Suchergebnisse

Transient expression is an easy, rapid and powerful technique for producing proteins of interest in plants. Recombinational cloning is highly efficient but has disadvantages, including complicated, time consuming cloning procedures and expensive enzymes for large-scale gene cloning. To overcome these limitations, we developed new ligationindependent cloning (LIC) vectors derived from binary vectors including tobacco mosaic virus (pJL-TRBO), potato virus X (pGR106) and the pBI121 vector-based pMBP1. LIC vectors were modified to enable directional cloning of PCR products without restriction enzyme digestion or ligation reactions. In addition, the ccdB gene, which encodes a potent cell-killing protein, was introduced between the two LIC adapter sites in the pJL-LIC, pGR-LIC, and pMBP-LIC vectors for the efficient selection of recombinant clones. This new vector does not require restriction enzymes, alkaline phosphatase, or DNA ligase for cloning. To clone, the three LIC vectors are digested with SnaBI and treated with T4 DNA polymerase, which includes 3' to 5' exonuclease activity in the presence of only one dNTP #dGTP for the inserts and dCTP for the vector#. To make recombinants, the vector plasmid and the insert PCR fragment were annealed at room temperature for 20 min prior to transformation into the host. Bacterial transformation was accomplished with 100% efficiency. To validate the new LIC vector systems, we were used to coexpressed the Phytophthora AVR and potato resistance #R# genes in N. benthamiana by infiltration of Agrobacterium. Coexpressed AVR and R genes in N. benthamiana induced the typical hypersensitive cell death resulting from in vivo interaction of the two proteins. These LIC vectors could be efficiently used for high-throughput cloning and laboratory-scale in planta expression. These vectors could provide a powerful tool for high-throughput transient expression assays for functional genomic studies in plants. ; This work was financially supported by grants from the Crop Functional Genomics Center and the National Research Foundation (Project No. 2010-0015105) of Ministry of Education, Science and Technology (MEST) of the Korean Government. ; OAIID:oai:osos.snu.ac.kr:snu2010-01/102/0000005113/9 ; SEQ:9 ; PERF_CD:SNU2010-01 ; EVAL_ITEM_CD:102 ; USER_ID:0000005113 ; ADJUST_YN:Y ; EMP_ID:A077085 ; DEPT_CD:517 ; CITE_RATE:2.046 ; FILENAME:첨부된 내역이 없습니다. ; DEPT_NM:식물생산과학부 ; EMAIL:doil@snu.ac.kr ; SCOPUS_YN:Y ; CONFIRM:Y

Abstract Background Transposable elements are major evolutionary forces which can cause new genome structure and species diversification. The role of transposable elements in the expansion of nucleotide-binding and leucine-rich-repeat proteins (NLRs), the major disease-resistance gene families, has been unexplored in plants. Results We report two high-quality de novo genomes (Capsicum baccatum and C. chinense) and an improved reference genome (C. annuum) for peppers. Dynamic genome rearrangements involving translocations among chromosomes 3, 5, and 9 were detected in comparison between C. baccatum and the two other peppers. The amplification of athila LTR-retrotransposons, members of the gypsy superfamily, led to genome expansion in C. baccatum. In-depth genome-wide comparison of genes and repeats unveiled that the copy numbers of NLRs were greatly increased by LTR-retrotransposon-mediated retroduplication. Moreover, retroduplicated NLRs are abundant across the angiosperms and, in most cases, are lineage-specific. Conclusions Our study reveals that retroduplication has played key roles for the massive emergence of NLR genes including functional disease-resistance genes in pepper plants. ; This work was supported by a grant from the Agricultural Genome Center of the Next Generation Biogreen 21 Program of RDA (Project No. PJ01127501) and by a grant from the Ministry of Science, ICT, and Future Planning (MSIP) of the Korean government through the National Research Foundation (NRF-2015R1A2A1A01002327) to DC.