Immunization with thetumor-associated antigen GA733 glycoprotein, which is highly expressed in colorectal cancer, is considered to be a promising strategy for cancer prevention and treatment. We cloned a fusion gene of GA733 and immunoglobulin Fc fragment (GA733-Fc), and that of GA733-Fc and an endoplasmic reticulum retention motif (GA733-FcK) into the Cowpea mosaic virus (CPMV)-based transient plant expression vector, pEAQ- HT . Agrobacterium tumefaciens (LBA4404) transformed with the vectors pEAQ- HT -GA733-Fc and pEAQ- HT -GA733-FcK was infiltrated into the leaves of Nicotiana benthamiana plants. To optimize harvesting of leaf to express therapeutic glycoproteins both spatially and temporally, protein expression levels at various leaf positions (top, middle, and base) and days post-infiltration (dpi) were investigated. The GA733-Fc and GA733-FcK genes were detected in leaves at 1–10 dpi using PCR. As assessed by western blot, GA733-Fc and GA733-FcK were expressed at the highest levels in the top leaf position at 5 dpi, and GA733-FcK was expressed more than GA733-Fc. The proteins were successfully purified from infiltrated N. benthamiana leaves using protein A affinity chromatography. ELISA verified that an anti-GA733 antibody recognized both purified proteins. Thus, a functional GA733-Fc colorectal cancer vaccine protein can be transiently expressed using a CPMV virus-based vector, with an optimized expression time and leaf position post-infiltration.

Many of the recessive virus-resistance genes in plants encode eukaryotic translation initiation factors (eIFs), including eIF4E, eIF4G, and related proteins. Notably, eIF4E and its isoform eIF(iso)4E are pivotal for viral infection and act as recessive resistance genes against various potyviruses in a wide range of plants. In this study, we used Clustered Regularly Interspaced Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated targeted mutagenesis to test whether novel sequence-specific mutations at eIF4E1 in Solanum lycopersicum (tomato) cv. Micro-Tom could confer enhanced resistance to potyviruses. This approach produced heritable homozygous mutations in the transgene-free E1 generation. Sequence analysis of eIF4E1 from E0 transgenic plants expressing Cas9 and eIF4E-sgRNA transcripts identified chimeric deletions ranging from 11 to 43 bp. Genotype analysis of the eIF4E1-edited lines in E0, E1, and E2 transgenic tomato plants showed that the mutations were transmitted to subsequent generations. When homozygous mutant lines were tested for resistance to potyviruses, they exhibited no resistance to tobacco etch virus (TEV). Notably, however, several mutant lines showed no accumulation of viral particles upon infection with pepper mottle virus (PepMoV). These results indicate that site-specific mutation of tomato eIF4E1 successfully conferred enhanced resistance to PepMoV. Thus, this study demonstrates the feasibility of the use of CRISPR/Cas9 approach to accelerate breeding for trait improvement in tomato plants.

Ethylene responsive element binding protein (EREBP) is a major group of the AP2/ERF family and plays significant roles in the regulation of abiotic and biotic stress responses. StEREBP is cloned from potato (Solanum tuberosum L.) and characterized to response in abiotic stress and hormone regulation. In this research, we constructed Chinese cabbage StEREBP overexpressing plants using the Agrobacterium-mediated transformation. Six transgenic plants are confirmed the T-DNA insertion by Southern blot analysis and RT-PCR analysis. Phenotype of transgenic plants is not different compared with control plant. The reverse transcription PCR (RT-PCR) showed that cold stress and ABA related genes are increased but ethylene related genes are decreased in StEREBP overexpressing Chinese cabbage plants. The StEREBP responded to abiotic factors and hormones suggested that they possibly had diverse roles in stress and hormone regulation of Chinese cabbage.

