Transformation Of BnSGS3 Into Oilseed Rape And Arabidopsis Thaliana And Research Of Resistance Of Transgenic Plants To Different Viruses

Virus disease is one of the main diseases in oilseed rape(Brassica napus). It can cause serious economic losses in the epidemic years. It has great importance of study in oilseed rape genome and discovering key resistance genes for resistance improvement. In recent years, the intron resistant genes have gradually been in a hot research. In reports, SGS3 is required for posttranscriptional gene silencing (PTGS) and is a functional gene which has resistance of viruses. But SGS3 gene in Brassica napus was never be reported up to now. In this study, we surveyed the viruses in oilseed rape fields and tested to definitude the variance of viruses infected oilseed rape varieties. Cucumber mosaic virus (CMV) CP gene was cloned and sequenced, and make homology alignment of other typical CMV CP gene sequences. SGS3 gene was cloned from oilseed rape, named BnSGS3.copy number and expression levels in various tissues of BnSGS3 in oilseed rape were detected by qRT-PCR. We transformed BnSGS3-overexpressing (BnSGS3-Ov) and BnSGS3-silenced (BnSGS3-Si) vectors into sgs3-14 mutant Arabidopsis thiana and oilseed rape (cv. Zhongshuang No.6) plants. These three viruses Oilseed rape mosaic virus (ORMV), Turnip mosaic virus (TuMV) and CMV were inoculated to the different plants. The viral symptoms and gene expression levels were detected. The main results obtained are listed as follows:1. In the 88 oilseed rape samples, the main virus that infected oilseed rape was TuMV, which detection rate is 100%.Detection rate of CMV and ORMV is respectly 76.7% and 54.5%. The 88 virus samples shows that compound infection situation is primarily. The CMV and TuMV compound infection rate is 45.4%。 TuMV and ORMV compound infection rate is 23.8%, and CMV, TuMV and ORMV compound infection rate is 30.7%. The PCR amplication and sequenced results showed that CMV CP gene has a total lengh of 657bp, clarified in subgroup I. The homology alignment suggested that CMV CP between oilseed rape and the other reported plants CMV CP sequences has sequence homogy of 76.5%～97.6%.2. SGS3 gene was cloned from oilseed rape and designed BnSGS3. Sequence analysis showed that BnSGS3 includes 2638bp, four introns and a 1824bp complete ORF. Sequence comparison by DNAMAN found that the homology between BnSGS3 and other SGS3 genes was 46.0%～76.5%. Similar to AtSGS3, BnSGS3 also had three domains: zinc-finger (ZF), rice gene X and SGS3 (XS) and coiled-coil (CC) domains, which shared 93.3%,78.3% and 73.3% identity with these domains of AtSGS3。3. The overexpressing vector BnSGS3-Ov was transformed into sgs3-14 mutant by Floral-dip.We got 8 positive plants via herbicide screening and PCR detection. ORMV, TuMV and CMV were inoculated to T3 BnSGS3-Ov transgenic plants, sgs3-14 mutant and WT plants to evaluate the disease resistance. The result revealed that after inoculation of CMV, three types of Arabidopsis showed virual disease symptom. The severity followed:sgs3-14> WT> BnSGS3-Ov. Leaves of sgs3-14 becomed yellow, mosaic, curl, lobus cardiacus fascicled, withered and died in 25 dpi (days of post inoculation). However, ORMV and TuMV gave a reverse trend:BnSGS3-Ov>WT>sgs3-14 plants.4. By qRT-PCR detection, BnSGS3 existed in one copy in oilseed rape (cv. Zhongshuang No.6). The native BnSGS3 expressed in all examined tissues with the highest expression in siliques, and flower, leaf, stem followed, the root has lowest expression level.ORMV, TuMV and CMV were inoculated in Zhongshuang No.6. The examination showed that the virus disease index is 50,34.67 and 33.49 respectively and that high susceptible to ORMV, moderate susceptibility to TuMV and CMV.5. Over-expressing vector BnSGS3-Ov and interfering vector BnSGS3-Si were transformed into oilseed rape (cv. Zhongshuang No.6) via Agrobacterium tumefaciens-mediated explant culture technique. A total of 58 transgenic oilseed rape plants were confirmed by PCR, including 33 BnSGS3-Ov and 25 BnSGS3-Si transgenic plants and positive rate received 48%. The relative transcript levels of BnSGS’3 in randomly selected 22 plants were determined by qRT-PCR. The results showed that the relative transcript levels of BnSGS3 among transgenic BnSGS3-Ov and BnSGS3-Si plants were 144%～ 581% and 13%～71% of the non-transgenic plants, respectively. ORMV, TuMV and CMV were inoculated in transgenic and non-transgenic oilseed rape, the detection results told that all three viruses induced BnSGS3 expression, but ORMV induced a dramatic increase in the BnSGS3-Ov plants, followed by TuMV and CMV. The induced expression level of BnSGS’3 showed correlations with viruses RNA level. Among which, ORMV and TuMV had positive correlations with BnSGS3 expression level, but CMV showed negative correlation. The results suggest that BnSGS3 inhibits CMV accumulation but promotes ORMV and TuMV accumulation. BnSGS3 should be used in different ways (up-and down-regulation) for breeding virus-resistant oilseed rape varieties.