CCACA碱基序列是玉米CMS-S线粒体orf355-orf77转录本剪切识别位点

玉米S型细胞质雄性不育系（CMS-S）及其近等基因恢复系是研究核 质互作机制的重要遗传资源和理想模式体系．目前认为,CMS-S花粉败育是由其线粒体内细胞质不育基因orf355-orf77表达的毒性蛋白引起,而核育性恢复基因Rf3可通过引发orf355-orf77转录本的降解而解除其毒性作用,使花粉育性得以恢复．本研究采用Northern杂交和3′-RACE技术确定了orf355-orf77转录本的剪切位点,并发现在育性恢复的花粉中,orf355-orf77转录本被剪切成小片段之后聚合了poly(A)序列,推测这一过程加速了mRNA分子的降解,是育性恢复的关键环节．利用生物信息学方法分析了orf355-orf77转录本6个剪切位点的侧翼序列,发现在剪切位点下游10个碱基的位置均含有5′-CCACA-3′序列,推测该序列受到特定功能蛋白的识别,然后募集核酸内切酶对其进行剪切．研究结果可为揭示玉米CMS S育性恢复机理提供重要的理论依据．     

Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic-nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function.     

甘蓝型油菜pol CMS育性恢复基因对orf224/atp6的转录调控

用 10个线粒体基因探针对波里马细胞质雄性不育 (polimaCMS)三系 1141A(pol) ,1141B(nap)和 1141R(pol)的花蕾线粒体RNA进行了Northern检测。结果表明 ,只有 3个探针atp6、orf2 2 4和orf2 2 2检测到转录本的差异。atp6在可育的 1141B中只转录产生一个丰度很高的 1 1kb转录本 ,在雄性不育的 1141A和pol胞质恢复系 1141R中 ,这个转录本的丰度明显减少并出现了分子量较大的 2个转录本 2 2kb、1 9kb转录本。与 1141A相比 ,恢复系1141R的 2 2kb和 1 9kb转录本丰度明显减少 ,并伴随着两个新的转录本 1 4kb和 1 3kb。表明orf2 2 4 atp6的表达与polCMS有关 ,并且其转录受到恢复基因Rfp的调控。同时通过对杂种F1 ( 1141A× 1141R)与另一个恢复系RS35 (pol)的比较证实 ,Rfp对orf2 2 4 atp6的调控与Rfp纯合与否无关。orf2 2 4 atp6在 1141A的苗期叶片中还转录产生育性恢复特异的 1 4kb转录本 ,这可能与细胞核基因型和相对低温条件有关。     

Nucleo-cytoplasmic interactions affect RNA editing of cox2, atp6 and atp9 in alloplasmic male-sterile rice (Oryza sativa L.) lines

RNA editing plays an important role in the regulation of mitochondrial gene expression in flowering plants. In this study, we examined RNA editing of the mitochondrial genes cox2, atp6 and atp9 in five isonuclear alloplasmic male-sterile lines (IAMSLs) of rice to investigate whether different cytoplasmic types affect RNA editing. Although many editing sites were conserved among the three genes, we found that the editing efficiency of certain sites was significantly different between different IAMSLs or between IAMSLs and their corresponding cytoplasmic donor CMS lines. Furthermore, several editing sites were found to be either present or absent in certain IAMSLs and their corresponding CMS lines. These results indicate that nuclear loci, as well as unknown editing factors within the mitochondria of different cytoplasmic types, may be involved in RNA editing, and they suggest that RNA editing in plant mitochondria is affected by nucleo-cytoplasmic interactions.     

Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of ‘Lead Rice’ and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6–orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of ‘Chinsurah Boro II’. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6–orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.     

10个线粒体基因在甘蓝型油菜NCa不育系统不同组织中的转录调控及其与育性的关系

用10个线粒体基因为探针,对NCα不育系、保持系和可育F1的苗期叶片、幼蕾及未成熟种子的线粒体RNA进行了Northern分析。结果表明,这10个线粒体基因除atp6外,其余9个基因在同一材料的不同组织中没有表达差异,都属于组成型表达的线粒体基因。其中,off139、orf222、atp1、cox1、cox2、cob、rm5S、rm26S等8个线粒体基因在不育系、保持系和可育F1的苗期叶片、幼蕾及未成熟种子中有着相同的表达,属于表达不受核基因型影响,没有组织特异性的类型：atp9基因分别在同一材料的不同组织中的转录也基本没有变化,但是在3个不同的材料间具有表达差异：可能属于表达受核基因型影响、没有组织特异性的线粒体基因。atp6基因也在3个材料的叶、蕾和种子中都产生相同大小的转录本,但是在各个材料的不同组织中存在着信号强度的差异,可能是属于表达既受核基因型影响、又有组织特异性的线粒体基因。Orf222和off139分别在不育系和可育F1幼蕾中产生相同大小和丰度的转录本,但是在保持系幼蕾中没有检测到转录本;orf222检测到的3条转录本分别为1．1kb、0．9kb、0．6kb,而off139检测到0．8kb和0．6kb两条带。atp9探针在不育系和保持系幼蕾中都检测到1条0．6kb的转录本,而在可育F1幼蕾中检测到0．6kb和1．2kb的转录本。讨论了orf222、off139、atp9基因的表达与NCα细胞质雄性不育的可能关系。