Cloning And Functional Characterization Of Rfo-like Gene From Eruca Vesicaria Subsp.sativa

Ogu CMS is one of the best cytoplasmic male steile line for Cruciferae crops.INRA of France and Pioneer of USA found restorer gene from Raphanus and applied for long-term patents.Several ago intergeneric hybrid between Ogu CMS Brassica napus and Eruca vesicaria subsp.sativa was obtained and male fertile plants were found after backcrossing with Eruca as the pertanal parent,suggesting that there might exist in Eruca male fertility restorer gene for Ogu CMS.If the fertility recovery gene can be found in the arugula genome,the above patent can be broken and the research process of rapeseed hybridization can be greatly promoted.This project is conducted from three aspects:First,we first designed the primer according to the conserved segment sequence of the Rfo-like gene of the cruciferous plant,and obtained the conservative segment sequence of the Arugula Rfo-like gene;on this basis,the primer was designed,and the upstream and downstream sequences of the gene were obtained through walking and the full-length sequence of the Arugula Rfo-like gene was obtained.Analysis of the obtained Arugula Rfo-like gene found that the downstream regions of the gene were very similar,but the differences in the upstream regions were large,so we designed primers FP6 and RP6according to the obtained upstream sequences to further clone them in the genomes of different varieties of arugula.Clustering the resulting Rfo-like gene sequences found that at least three upstream sequences existed.The two obtained Rfo-like sequences BJ4-1 and BJ1-5 were constructed onto the p Cambia2300-35S-NOS plant overexpression vector,and the Ogu CMS cabbage type rape was infested by agrobacterium transformation method,and the fertility recovery was observed when the flowers bloomed.Second,using the internal sequence primers of the Rfo-like gene of arugula,taking the DNA of the hybrid offspring of Ogu CMS cabbage-type rapeseed×arugula as the template,it was found that 6 single plants（2 fertile and 4 sterile）could clone the unique bands of arugula and similar in size to the Rfo-like gene.We are ready to perform clonal sequencing,which is then compared with the Rfo-like gene sequence obtained by arugula cloning and corresponding its sequence to its male fertility to determine the fertility recovery gene sequence.Phylogenetic analysis indicated that there exist at least three types of sequences for Eruca Rfo-like gene.Two forms of the Eruca Rfo-like gene,BJ4-1and BJ1-5,were ligated into p Cambia 2300-35S-NOS to transform Ogu CMS Brassica napus to test their functions for male fertility restoration.Eruca-specific fragments were amplified from offsprings（2 male fertile and 4 male sterile）of Ogu CMS B.napus×Eruca.Sequencing will be carried out to decide what sequence is for the male fertility restoration.Thirty intergeneric F₁hybrids were obtained by pollinating Ogu CMS Brassica oleracea var.italica.Morpholocally some F₁plants were between the parents,but more F₁plants were morpholocally like the paternal parent Eruca.To this result we postulated that it might be due to elimination of chromosomes of the maternal parent and doubling of chromosomes of the paternal parent,but this hypothsis still needs more convincing evidence.Among the intergeneric F₁hybbrids between Ogu CMS Brassica oleracea var.italica and Eruca one plant is especially like the paternal parent Eruca.PCR products were obtained and sequencing indicated that its Rfo-like sequence is very similar to BJ1-5 from Eruca,indicating that we have got true hybrids between Ogu CMS Brassica oleracea var.italica and Eruca.More Eruca lines will be used to backcross the F₁hybrids to test their ability for male fertility restoration.