Identification Of The GRF Genes In The Pathways Of Mitochondrial Retrograde Regulation Of Brassica Juncea CMS System

The signal transduction from mitochondrion to the nucleus is usually named Mitochondrial retrograde regulation(MRR),generally exsiting in plant,animal and eukaryote.Mitochondrial retrograde regulation can be induced by variety stress,including biotic stress,abiotic stress and mitochondrial respiration inhibitors(antimycin A).Alternative oxidasela(AOX1a)is a nuclear gene encoding mitochondrial proten,which is regarded as a report gene of mitochondrial retrograde regulation and can be induced by antimycin A(AA).The study of Arabidopsis th.aliana recently showed that transcription factors(TFs)play important role in MRR.For example,AtMYB29 AtNAC013.Cytoplasmic Male Sterility(CMS)of Brassica juncea is mtDNA Mutant.we can use it to study Mitochondrial reverse signal with its maintenance(MF)and revertant(REV).Formerly,we found that AtGRF3 is a homologue of 14-3-3 gene Bmhl/2 in yeast,and AtGRF3 is a negative regulator of Mitochondrial retrograde regulation.Furthermore,we found some 14-3-3 genes exist difference in REV/CMS expression profile of Brassica juncea.Thus,our research aim at study the function of 14-3-3 genes in Mitochondrial reverse signal,taking advantage of VIGS to silence and verificate the function of 14-3-3 genes and by the way of yeast two hybrid to study the interaction between 14-3-3 proteins and TFs.The main results are as follows:1.Mitochondrial reverse signaling pathway changes in CMS plantsCompared with MF and REV plants,the expression level of AOXla in CMS plants is lower significantly under antimycin A treatment.Moreover,the induced expression of AOXla in Brassica rapa CMS&MF.Rapha1nus Sativus CMS&MF and Nicotiana tabacum CMS&MF was similar to that in Brassica juncea.These results verified that Mitochondrial reverse signaling pathway changes in CMS plants.2.Identification and screening of candidate genes(BjuGRF2,BjuGRF3)in Mitochondrial reverse signaling pathway of Brassica junceaWe identified the 14-3-3 genes in Brassica juncea,Brassica rapa and Brassica nigra based on the sequence homology and structure domains referring to Arabidopsis thaliana,and in total we identified 37 genes in Brassica juncea,21 genes in Brassica rapa and 26 genes in Brassica nigra.Expression profiles of 14-3-3 genes in Brassica juncea MF,CMS and REV plants were analysed using qRT-PCR.The results indicate that BjuGRF2(BjuB026023)and BjuGRF3(BjuA026371)is highly expressed in CMS plants than in MF and REV plants.Under the treatment with AA,we found that the expression of BjuGRF1 in CMS plants is lower than control,while the expression of BjuGRF2 in MF and REV plants is higher than control;at the same time,it shows no difference of BjuGRF3 expression between AA treatment and control in CMS plant,but the expression of BjuGRF3 in MF and REV plants is higher than control3.BjuGRF2 and BjuGRF3 is negative regulator of Mitochondrial retrograde regulation in Brassica junceaWe constructed the VIGS vector of BjuGRF2,BjuGRF3 seperately and successfully silenced the gene expression of BjuGRF2 and BjuGRF3.Under antimycin A treatment.the expression of AOXla in BjuGRF2 silenced plants is induced remarkebly,but the induced expression of AOX1a is lower in Mock plants,which indicate that BjuGRF2 negativly regulates MRR.The induced expression of AOX1a in BjuGRF3 silenced plants is higher than Mock plants with AA treatment,it reveals that BjuGRF3 negativly regulates MRR.4.14-3-3 protein(BjuGRF2,BjuGRF3)interacts with transcription factors in Brassica junceaYeast two hybrid results showed that BjuGRF1 interacts with BjuMYB29.and BjuGRF3 interactw with BjuNAC013.This may sugested that 14-3-3 proteins through the interaction with transcription factors negatively regulate the expression of AOXla in Mitochondrial reverse signal pathway.