水稻苗期致死突变体的鉴定及其基因定位

Identification and Gene Mapping of a Seedling Lethal Mutant in Rice

肽链释放因子RF1通过识别终止密码子在翻译终止过程中起重要调控作用。获得水稻苗期致死突变体,明确OseRF1-1表达模式,为进一步研究OseRF1-1功能奠定基础。以粳稻花之舞和T-DNA插入突变体为材料,利用Tail-PCR、石蜡切片技术、Southern杂交和Northern杂交技术鉴定一个肽链释放因子eRF1-1的T-DNA单拷贝插入突变体ls。采用DNAMAN6.0软件和SMART对OseRF1-1进行生物信息学分析,利用RT-qPCR方法分析OseRF1-1的组织特异性表达。通过PEG介导方法转化拟南芥,明确OseRF1-1的亚细胞定位。结果显示,ls突变体植株在3-5叶分蘖期迅速死亡;ls突变体叶鞘与分蘖节连接区域褐化,分蘖节越往下部位症状较严重;Tail-PCR分析结果显示,ls突变体T-DNA插入时丢失了T-DNA的右边界和NOS的终止子序列导致GUS和OseRF1-1融合转录,而GUS的终止密码子仍然存在,进而引起OseRF1-1不能进行正常翻译;ls突变体转录出GUS和OseRF1-1的融合转录子;OseRF1-1在各组织中均检测到表达且在穗中高表达;该基因定位于细胞核和细胞质。ls突变体导致水稻苗期致死可能是由于T-DNA插入OseRF1-1导致该基因不能正常转录引起。

Peptide release factor 1(RF1)plays a key role in the regulation of the translation termination by recognizing stop codons.Rice seedling lethal mutant was obtained,the expression profile of OseRF1-1 was clarified,and a basis for further function analysis of OseRF1-1 was provided.Using the Japonica rice cv.Huazhiwu and T-DNA insertion mutant as materials,Tail-PCR,paraffin section technology,Southern blotting and Northern blotting technologies were used to identify a T-DNA single-copy insertion mutant of the peptide release factor eRF1-1,named ls.DNAMAN6.0 software and SMART were applied to analyze the OseRF1-1’s bioinformatics characteristics,and RT-qPCR was used to analyze the tissue-specific expression of OseRF1-1.Transforming Arabidopsis thaliana by a PEG-mediated approach was to clarify the subcellular localization of OseRF1-1.The results suggested that the ls mutant rapidly died at the 3rd to 5th leaf tillering stages.Paraffin section showed that the junction region between leaf sheath and tiller node in ls mutant was browning,and the symptoms were more serious in the lower part of tiller nodes.Tail-PCR analysis showed that the right border of the T-DNA and the NOS terminator sequence in ls mutant were lost,resulting in a fusion transcript of GUS and OseRF1-1 in ls mutant.However,the stop codon remained in GUS,which led to the OseRF1-1 not translated normally.OseRF1-1 was expressed in all tissues tested,especially highly expressed in panicles.This OseRF1-1 gene was localized in nucleus and cytoplasm.Above results indicate that ls mutant causing rice seedlings lethal may be resulted from the abnormal transcription of OseRF1-1 gene by T-DNA insertion.