The sequencing of radish genome aids in the better understanding and tailoring of traits associated with economic importance. In order to accelerate the genomics assisted breeding and genetic selection, transcriptomes of 33 radish inbred lines with diverse traits were sequenced for the development of single nucleotide polymorphic (SNP) markers. The sequence reads ranged from 2,560,543,741 bp to 20,039,688,139 bp with the GC (%) of 47.80–49.34 and phred quality score (Q30) of 96.47–97.54%. A total of 4951 polymorphic SNPs were identified among the accessions after stringent filtering and 298 SNPs with efficient marker assisted backcross breeding (MAB) markers were generated from the polymorphic SNPs. Further, functional annotations of SNPs revealed the effects and importance of the SNPs identified in the flowering process. The SNPs were predominantly associated with the four major flowering related transcription factors such as MYB, MADS box (AG), AP2/EREB, and bHLH. In addition, SNPs in the vital flowering integrator gene (FT) and floral repressors (EMBRYONIC FLOWER 1, 2, and FRIGIDA) were identified among the radish inbred lines. Further, 50 SNPs were randomly selected from 298 SNPs and validated using Kompetitive Allele Specific PCR genotyping system (KASP) in 102 radish inbred lines. The homozygosity of the inbred lines varied from 56 to 96% and the phylogenetic analysis resulted in the clustering of inbred lines into three subgroups. Taken together, the SNP markers identified in the present study can be utilized for the discrimination, seed purity test, and adjusting parental combinations for breeding in radish.

The ‘common’ strawberry (Fragaria × ananassa) is a popular fruit in Korea and is often consumed fresh. Polyploidy is common in horticultural crops, and Fragaria × ananassa is octoploid, having 8 sets of each chromosome; however, the molecular genetic information of octoploid strawberry is still insufficient to develop effective markers for agronomic traits. In this study, a set of high-quality single nucleotide polymorphisms (SNPs) was collected via the genotyping-by-sequencing (GBS) method to genotype the strawberry inbred lines developed in Korea. We used a population of 89 strawberry inbred lines, which were derived from nine strawberry cultivars. The FANhybrid_r1.2 octoploid assembly was used as a reference genome. The average length of reads was 87,458,702 bp, and a total of 437,058 SNPs were collected. Several filtering criteria (SNP quality, read depth, and genotype quality) were used to identify 20,923 potential SNPs. Finally, 2863 high-quality SNPs were selected for the study. The genetic diversity of the inbred lines was analyzed by phylogenetic tree and structure analysis using the SNP genotype data. This is the first report of genotyping strawberry inbred lines using the GBS technique, and can be used to accelerate strawberry molecular breeding.

The main goal of this research was to determine optimum pH conditions for coupling between protein A and epoxy-activated Sepharose beads for purification of monoclonal antibodies (mAbs) expressed in plants. To confirm the effect of pH conditions on purification efficacy, epoxy-activated agarose beads were coupled to protein A under the pH conditions of 8.5, 9.5, 10.5, and 11.5 (8.5R, 9.5R, 10.5R, and 11.5R, respectively). A total of 300 g of fresh leaf tissue of transgenic Arabidopsis expressing human anti-rabies mAb (mAbP) SO57 were harvested to isolate the total soluble protein (TSP). An equal amount of TSP solution was applied to five resin groups including commercial protein A resin (GR) as a positive control. The modified 8.5R, 9.5R, 10.5R, and 11.5R showed delayed elution timing compared to the GR control resin. Nano-drop analysis showed that the total amount of purified mAbPSO57 mAbs from 60 g of fresh leaf mass were not significantly different among 8.5R (400 μg), 9.5R (360 μg), 10.5R (380 μg), and GR (350 μg). The 11.5R (25 μg) had the least mAbPSO57. SDS–PAGE analysis showed that the purity of mAbPSO57 was not significantly different among the five groups. Rapid fluorescent focus inhibition tests revealed that virus-neutralizing efficacies of purified mAbPSO57 from all the five different resins including the positive control resin were similar. Taken together, both pH 8.5 and 10.5 coupling conditions with high recovery rate should be optimized for purification of mAbPSO57 from transgenic Arabidopsis plant, which will eventually reduce down-stream cost required for mAb production using the plant system.

