Cloning And Overexpression Vector Construction Of Ogura CMS Restorer Genes From Eruca Vesicaria Subsp.sativa

Cytoplasmic male sterility(CMS)is a common maternal genetic phenomenon and is important for heterosis utilization.It is generally believed that interaction between mitochondrial chimeric gene and nuclear restorer gene is the main reason for fertility restoration of CMS.Ogu CMS of Raphanus has the advantage of complete sterility and easy to be maintaine.After the improvement of low temprature chlorosis and nectary degenerationand,Ogu CMS has become the most ideal way of heterosis utilizationbest male sterility for rapeseed.The bottleneck for Ogu CMS utilization is with the restorer.It is believed that restorer gene for Ogu CMS occurred only in Raphanus and cannot be found in the Brassica genu.INRA of France found restorer from Raphanus and patented the restorer gene.Pioneer-Hibridof US abtained double-low rapeseed restorer materials and also applied for international patents.Ogu CMS for rapeseed has been imported into China since last 80 s and some restorer lines were found from Raphanus.However it is difficult to overcome the patent barriers since these restorer lines were also from Raphanus.In previous studies intergeneric hybrids were produced between Ogu CMS Brassica napus and Eruca vesicaria subsp.sativa.In this research different Eruca lines were used to pollinate the male sterile hybrid offsprings.Some offsprings were all male fertile,some all sterile.Some Eruca lines produced both fertile and sterile plants and fitness test indicated it is consistent with the 1:1 segregation,suggesting that there were one pair of dominant restorer genes in Eruca.Sequnces for Raphanus restorer(Rfo)and Brassica Rfo-like genes were downloaded and aligned to find the conserved regions.Conserved fragment of the Eruca Rfo-like genes were amplified using primers beased on sequences of conserved regions.5’-and 3’-walking were carried out and the full length of Rfo-like gene was amplified from Eruca genomic DNA.Sequence alignment indicated that the Eruca Rfo-like genes were highly similar at the translate region.There were deletions in three regions of Eruca Rfo-likes compared with Raphanus Rfo.Phylogeny analysis indicated that Raphanus Rfo clustered into one major branch,and the Eruca Rfo-likes clustered into another major branch,in which sequences of Eruca BJ-1 clustered into one branch and sequences from other Eruca lines clustered into another branch.The two Eruca branches were represented by BJ1-5(Es Rfo-like1)and BJ4-1(Es Rfo-like4),respectively.The similarity between genomic sequences of Es Rfo-like1 and Es Rfo-like4 was 93.05%.Coding sequences of Es Rfo-like1 and Es Rfo-like4 showed similarity of 88.55% and 87.46% with Raphanus genomic Rfo.The similarity between deduced amino acid sequences of Es Rfo-like1 and Es Rfo-like4 was 89.40%.Deduced amino acid sequences of Es Rfo-like1 and Es Rfo-like4 showed similarity of 81.42% and 80.70% with Raphanus genomic Rfo.Structures prediction carried out using NCBI and Interpro indicated that Raphanus Rfo,Es Rfo-like1 and Es Rfo-like4 had 17 个 PPR repeats,and the major difference accured at the third PPR repeats,i.e.,Raphanus Rfo had 35 amino acids while Es Rfo-like4 and Es Rfo-like1 had 31 and 32 amino acids,respectively.Es Rfo-like4 and Es Rfo-like1 were ligated to plasmid by homologous recombination to produce p Cambia 2300-35S-Es Rfo-like-nos.Engeering Agrobacteria was produced by introduction of p Cambia 2300-35S-Es Rfo-like-nos.In the future Ogu CMS B.napus will be transformed with above Agrobacteria to comfirm the restorer function of Es Rfo-like4 and Es Rfo-like1.