从茎瘤芥胞质雄性不育系(CMS)线粒体中克隆的T基因是一个CMS相关基因,它能够通过选择性剪接产生T1170和T1243两个转录本,而T1243是T基因在转录过程中保留了内含子TinⅡ的产物.本实验室前期的实验结果表明,在模式植物拟南芥中过表达T1243(OE-T1243)后,植株表现出明显的雄性不育表型,而OE-T1170植株的表型与野生型无异,然而这一现象的机制却不明确.本研究通过花药石蜡切片进一步确认了OE-T1243植株异于OE-T1170植株的花粉败育表型.对15个已知花药发育相关基因的表达分析表明,与野生型相比,OE-T1243植株中nozzle/sporocyteless,barely any meristem 1和2发生了不同程度的表达下调,而在OE-T1170中未发生明显变化.为了进一步研究TinⅡ内含子的潜在功能,将其构建于(60)35S基本启动子区域上游,来测试对下游GUS基因的启动效果.对转化植株的分析表明,TinⅡ具有增强子功能,它可以显著增强GUS基因的表达.表明TinⅡ内含子能够作为一个增强子影响CMS相关基因T的功能.
The T gene,which was cloned from the mitochondria of tumorous stem mustard(Brassica juncea var.tumida),is a cytoplasmic male sterility(CMS)-related gene that can produce two transcripts,T1170 and T1243.The latter is transcribed with the uncleaved intron TinII.In our previous study,transgenic Arabidopsis thaliana plants over-expressing the T1243 transcript(OE-T1243)showed a severe male-sterile phenotype,whereas OE-T1170 plants did not.However,the functional mechanism of the T gene in B.Juncea remained unknown.In this study,microscopic analyses of paraffin sections of anthers confirmed that OE-T1243 plants did not produce normal pollen,whereas OE-T1170 plants did.We analyzed the transcription of 15 anther development-related genes and found that transcript levels of nozzle/sporocyteless and barely any meristem 1 and 2 were markedly lower in OE-T1243 plants than those in wild type,while the transcript levels of these genes in OE-T1170 plants were unchanged.To investigate the potential roles of TinII,we inserted the TinII sequence upstream of a minimal region(60)of the cauliflower mosaic virus 35S promoter fused to the 5′end of theβ-glucuronidase(GUS)reporter gene.Analysis of the transgenic plants suggested that TinII acted as an enhancer to significantly increase GUS expression.The potential action mechanism is that the TinII intron acts as an enhancer to affect the function of the CMS-related gene T.
JIN ZhuPingWU LingLingCAO JiaShuCHEN ZhuJunPEI YanXi
