Investigation Of Interaction Mechanism Of The EIF(iso)4E Gene To TuMV In Brassica Rapa L.

Chinese cabbage(B.rapa L. ssp. pekiensis) originated in China, is one of the most important vegetable crops in China. The virus disease is one of the three main diseases in cabbage production, which affects the production of Chinese cabbage. Through identification test, Turnip mosaic virus(TuMV) is the main pathogen causing virus disease.Based on the recessive resistance gene retr02(encoded eIF(iso)4E protein), we have developed two functional markers for marker-assisted selection breeding. In addition, we also analysed the interaction mechanism of TuMV VPg to eIF(iso)4E through yeast two hybrid, and the results are as follows:1. The retr02 and Retr02 sequences have been aligned, an additional G at the junction of exon 1 and intron 1, which possessed a stop codon in intron 1, resulting in a much truncated, non-functional protein. Based on this site, a KASP_retr02 marker and a dCAPS-BslI have been developed, which are easily scored to determine the TuMV resistance of the F2 population(142 individuals), and the results of the dCAPS-BslI and KASP_retr02 marker confirmed those obtained with ELISA. The markers were used to detect the 118 highly inbred lines for marker-assisted selection in breeding programs, and only two individuals were shown to be resistant, while the remaining 116 were found to be susceptible. Although the ‘80186’ line was found to be resistant to TuMV, it was not digested by BslI in association with the dCAPS marker. It is therefore likely that it possesses alternative resistance genes or mechanisms against TuMV. The two markers above can get the same results, but in practice, the dCAPS marker is likely to be less popular than the KASP marker in breeding programs. Conversely, KASP technology delivers extremely high levels of assay robustness and accuracy with notable savings in cost and time.2. There are three copies about eIF(iso)4E gene in cabbage, i.e. eIF(iso)4E.a、eIF(iso)4E.b and eIF(iso)4E.c. After identifying them, the eIF(iso)4E.b was a pseudogene. The pGADT7 and pGBKT7 vectors have been constructed with the eIF(iso)4E.a/c and TuMV-C4/CDN1 VPg, respectively. The yeast two-hybrid results showed that pGBKT7:C4 VPg could interact with pGADT7:eIF(iso)4E.a, without pGADT7:eIF(iso)4E.c; the pGBKT7:CDN1 VPg could interact with pGADT7:eIF(iso)4E.c, without pGADT7:eIF(iso)4E.a. In addition, the different TuMV isolates VPg could interact with different eIF(iso)4E copies in B. rapa. In the process of TuMV evolution, the plants could generate the relative resistance genes to prevent the TuMV reproducing.3. Through analyzing the domain tertiary structure of eIF(iso)4E, the protein contains the cap-reception site. There are 20 various amino acids between eIF(iso)4E.a and eIF(iso)4E.c, 17 of which are located in the cap-reception site(account for 85%). In the interaction of TuMV VPg to the eukaryotic initiation factors(eIF(iso)4E), the cap-reception site of eIF(iso)4E plays a vitally important role to the virus infection.4. Through alignment the TuMV-C4 VPg with TuMV-CDN1 VPg sequences, there were five different amino acids(52 I/L, 97 E/K, 101 N/D, 105 N/D, and 117 I/V). Using the SOE-PCR, the TuMV-C4 VPg as the template, we have got five directed-site mutants, and the pGBKT7 vectors have been constructed with the mutants. Through the yeast two-hybrid analysing the interaction of the pGBKT7:C4 VPg to pGADT7:eIF(iso)4E.a, the first、second、forth and fifth sites of the C4 VPg were vitally important; the interaction of the pGBKT7:C4 VPg to pGADT7:eIF(iso)4E.c, the first site of the C4 VPg was really significant. In the evolution process of TuMV and cabbage, as for the different TuMV isolates, the crucial amino acid in the eIF(iso)4E was also various. With the development of the TuMV, the eIF(iso)4E in cabbage evolved as well. The research would lay the foundation for the co-evolution between TuMV and eIF(iso)4E.