在转基因水稻植株中蜡质基因第1内含子对基因表达影响的分析

Analysis of Influence of Wx Intron 1 on Gene Expression in Transgenic Rice Plant

为阐明水稻Wx基因第 1内含子在整体植株的胚乳发育阶段是否确有增强基因表达的功能 ,以及弄清高和中、低直链淀粉含量的水稻品种Wx基因第 1内含子 112 6个碱基之间有差异的 16个碱基中哪几个碱基影响了该内含子的正常剪接从而降低了基因的表达水平 ,我们分别用高直链淀粉含量品种的Wx基因翻译起始密码子ATG上游 3.1和 2 .1kb片段与GUS基因编码区融合构建成嵌合质粒 ,并在此基础上 ,(1)去除嵌合质粒中Wx基因的第 1内含子 ;(2 )将嵌合质粒Wx基因的第 1内含子中 (3.1kb)与中、低直链淀粉含量的水稻品种Wx基因第 1内含子有差异的 6个碱基以中、低直链淀粉含量的水稻品种的碱基替换。将上述改造过的几种质粒分别转化粳稻品种中花 11,测定转化植株未成熟种子胚乳中的GUS活性。结果表明第 1内含子的缺失或此内含子的 5′端剪接点上的碱基G以T替换均造成GUS活性的急剧下降 ,说明第 1内含子在植株体内的确有增强基因表达的功能 ,而且在中、低直链淀粉含量的水稻品种中Wx基因第 1内含子 5′端剪接点上自然存在的G→T突变是造成这些品种中该内含子剪接不正常。

Our previous studies demonstrated that: (1) the first intron of rice Wx gene could enhance transient expression of reporter gene in rice protoplasts; (2) the first intron of rice Wx gene could be normally spliced in rice cultivars with high amylose content whereas it was spliced aberrantly in those with intermediate/low amylose content; (3) there are 16 single base differences in Wx intron 1 between rice cultivars with high and with intermediate/low amylose content. In order to confirm if this intron could indeed enhance the gene expression at endosperm development stage of rice plant, and to identify which one of these 16 bases plays important role in Wx intron 1 normal splicing, we have constructed two chimeric genes, containing Wx gene promoter with intron 1 ( 3.1 or 2.1 kb) and the GUS gene. Then, several constructs were reorganized on the basis of these two chimeric genes: (1) the Wx intron 1 was removed from two chimeric genes; (2) 6 single base mutations belonging to these 16 different bases within Wx intron 1 were introduced individually into chimeric genes (3.1 kb) (Fig.1). These nine constructs were introduced, respectively, into rice plants, and the GUS activities in the immature seeds of transgenic plants were determined (Fig.2). Results show that either the deletion of intron 1 or a single base mutation (G→T ) at the 5′ splice site of Wx intron 1 decreased the expression of GUS gene. These results indicate that in vivo the intron 1 indeed enhances the gene expression, and the G→T mutation in Wx intron 1 of rice cultivars with intermediate/low amylose content is, probably the key factor causing abnormal splicing of this intron in these cultivars.