De novo Transcriptome Analysis to Identify Anthocyanin Biosynthesis Genes Responsible for Tissue-Specific Pigmentation in Zoysiagrass (Zoysia japonica Steud.)

학위논문 (박사)-- 서울대학교 대학원 : 식물생산과학부, 2015. 8. 허진회.조이시아속 잔디(Zoysiagrass, Zoysia sp.)는 전 세계적인 온난기후에서 주로 서식하고 균일한 녹색을 띄고 있다. 그러나 몇몇 잔디 품종에서 이삭 및 포복경에 붉은 색소를 띄는데 이는 심미적 가치를 떨어뜨린다. 두 개의 조이시아속 잔디 품종인 안양중지(AJ)와 그린조아(GZ)는 이삭과 포복경에서 각각 보라색과 녹색을 띄는데 시아니딘과 페츄니딘이 착색의 주 성분임을 알 수 있었다. 두 잔디 품종간에 서로 다르게 발현되는 유전자를 확인하기 위하여 신규전사체 분석을 수행하였다. 이중 두 개의 안토시아닌 생합성 관련 유전자인 안토시아니딘 합성 효소 ZjANS1(Z. japonica anthocyanidin synthase 1)와 디하이드로플라보놀 4-환원 효소 ZjDFR1(Z. japonica dihydroflavonol 4-reductase 1)이 안양중지 이삭에서 높게 발현되었다. 또한 이 유전자들은 여러 다른 조이시아속 잔디 품종들 중 보라색을 띄는 잔디 품종에서 높게 발현되었고, 반면 녹색을 띄는 잔디 품종에서는 낮게 발현되었다. 잔디의 ZjDFR1과 ZjANS1 유전자가 암호화하는 효소 활성이 실제 안토시아닌 생합성에 작용하는 것을 확인하였다. 또한 ZjDFR1과 ZjANS1 유전자를 각각 DFR과 ANS 유전자가 없는 애기장대 돌연변이체에 형질전환하여 T1 형질전환 개체에서의 안토시아닌 생합성을 확인하였다. 이를 통하여 잔디의 DFR, ANS 유전자의 기능을 확인하였고 이는 단자엽작물이 아닌 애기장대에서도 기능하는 것을 확인할 수 있다. 본 연구를 통하여 잔디에서의 ANS와 DFR 유전자의 높은 발현이 조직 특이적인 안토시아닌 생합성을 통한 착색에 관여함을 알 수 있었다.ABSTRACT i CONTENTS iii LIST OF TABLES vi LIST OF FIGURES viii LIST OF ABBREVIATIONS xii LITERATURE REVIEW 1 Zoysia japonica 1 Anthocyanin Biosynthesis 3 Next Generation Sequencing (NGS)-based RNA-Seq 5 REFERENCES 7 CHAPTER I. De novo Transcriptome Assembly and Differential Gene Expression Profiling of Anthocyanin Biosynthesis in Zoysiagrass (Zoysia japonica Steud.) ABSTRACT 15 INTRODUCTION 17 MATERIALS AND METHODS 21 RESULTS 33 Purple Pigmentations of Zoysiagrass are Related to Anthocyanin Accumulation 33 De novo Transcriptome Analysis of Zoysiagrass 40 Gene Ontology (GO) Term Analysis and Profiling of Differentially Expressed Genes (DEGs) 47 Anthocyanin Biosynthesis Genes in Zoysiagrass 54 Spatiotemporal Expression of Anthocyanin Biosynthesis Genes in Zoysiagrass 57 ZjDFR1 and ZjANS1 Confer Different Anthocyanin Accumulation in Zoysiagrass 63 Isolation of Genes Encoding DFR and ANS Protein from Zoysiagrass 69 DISCUSSION 80 REFERENCES 84 CHAPTER II. Functional Characterization of Dihydroflavonol 4-Reductase and Anthocyanidin Synthase in Zoysia japonica ABSTRACT 92 INTRODUCTION 94 MATERIALS AND METHODS 96 RESULTS 100 Sequence Analysis and DNA Methylation Status of ZjDFR1 and ZjANS1 Promoter Regions 100 Dihydroflavonol Reductase and Leucocyanidin Oxygenase Activities of Recombinant ZjDFR1 and ZjANS1 Proteins 107 Overexpression of ZjDFR1 and ZjANS1 Induces Anthocyanin Biosynthesis in Arabidopsis 110 DISCUSSION 117 REFERENCES 122 ABSTRACT IN KOREAN 125Docto