Eun Su Lee, Jinhee Kim, Jong Pil Hong, Do-Sun Kim, Minkyong Kim, Yun-Chan Huh, Chang-Gi Back, Jundae Lee, and Hye-Eun Lee. Korean J. Breed. Sci. 2018;50:424-33. https://doi.org/10.9787/KJBS.2018.50.4.424

Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus) is an economically important crop in South Korea. Although efforts have been made to develop disease-resistant cultivars, defense signal pathways in watermelon are still unknown. GNK2 domain containing proteins in plants are involved in oxidative stress and salt stress, and in defense signaling responses to pathogens and viruses. Arabidopsis thaliana plasmodesmata-located protein 5 (PDLP5) controls cell-to-cell communication and defense signaling. We identified five Arabidopsis PDLP5 (AtPDLP5) homologous in watermelon, using the Cucurbit Genomics Database. The five putative watermelon PDLPs had two GNK2 domains and showed high sequence identities to AtPDLP5. Their mRNAs were expressed in three watermelon cultivars, PI189225, Au-Producer, and 920533. Each mRNA was differently expressed after infection of Colletotrichum orbiculare, Didymella bryoniae, and Pseudomonas syringae. In addition to pathogen infection, their expression patterns were altered when subjected to abiotic stresses (drought, salt, and cold) or salicylic acid treatment. Cla006974 mRNA was absent in the 920533 cultivar infected with D. bryoniae. Based on these findings, we propose that five putative watermelon PDLPs are involved in defense against fungal pathogens and abiotic stresses. In addition, Cla006974 could be useful as a marker for selecting Gummy stem blight-resistant cultivars via molecular screening.

To obtain intact and full-length RNA transcripts of onion (Allium cepa), long-read sequencing technology was first applied. Total RNAs extracted from four tissues; flowers, leaves, bulbs and roots, of red–purple and yellow-colored onions (A. cepa) were sequenced using long-read sequencing (RSII platform, P4-C2 chemistry). The 99,247 polished high-quality isoforms were produced by sequence correction processes of consensus calling, quality filtering, orientation verification, misread-nucleotide correction and dot-matrix view. The dot-matrix view was subsequently used to remove artificial inverted repeats (IRs), and resultantly 421 IRs were removed. The remaining 98,826 isoforms were condensed to 35,505 through the removal process of redundant isoforms. To assess the completeness of the 35,505 isoforms, the ratio of full-length isoforms, short-read mapping to the isoforms, and differentially expressed genes among the four tissues were analyzed along with the gene ontology across the tissues. As a result, the 35,505 isoforms were verified as a collection of isoforms with high completeness, and designated as draft reference transcripts (DRTs, ver 1.0) constructed by long-read sequencing.

The expression and glycosylation patterns of anti-colorectal cancer therapeutic monoclonal antibody (mAb) CO17-1A recognizing the tumor-associated antigen GA733-2, expressed in human colorectal carcinoma cells, were observed in the leaf and stem tissues of primary (0 cycle), secondary (1 cycle), and tertiary (2 cycle) growths of seedlings obtained from the stem cut of T2 plants. The bottom portion of the stem of T2 seedlings was cut to induce the 1 cycle shoot growth, which was again cut to induce the 2 cycle shoot growth. In the 1 and 2 cycle growths, the periods for floral organ formation (35 days) was shorter than that (100 days) for the 0 cycle growth. The genes of heavy and light chains of mAb CO17-1A existed at the top, middle, and basal portions of the leaves and stem obtained from the 0, 1, and 2 cycle plants. The protein levels in the leaves and stem tissues from the 1 and 2 cycles were similar to those in the tissues from the 0 cycle. The glycosylation level and pattern in the leaf and stem did not alter dramatically over the different cycles. Surface plasmon resonance (SPR) confirmed that mAbs CO17-1A obtained from leaf and stem tissues of the 0, 1, and 2 cycles had similar binding affinity for the GA733-2 antigen. These data suggest that the shoot growth by bottom stem cutting is applicable to speed up the growth of plant biomass expressing anti-colorectal cancer mAb without variation of expression, glycosylation, and functionality.

