It is generally believed that the meaning of “Yi (泗)” in the Book of Songs has been nasal fluids since Mao Heng, and has been explained from the perspective of borrowing, phonetic and ideographic word-formation, etc. However, there are still seven questions about the exact meaning of “Si (泗)”, which arouses different doubts about it. From the perspectives of handed-down documents and unearthed documents, variant relationships, literary meanings, contexts, dialects and so on, the word “Yi (泗)” of “Ti Si Pang Tuo (涕泗滂沱)” should be “Yi (㳑)” meaning that the water is full and flowing out. Furthermore, “Yi (泗)” has heterogeneous relationships with “Yi (益)”, “Yi (㳑)”, “Yi (溢)”, “Yi (洫)” and “Yi (泆)”, and has homologous relationships with “Ti (涕)”, “Yi (洟)” and “Ti (嚏)”, and has an isomorphic relationship with “Si (泗)” of “Si Shui (泗水)”. These relationships can be used to collate unearthed and handed-down documents.

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the β-casein gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5’ arm region and 1.8 kb or 0.64 kb of 3’ arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of endostatin gene and inserted into exon 7 of the β-casein gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3’ arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine β-casein gene in the mammary gland.

Bromoviridae과 Cucumovirus속에 속하는 대표 바이러스인 오이모자이크바이러스 (Cucumber mosaic virus: CMV)는 많은 경제적으로 중요한 원예작물 및 관상식물들에 심한 손실을 초래하는 바이러스이다. 다중염기서열 비교는 현재까지 서브그룹1에 속하는 모든 CMV 계통들에서 3'말단부의 보전적 염기서열들이 존재하고 있음을 보여주었다. 이런 관찰에 기초하여, 우리는 CMV RNA3와 상동성을 가지는 162 bp 상보적 DNA를 포함하며 CMV감염에 대하여 식물 유래 RNA간섭 현상을 유도할 수 있는 도치된 반복 구조를 가지는 머리핀RNA (pIR-CMVNCR)를 발현시킬 수 있는 벡터를 제작하였다. 아그로박테리움을 이용하여 IR-CMVNCR의 일시 발현은 CMV 감염을 저해하였으며, 아그로박테리움이 접종된 식물의 상엽에서는 CMV 병징이 발현되지 않았다. 또한 RT-PCR결과는 아그로박테리움이 접종된 식물의 접종엽 및 상엽에서 CMV 서브유전자 4를 포함하는 CMV RNA들이 모두 검출이 되지 않았다. CMV 유래 작은 저해 RNA들의 축적이 관찰되었으며, 이의 결과의 의미는 아그로박테리움에 의해 IRCMVNCR을 일시 발현시킨 야생담배 (Nicotiana benthamiana)의 접종엽에서 RNA 간섭 현상이 유도되어 CMV 감염을 억제시키는 것으로 판명되었다.

A strain of Bacillus thuringiensis, named Bt 1-3, was isolated from Korean soil sample and it showed high insecticidal activity against Plutella xylostella. Bt 1-3 was deterimined to belong to ssp. aizawai (H7) by an H antiserum agglutination test and produced bipyramidal-shaped crystal proteins. PCR analysis with specific cry gene primers showed that Bt 1-3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2Ab genes. In addition, this isolate showed high uptake rate of foreign plasmid by electroporation. Based on these characteristics of Bt 1-3, we tried to construct a spore-free Bt 1-3 mutant by knock-out sigG gene, which is known as a key transcription factor during sporulation. First, we constructed a basal vector, named pDST, consisting of erythromycin resistant gene (EmR), partial polyhedrin gene and temperature sensitive origin of replication gene (Orits). Subsequently, according to the chromosomal DNA sequence of Bt subsp. konkukian 97-27, we amplifed upstream and downstream regions of Bt 1-3 sigG, and cloned into pDST (pDST-G). So far, several EmR colonies were obtained by electroporating into the wildtype Bt 1-3 and crossover by homologous recombination is going on.

