Identification And Function Analysis Of Hau CMS-associated Gene In Brassica Juncea

Cytoplasmic male sterility(CMS) is one of the most important ways to produce hybrid seeds in rapeseed. For decades, polima and ogura systems are widely used CMS types around the world. The polima CMS discovered by professor Fu is the predominant system used in China, and the ogura CMS is widely used in several European countries. However, the homogenization of cytoplasm is not good for rapeseed production potentially. Therefore, characterization and identification of novel CMS system is beneficial for rapeseed production. In this study, we isolated and sequenced the specific mitochondrial fragments of hau cyoplasm, and then identified and analysed the transcripts and translated products of the specific chimeric open reading frame (ORF) by northern blotting and western blotting respectively. In order to further analyse the machanism of this orf, we transformed this orf to E. coli cells and wild type Arabidopsis. The main results are listed here:1. The specific mitochondrial genomic region of hau mitotype The flanking sequences of several mitochondrial genes in the male-sterile line were compared with those in the maintainer line. A chimeric fragment was found to be located downstream of the atp6gene in hau cyoplasm. A novel chimeric gene (named orf288) was found to be located downstream of the atp6gene in hau CMS sterile line. The BLAST searches indicated that this fragment shares high similarity (99.5%) with orf263in alloplasmic male-sterile Brassica lines (tour cyoplasm). There were four point mutations:one deletion at position764bp and three nucleotide changes at485bp,785bp and802bp in orf288. Multiple alignments of orf286and orf288showed that orf286from the mitochondrial genome of B. napus shares93%nucleotide sequence identity with orf288.2. The expression analysis of atp6/orf288region in hau cytoplasm The RT-PCR and northern blotting analysis revealed that orf288is cotranscriped with atp6in hau male-sterile line. This chimeric gene (orf288) was predicted to encode a32kD protein with a triple transmembrane region. To confirm that this gene was actually translated, a peptide antigen of ORF288was synthesized and used to produce a polyclonal antibody. A band of approximately32kD was detected exclusively for the male-sterile protein samples extracted from mitochondrial and buds. Western blotting analysis showed that this predicted open reading frame was translated in the mitochondria of male-sterile plants.3. ORF288is toxic to E.coli host cells It has been widely accepted that mitochondria have evolved from an a-proteobacterial endosymbiont into vital eukaryotic organelles. Several researchers found that CMS-associated genes are toxic to host E.coli cells. To examine the function of orf288, its coding sequence was cloned into the expression region of a PET32a vector, and IPTG was used to induce its expression in E. coli. The growth of the host bacteria was repressed significantly with the expression of ORF288. This result means that ORF288is toxic to host cells. Therefore, we predicted that it may affected the function of plant mitochondria, and then suppressed the development of stamens.4. Expression of orf288in Arabidopsis significantly impairs the development of anthers To investigate the association of this chimeric gene with the male abortion of the hau CMS sterile line, four constructs were prepared and transformed into A. thaliana.(1) The VNO construct contained a35S promoter and orf288, which was fused to the mitochondrial transit peptide sequence of the nuclear coxIV gene of yeast for mitochondrial targeting.(2) VN3:The fused ORF that was driven by the AP3promoter was specifically designed to investigate its effects on floral organs. VNII vector was constructed, in which orf288was driven by the AP3promoter but lacked the coxIV presequence.(4) AGUS construct with the β-glucuronidase (GUS) gene driven by the AP3promoter was transformed into the plants as control. Almost80%of transgenic plants with VN3and VNII fragemts failed to develop anthers. 5. The subcellular localization of ORF288The transformation experiments indicated that orf288can affected development of stamens in transgenic plants. The trangenic plants even with VNII aslo develped no pollen. Therefore, we predicted that orf288is functional in the cytoplasmic matrix or is targeted to the mitochondria without the assistance of an external transit peptide. PredSL and TargetP were used to predict the subcellular localization of ORF288and the result showed that the N-terminal sequence could have its own signal function. To confirm that ORF288is able to anchor to the mitochondria, the entire ORF was fused with GFP, and the fusion protein was expressed transiently in Arabidopsis protoplasts using the PEG transfection method. The result indicated that without the external signal presequence, ORF288that is expressed in the cellular matrix additionally targets itself to the mitochondria.