The goal of marker-assisted backcrossing is to reduce the number of generations significantly by using genome-based molecular markers. Among other types of molecular markers, SNP (single nucleotide polymorphism) is mostly used in genetic diversity analysis due to its abundance. To develop high-throughput SNP marker for MAB system, we selected 20 Chinese cabbage lines each represent traits as inner leaf color, disease resistance, head type and maturity etc. Then, we sequenced the transcriptomes of 20 lines by using Illumina Hiseq2000. The average transcriptome size was 1.37 Gbase, and the average of short reads mapping rate was about 62.15% (30xcoverage). We identified 13,976 SSR markers and 380,198 SNPs by aligning contigs of 20 Chinese cabbage lines. To develop SNP marker set, we chose 409 SNPs that covers the whole Brassica rapa transcriptome. The filtering criteria were depth, polymorphism, segregation ratio, lack of adjacent SNP and copy number. We positioned the selected SNP markers to the Chinese cabbage linkage map. Clustering dendrogram was produced using SNP marker and three different clusters were generated. The result showed that the genotyping data is partially linked to the phenotyping data. We assume that the developed SNP marker set can be applied in the Chinese cabbage MAB system soon.

Numerous studies using single nucleotide polymorphisms (SNPs) have been conducted in humans, and other animals, and in major crops, including rice, soybean, and Chinese cabbage. However, the number of SNP studies in cabbage is limited. In this present study, we evaluated whether 7,645 SNPs previously identified as molecular markers linked to disease resistance in the Brassica rapa genome could be applied to B. oleracea. In a BLAST analysis using the SNP sequences of B. rapa and B. oleracea genomic sequence data registered in the NCBI database, 256 genes for which SNPs had been identified in B. rapa were found in B. oleracea. These genes were classified into three functional groups: molecular function (64 genes), biological process (96 genes), and cellular component (96 genes). A total of 693 SNP markers, including 145 SNP markers [BRH-developed from the B. rapa genome for high-resolution melt (HRM) analysis], 425 SNP markers (BRP-based on the B. rapa genome that could be applied to B. oleracea), and 123 new SNP markers (BRS-derived from BRP and designed for HRM analysis), were investigated for their ability to amplify sequences from cabbage genomic DNA. In total, 425 of the SNP markers (BRP-based on B. rapa genome), selected from 7,645 SNPs, were successfully applied to B. oleracea. Using PCR, 108 of 145 BRH (74.5%), 415 of 425 BRP (97.6%), and 118 of 123 BRS (95.9%) showed amplification, suggesting that it is possible to apply SNP markers developed based on the B. rapa genome to B. oleracea. These results provide valuable information that can be utilized in cabbage genetics and breeding programs using molecular markers derived from other Brassica species.

This study were carried out for selection of proper transformation variety and development of efficient regeneration and transformation methods. The number of shoot in commercial varieties of gourd plant were 0 ~ 7.3. and fusarium wilt resistant pure lines were 2.0 ~ 6.5 per dish containing on MS medium supplemented with 3 mg/L BA. The shoot regeneration frequency of fusarium wilt resistant pure lines were wide variation on the deviation. The expression of GFP was high 67% and 100% at the co-cultivation with Agrobacterium . The effective shoot regeneration plant hormone were combination BA and 2,4-D. The number and elongation condition of shoot was good after 4 weeks change with MS medium supplemented with 1 mg/L BA. Effective callus production plant hormone were combination of 3 mg/L BA and 0.1 mg/L 2.4-D. 서 론 박은 아프리카 대륙이 원산지로 아시아로 전래 되었으며 원래 단단한 껍질을 갖는 박과 식물의 과실을 통칭하는 말로도 사용되어 왔는데 현재는 bottle gourd를 의미한다(Erickson et al. 2005). 수박은 저온에 민감하여 장해를 입기 쉬우며 수박과일 썩음병, 세균점무늬병 및 덩굴쪼김병 등 토양 전염성 병원균 감염에 의한 피해가 크다(Lee 1989, 1994). 토지 집약적 재배를 해야 하는 우리나라에서는 접목이 수박재배에 보편적으로 이용되기 시작하면서 대목용 박이 상업적으로 중요하게 생산되고 있다. 박과 작물에서 대목의 이용은 고품질 박과 작물 재배를 위한 주요한 육종 기술로 병해충 저항성 및 저온 신장성, 내건성 및 내습성 등의 특성 개량을 위하여 이용되고 있다(Lee 1994; Edelstein et al. 1999; Park et al. 2005) 최근에는 접목기술을 이용하는 작물에서 대목을 형질전환 재료로 사용하면서 접수에서 수확하는 과실은 형질전환체가 아니라서 형질전환 기술을 이용하되 최종산물은 형질전환체가 아니라 소비자에게는 거부감이 없어서 새로운 육종기술로 주목 받고 있다(Victor et al. 2012). 박 대목의 형질전환에서의 문제점은 형질전환 효율이 아주 낮은 것이며, 또한 형질전환 재료로 이용되고 있는 박 G5 품종은 직접적으로 대목으로 이용하기에는 농업적인 특성이 좋지않은 문제점을 가지고 있다(Han et al. 2005). 본 연구에서는 대목용으로 이용되는 박의 형질전환 효율을 높이고 농업적 특성이 우수하여 접목에 이용 가능한 대목용 박 대목 품종인 국립원예특작과학원에서 개발한 덩굴쪼김병 저항성이 있는 순계계통과 상용품종을 이용하여 실험을 수행하였다. 또한 재분화 효율을 증진시킬 수 있는 생장조절제를 선발하고자 하였으며, 선발된 생장조정제를 가지고 형질전환을 통하여 형질전환 효율을 검정하였다. 형질전환 효율을 높이기 위한 방법으로 캘러스 단계를 통해 신초를 유도할 수 있는 생장조정제 조합을 선발하였다.