Accurate chromosome segregation is critical to ensure genomic integrity during cell division. This process is facilitated by the kinetochore, a multiprotein structure that is assembled on centromeric regions of chromosomes. The kinetochore establishes a mechanical link between the chromosomes and spindle microtubules and modulates cell cycle progression by regulating spindle assembly checkpoint (SAC). Defects in this process result in an aneuploidy, leading to miscarriages, infertility and various genetic disorder such as Down’s syndrome. Although the numerous kinetochore proteins have been identified and studied, the mechanisms that engaged in kinetochore assembly and chromosome segregation are poorly understood. Here we investigated the function of kinetochore protein Zwint-1 on homologous chromosome segregation during oocyte meiotic maturation. We found that Zwint-1 was localized at the kinetochore during meiotic maturation. Knockdown of Zwint-1 caused premature polar body extrusion, indicating acceleration of meiosis I. Interestingly, Zwint-1 knockdown impaired the recruitment of Mad2 at the kinetochores. However, BubR1 localization at the kinetochores was not affected by Zwint-1 knockdown, suggesting that Zwint-1 selectively regulates the recruitment of SAC components into the kinetochores. We also found that Zwint-1 knockdown abrogated chromosome alignment and segregation, thereby resulting in a high incidence of aneuploidy. These chromosomal defects were mostly due to the abnormal kinetochore-microtubule (kMT) attachments. Intriguingly, chromosome misalignment mediated by SAC inactivation was repaired, when anaphase onset was delayed by treating oocytes with proteasome inhibitor MG132. However, surprisingly, chromosomal defects following Zwint-1 knockdown were not restored by delaying anaphase onset. This result suggests that chromosomal defects induced by Zwint-1 knockdown are less likely associated with the failure of SAC activation. In addition, we observed that Aurora B/C kinase activity was not affected by Zwint-1 knockdown. Nevertheless, the meiotic defects induced by Zwint-1 knockdown were similar to those observed in Aurora B/C inhibition, suggesting that Zwint-1 is a downstream effector of Aurora B/C kinase during meiosis. Consistent with this, in Zwint-1 knockdown oocytes chromosomal defects following Aurora B/C inhibition were not restored when Aurora B/C inhibitor was removed, whereas the defects were well rescued in control oocytes after removing Aurora B/C inhibitor. This result suggests that the role of Aurora B/C kinases that correct erroneous kMT attachment is primarily regulated by Zwint-1. Collectively, our results demonstrated for the first time that Zwint-1 is an essential downstream effector of Aurora B/C kinase that corrects erroneous kMT attachment and regulates SAC activity, which ensures accurate homologous chromosome segregation during oocyte meiosis.

Tissue-specific promoters are a very useful tool for manipulating gene expression in a target tissue or organ; however, their range of applications in other plant species has not been determined, to date. In this study, we identified two late pollen-specific rice promoters (ProOsLPS10 and ProOsLPS11) via meta-anatomical expression analysis. We then investigated the expression of both promoters in transgenic rice (a homologous system) and Arabidopsis (a heterologous system) using ProOsLPS10 or ProOsLPS11::GFP-GUS constructs. As predicted by microarray data, both promoters triggered strong GUS expression during the late stages of pollen development in rice, with no GUS signals detected in the examined microspores and sporophytic tissues. Interestingly, these promoters exhibited different GUS expression patterns in Arabidopsis. While in Arabidopsis, the OsLPS10 promoter conferred GUS expression at the uni- and bi-cellular macrospore stages, as well as at the shoot apical region during the seedling stage, the OsLPS11 promoter was not active in the pollen at any stage, or in the examined sporophytic tissues. Furthermore, by performing a complementation analysis using a sidecar pollen (scp) mutant that displays developmental defects at the microspore stage, we found evidence that OsLPS10, which can be an applied promoter expressed in Arabidopsis, is useful for directing gene expression in the early stages of pollen development. Our results indicate that the OsLPS10 and OsLPS11 promoters can drive the expression of target genes during the late stages of pollen development in rice, but not in Arabidopsis. Our results also emphasize the necessity of confirming the applicability of an established promoter to heterologous systems.

