Study On The Cleavage Mechanism Of The Transcripts Of Orf355-orf77in S-type Cytoplasmic Male Sterility (CMS-S)Mitochondria

Cytoplasmic male sterility (CMS) is a common phenomenon in flowering plants, which can not produce functional pollen, but the pistil is normal. CMS materials have widly range of restorers and can keep sterility by their maintainer lines, thus they have been widely used in hybrid breeding and hybrid productions. In addition, CMS materials in maize are very precious genetic resources and can be applied in the study of nuclear-cytoplasmic interactions.S cytoplasmic male sterility (CMS-S) belongs to the three main cytoplasmic male sterile types in maize. One of the previous studies has suggested that the sterility character was cused by a chimeric open reading frame called orf355-orf77in mitochondria. The expression of orf355-orf77was tissues specific, only accumulated in pollen. The expression of the transcript was significantly reduced in restorer line. At the same time, smaller RNA fragments appeared. So the formation of these small pieces of RNA will be helpful to clarify its restoration mechanism. In this study, we use sterility line S-Mo17rf3rf3(rf3) and restorer line S-Mo17Rf3Rf3(Rf3) as research materials. Results are listed as follows:(1) Through the method of3’RACE, the cleavage sites were identified in the1.6kb transcript, the cleavaged products in Rf3were more than those in rf3. Shear segments were added to poly (A) tails, poly (A) tails could accelerate the degradation of RNA molecules in mitochondria, so these results supported the hypothesis:the orf355-orf77transcript was degradated after being sheared into fragments;(2) Among the six shear sites, the flanking regions of three contained a conserved sequence,5’-CCACA-3’;(3) About90nt bases including the cleavage sites were used for RNA-protein affinity chromatography analysis. RNA binding proteins, which could bind poly(A) and molecular chaperones were identified;(4) Prokaryotic expression vector pGEX-6p-1and pTYB1were used respectively to express the orf355N terminal and the or/355C terminal. At10℃,0.05mmol/L IPTG induction condition, we obtained high concentration of orf35JC soluble protein; we employed prokaryotic expression protein to prepare antibodies, results of Western Blot showed Rf3and rf3without any stripes;On the basis of above experiments, we speculated molecular mechanism on CMS-S fertility restoeration:orf355-orf77open reading frame happened in complicated shear process, leading to the degradation in orf355-orf77transcipt. But this shear process almost did not exist in rf3. Rf3contained more mRNA sequence snippets followed by poly (A).5’-CCACA-3’conservative sequence, was found near the splice sites. The sequence was a cleavage protein recognition sequence. Putative TCP-1/cpn60, one of the molecular chaperonin family protein, participated in the shear process, with high abundance ratios protein. On the contrary, the protein concentration was very low in rf3. This kind of molecular chaperonin protein assisted the PPR proteins to function. Using orf355antibodies in Western Blot, both Rf3and rf3had no protein band appearing. The result showed that fertility restoration did not enter into the translation process, Pollen fertility restoration was just regulated at the post-transcriptional level.