Proteomic Studies On Hau Cytoplasmic Male Sterility In Brassica Napus

Rapeseed is one of the major oilseed crops.Cytoplasmic male sterility(CMS)is one of the most important ways to utilize the heterosis of rapeseed.The hau CMS was originally found as a spontaneous male sterile mutant in B.juncea in the experimental field of HuaZhong Agricultural University in 1999.The petals of CMS plants in B.juncea or B.napus nuclear background were small and shrinked,and the anthers were replaced by thickened petal-like structures and pollen grains were not detected by acetic red dyeing.Hau CMS line is completely and stable abortive,which is not influenced by environmental factors such as temperature.It is a new type of CMS systems,and it has broad application prospects in the breeding and production of B.napus.The sterile,maintainer and restorer lines of hau CMS system in B.napus were used in this study.We tested the protein expression pattern of the sterility related gene orf288 by Western Blot.Combined iTRAQ with bioinformatic analysis,we identified candidate proteins which are related to the male sterile of hau CMS.We screened for ORF288-interacting proteins by yeast two-hybrid assay using ORF288 and ORF288103-288 as baits,respectively.The main results are displayed as follows: 1.Western Blot analysis of the sterile protein ORF288Western blot was performed to validate the expression pattern of the sterile protein ORF288.We extracted flower bud proteins from sterile lines,maintainer lines,restorer lines of Brassica,and wild-type,transgenic plants of Arabidopsis,respectively.The expression of ORF288 showed significant differences which indicated that orf288 gene in sterile lines and restorer lines can be translated into protein,and according to the molecular weight of this protein,the translation may starts from the second ATG of the transcription.2.Yeast two-hybrid(Y2H)assay for screening ORF288-interacting proteinswe screened for ORF288-interacting proteins by yeast two-hybrid assay with ORF288 and ORF288104-288 as baits,respectively.22 candidate proteins which were identified from the cDNA library of Brassica napus possiblely interacted with ORF288,three of them are located in mitochondria or ribosome.And 79 candidate proteins possiblely interacted with ORF288103-288,twelve of them are located in mitochondria,ten in ribosome.At present,point-to-point analysis had not yet get the target protein.3.iTRAQ-based quantitative proteomics and bioinformatics analysis of Brassica napus hau CMS floral budsBy observing the semithin sections of the anthers,we confirmed that the abnormal anther development in the hau CMS line is arrested at the differentiation of the archesporial cells.Then,combined with the iTRAQ and bioinformatics analysis,we identified 186 candidate proteins which are related with the male sterile of hau CMS.Most of the differentially expressed proteins between the sterile line and maintainer line are associated with the proteasome and the autophagy pathways.Then a quantitative real-time PCR was performed to confirm the transcript levels of these protein.Of the selected proteins,as we can see,the consistency is poor,indicated that post-translational modifications play an important role in regulating cytoplasmic male sterility in the Brassica napus.In conclusion,the sterile protein ORF288 may interacts with an unknown factor,and influence the proteasome and the autophagy pathways,then protein degradation disorder,ROS accumulation,mitochondria vacuolization,carbohydrate and energy metabolism disorder,finally result in cytoplasmic male sterility.This study will greatly accelerate the understanding of the molecular mechanism of CMS.