Finally, the plasmids were extracted from the yeast colony with an E.Z.N.A. After that, the SD/-Trp medium was used to screen the recombinant clones. 2017) was linearized with StuI and SmaI then, fragments and linearized pCB301 vector were co-transformed into the yeast strain W303-113. coli– Agrobacterium Shuttle Vector pCB301 (Sun et al. Then, using that cDNA as a template, we used Prime STAR Max DNA Polymerase (R045A, TaKaRa) to amplify five fragments that covered the whole SMV genome, and PCR products were purified with AxyPrep™ DNA Gel Extraction kit (AP-GX-50, Axygen, Wujiang, China). Next, we used PrimeScript II 1st strand cDNA synthesis kit (6210A, TaKaRa, Dalian, China) to carry out reverse translation of total RNA into cDNA. coli in future Agrobacterium-based transformation experiments.įirst, we used TRIZOL method to extract total RNA from fresh soybean leaves infected by SMV. Perhaps this experimental demonstration will enable us to skip the steps of screening recombination clones with E. The PCR and sequencing results indicated that all the recombinant clones contained in the selected Agrobacterium colonies were constructed correct. The LR recombination reaction was successfully carried out between the donor vectors and pCB301- attL-SC15P, and the recombinant reaction product was transformed directly into Agrobacterium. To verify whether or not the Agrobacterium inhibitory sequence can be used for screening a recombinant clone in Agrobacterium, the vector pCB301- attL-SC15P was constructed by inserting this sequence between the attL1 and attL2 sequences from the Gateway cloning system, and by ligation with the pCB301 frame. While the presence of P1-encoding sequence is not a sufficient condition to inhibit the growth of Agrobacterium, the deletion of other sequences will reduce the degree of Agrobacterium growth inhibition. Experiments investigating recombination between the SC15 sequences and other strains indicated that the specific sequence underpinning this growth inhibition effect is located in the sequence encoding the P1 protein. 2012).ĭuring our construction of SMV-based clone vectors, we found that one of the SMV physiological strains used, SC15, could significantly inhibit the growth of Agrobacterium. coli, but whether there exists a lethal virus protein to Agrobacterium remains unknown (Ali et al. The P3 and CI proteins of some viruses were reportedly toxic to E. To date, however, the interaction between any Potyvirus and Agrobacterium has not been reported on. In addition, its transmission can happen by HC-Pro and CP proteins interacting with the putative receptor in aphid stylets (Seo et al. Exactly as any other viruses, much of the life history of SMV includes its replication, assembly, and transportation that are achieved by interacting with host effectors. In China, the SMV isolations have been divided into 21 (SC1 to SC21) strains by 10 different soybean cultivars (Li et al. This virus mainly infects soybean plants and leads to mosaic and necrosis symptoms on leaves (Hill et al. The soybean mosaic virus (SMV) belongs to Potyvirus genus, whose genome encodes 11 mature proteins: P1, HC-Pro, P3, P3N-PIPO, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, Nib, and CP (Chung et al. Instead, the recombinant clones used in Agrobacterium infection experiments usually must be screened in advance in other cells (e.g. However, Agrobacterium is still rarely used directly as the host cell for recombinant clone screening. The results from the LR recombination reaction with pCB301- attL-SC15P and Agrobacterium transformation showed the valuable application potential of the Agrobacterium inhibitory sequence to serve as a negative screening factor for effective recombinant clone screening in Agrobacterium.Īgrobacterium is the only cellular organism on Earth naturally able to transfer DNA between itself and plants, making it an important tool in plant transgenic techniques, such as genetic transformation and transient expression (Schell and Van 1977 Leuzinger et al. A vector (pCB301- attL-SC15P) compatible with the Gateway cloning system was constructed using this Agrobacterium inhibitory sequence. But the lack of other protein encoding sequences, except for the sequence encoding coat protein, should reduce the ability of SC15 to suppress Agrobacterium growth. Recombinant and truncated virus vector experiments showed that the polymorphism of a P1 protein coding sequence of SC15 leads to the growth inhibition of Agrobacterium. In particular, the clone vectors constructed with SMV SC15 significantly suppressed the growth of Agrobacterium. While constructing soybean mosaic virus (SMV) clone vectors, we found that transformed Agrobacterium grew significantly different depending on the viral strains used. Infectious clone vectors used widely in genetic research.
0 kommentar(er)