Based on the sequences of Brassica rapa ssp. pekinensis, we carried out a single-nucleotide polymorphism (SNP) study for genes controlling disease resistance (clubroot, turnip mosaic virus and soft rot) and leaf traits in B. rapa leafy genotypes ‘VC40’ and ‘SR5’. In total, 7645 SNP markers were obtained based on the annotation of a B. rapa database for disease resistance. Among these 7645 SNP markers, 141 were related to 125 genes linked to leaf traits. From these 141 SNP markers, 63 were screened and High Resolution Melt (HRM) primers were designed for genomic studies. A total of 20 polymorphic SNP primers were finally obtained. Eventually, these markers will be further used for the detection of quantitative trait loci and mapping studies.

ABSTRACTLimited functional genomics resources and whole genome association studies could be substantially improved in pepper (Capsicum annuum L., Solanaceae) through the application of a molecular approach for characterization of gene content and identification of molecular markers. We performed 454 GS‐FLX Titanium massive parallel pyrosequencing of polyA‐selected and normalized cDNA libraries generated from a single pool of transcripts obtained from two pepper varieties, the highly pungent Saengryeg 211 and the nonpungent Saengryeg 213, for de novo transcriptome assembly, functional annotation, and in silico discovery of potential molecular markers. A single 454 pyrosequencing run generated 361,671 and 274,269 reads totaling 164.49 and 124.60 Mb of sequence data, which assembled into 23,821 and 17,813 isotigs and 18,147 and 15,129 singletons for both varieties, respectively. These reads were organized into 20,352 and 15,781 ‘isogroups’ for both varieties. Assembled sequences were functionally annotated on the basis of homology to genes in multiple public databases and assigned with gene ontology (GO) terms. Analysis of sequence variants identified a total of 11,584 and 9641 potential single nucleotide polymorphisms (SNPs), which eventually resulted in 665 and 632 genotype‐specific SNPs for both the varieties, respectively, after examining SNP frequency distribution for each mapped unigenes. High‐throughput transcriptome profiling and large scale of polymorphic marker discovery using next generation sequencing (NGS) of two pepper varieties provides valuable information for functional genomics resources, which will help to further improve the pepper‐breeding efforts with respect to genetic linkage maps, quantitative trait locus (QTL) mapping, and marker‐assisted trait selection.

The protein-protein interaction between VPg (viral protein genome-linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso) 4E. The seven amino acids in the cap-binding pocket of eIF(iso) 4E were selected on the basis of other previous results and comparison of protein models of cap-binding pockets, and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso) 4E interrupted its interaction with TuMV VPg. All eIF(iso) 4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso) 4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso) 4E W95L, W95L/K150E and eIF(iso) 4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T-1 and T-2 transformants demonstrated that the over-expression of the eIF(iso) 4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for the engineering of broad-spectrum resistance in Chinese cabbage.