Cambial meristematic cells (CMCs) are innately undifferentiated cells located in the meristems of plant with function as a stem cell to renew itself or replace specialized tissues. Another interesting feature of plant stem cells is controlling the plant defense in response to various stresses. Several groups have studied for stem cell triggered immunity signaling however, the molecular basis of the stem cell triggered immunity remains unclear. We previously obtained deferentially expressed 563 stem cell specific gene profiles from transcriptome analysis between two different cell types, CMC and dedifferentiated dells (DDCs) of yew tree (Taxuscuspidate). In a line of comparative genomics approach, we have selected 30 Arabidopsis homologous immune regulator candidate genes that showed significantly enriched GO terms ; at “response to stress” and “defense response”. We obtained one of homologous knock-out (KO) Arabidopsis mutant line on the locus At1G71110 whose cognate yew homologous gene showed predominantly expressed in CMCs compared to DDCs (20 times higher). For the assessment of basal disease resistance KO mutant plants were inoculated with Pseudomonas syringae pv. tomato (Pst) DC3000 and counted pathogen isolated from inoculated leaves. Interestingly, the KO mutant plants were not compromised in basal disease resistant, however, the hypersensitive response was significantly enhanced in the mutant compared to wild type in response to PstavrB, suggesting R-gene mediated defense response involved. We also investigated there sponse to the small reactive redox molecules such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) that associated significantly in plant immune response. Notably the KO mutant plants exhibited hypersensitivity specifically under nitrosative stress condition derived by S-nitrosiglutathione (GSNO), anitrioxide (NO) donor. Taken all together, putative endomembrane components At1G71110 may play a pivotal role in R gene mediated plant immune system. To further investigate its role(s) and molecular signaling network various defense gene expression profiles and functional genomics approach are ongoing for the long term aim of muti-stress tolerant crop development

Flowering is one of the most important developmental programs that plants use to ensure survival and reproductive success. The timing of flowering is under the control of several interdependent pathways. The molecular and genetic background of the interaction between environmental factors and the floral transition in these cultivars are still not reported. TaVRT2 expression is up-regulated in the winter genotypes during the vegetative phase and in photoperiod-sensitive genotypes during short days, and is repressed by vernalization to a level that allows the transition to the reproductive phase. Protein-protein interaction studies revealed that TaVRT-2 interacts with proteins encoded by two important vernalization genes (TaVRT-1/VRN-1 and VRN-2) in wheat. These results support the hypothesis that TaVRT-2 is a putative repressor of the floral transition in wheat. This gene is located on the short arm of homologous group 7 chromosomes in hexapolid wheat. The TaVRT2 acts as a repressor of the floral transition in wheat. We found that the flowering suppressor of flowering suppressor gene might be located on the short arm of chromosome 7D using several chromosomal substitution or aneuploid lines. The genetic map has been constructed from segregation of 370 SSR loci using 210 recombinant inbred lines (RILs), which was established at F7 eneration by the single-seed descent method from F2 family derived from Mironnovavskaya 808 and Chinese Spring. To perform mapping on the TaVRT2 on the homologous group 7 chromosomes, three homogous genes, TaVRT-1, TaVRT-2, TaVRT-3, are isolated from two common wheat cultivars; Chinese Spring (CS), Mironnovavskaya 808 (M808) and nucleotide polymorphisms between two cultivars are detected.

Research has been in progress for more than a decade to production of useful proteins by genetic modification in cattle. However, the levels of protein production in transgenic cattle have been reported very low. To enhance protein production in transgenic animal, we tried homologous recombination to donor cells for production of transgenic clone cattle through nuclear transfer procedure. Thus, we constructed the two targeting vectors of human thrombopoietin (TPO) at bovine -casein locus using homologous recombination with 13.6 kb and 9.6 kb homology. In two targeting vectors, positive selection was through the neomycin resistance gene and negative selection was by the diphtheria toxin (DT). Gene targeting was attempted in bovine embryonic fibroblasts (bEF) and bovine ear skin fibroblasts (bESF). To determine the most appropriate concentration of neomycin for bEF and bESF, G4l8 resistance was confirmed by culturing the cells in various concentrations of the drug and both of the cells were optimally selected at of neomycin. The transfected bEF and bESF by the targeting vectors were colonized efficiently at the ratio of DNA to transfection reagent such as :2 and :. Comparing number of healthy clones from passage 4 to passage 8, bESF (17%) persist in culture for much longer than bEF (6%). The two gene-targeted bESF clones of 30 random-integrated clones with 9.6 kb homology length were confirmed, however, nothing was out of 72 random integration clones with 13.6 kb homology length, The DT also worked more efficiently in clones transfected with the vector of 9.6 kb homology length. Our data suggests that the choice of donor cell for long culture period should be considered to obtain targeted cell clone, and the gene-targeting frequency and the DT working efficiency are dependent on the length of target homology.

단백질분해효소를 생산하지 않는 균주 B. subtilis MT-2의 염색체 DNA를 추출한 다음, B. subtilis AC819 균주에 상동성 유전자재조합을 이용하여 competent cell 형질전환을 시켰다. 얻어진 형질전환체를 B. subtilis HL-1이라고 명명하였으며, 그 표현형은 histidine 요구성, streptomycin 내성, tetracyclin 내성을 나타내면서 단백질 분해효소를 생산하지 않았다. 플라스미드 pUB11