Sequence analysis of pepper BAC clones linked to the markers on the pepper chromosome number 2

Thesis(masters) --서울대학교 대학원 :식물생산과학부(원예과학전공),2008. 2.Maste

유기성폐기물의 통합처리 및 바이오매스 이용기술

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Immune Profiling of Advanced Thyroid Cancers Using Fluorescent Multiplex Immunohistochemistry

Background: Advanced thyroid cancers, including differentiated thyroid carcinoma (DTC) with distant metastasis, and anaplastic thyroid carcinoma (ATC), are associated with poor clinical outcomes and limited treatment options. This study aimed to determine the immune profiles of advanced thyroid cancers using fluorescent multiplex immunohistochemistry (F-MIHC) and multispectral imaging (MSI). Methods: Twenty-eight tissue samples were collected from 12 patients who had DTC with distant metastasis and from 16 with ATC. The samples were assessed using F-MIHC and MSI with antibodies against the cell surface molecules, cluster of differentiation (CD)4, CD8, programmed cell death-1 (PD-1), PD ligand 1 (PD-L1), forkhead box protein 3, and cytokeratin (CK). The expression of PD-L1 was evaluated using tumor proportion score (TPS) and combined positive score (CPS). Results: Significantly, more PD-L1-positive tumor cells (CK+PD-L1+) per mm2 were found in ATC samples than in DTC samples (183.5 vs. 0.03, p < 0.001). Lymphocyte infiltration was significantly increased in ATC compared with DTC, with significantly more PD-L1- or PD-1-positive lymphocytes in ATC samples than in DTC samples. The TPS and CPS for PD-L1 expression were negative in all DTC samples but positive in 81% and 94% of ATC samples, respectively. Conclusions: Immune profiling revealed significant differences between advanced DTC and ATC, particularly in terms of PD-L1 expression and lymphocyte infiltration. Therefore, immune profiling using F-MIHC and MSI can provide invaluable information regarding tumor microenvironments, which could help select candidates for immunotherapy

Genetic profiles of aggressive variants of Papillary thyroid carcinomas

Aggressive variants of papillary thyroid carcinoma (PTC) have been described with in-creasing frequency and are associated with unfavorable clinical outcomes. However, limited data exist on the comprehensive genetic profile of these variants. We performed targeted next-generation sequencing in 36 patients with aggressive variants of PTC and compared it to PTC from The Cancer Genome Atlas (TCGA) project and poorly differentiated thyroid cancers (PDTCs)/anaplastic thyroid cancers (ATCs) from the Memorial Sloan Kettering Cancer Center (MSKCC). BRAF mutation was the most prevalent (89%) in aggressive variants of PTC compared to that in other thyroid can-cers. RAS mutation was identified in one patient (3%), which was less frequent than in others. TERT promoter mutation (17%) ranged between that of PTCs (9%) and PDTCs (40%). Tumor suppressor genes, ZFHX3, TP53, and CHEK2, were mutated in 14%, 3%, and 6% of aggressive variants of PTC, respectively. The mutation rate of TP53 (3%) was significantly higher than that of PTCs (0.7%) and lower than that of ATCs (73%). Mutations in three functional groups, histone methyl transferases, SWI/SNF chromatin remodeling complex, and the PI3K/AKT/mTOR pathway, were present in 11%, 14%, and 11% of samples, respectively. In conclusion, aggressive variants of PTC had higher BRAF and lower NRAS mutation prevalence than other thyroid cancers. The prevalence of mutations in the TERT promoter, TP53, and genes encoding three functional groups ranged between that of PTCs and PDTCs/ATCs