different growth periods. The hybrid plant A4-1 displayed GUS expression in flowers and leaves at the flowering stage. This result implied that the gene trap system was able to identify the specific organs that were GUS positive at specific stage.
6. Of transgenic TO lines and T] generations, some plants showed morphological aberrations, including dense spike (Ds31-l), yellow leaf (Ds147), slender (Ds9), albino, stripe, early tillering, growth promotion, leaf with big angle. Further molecular analysis is in necessity to testify whether these morphological aberrations are linked to T-DNA insertion or not.
引文
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