The Application of Genomic Approaches in Studying a Bacterial Blight-Resistant Mutant in Rice

Rice bacterial blight disease (BBD), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the serious diseases in most rice production regions. In this report, we screened for resistance mutants from the mutation pool of TNG67 variety derived by sodium azide (SA) mutagenesis with phenotype investigation and assisted with fluorescent detection. SA0423 is a mutant of broad range resistance against Xoo for many years; the resistance was studied following the concept of central dogma. The inheritance of resistance was characterized, and three QTLs were mapped onto the genome of SA0423 using simple sequence repeat (SSR) markers and R/qtl by genomic approach. In transcriptomic approach, only one differential expression QTLs (eQTLs) were identified; two differentially expressed proteins (pQTLs) were identified and genetically characterized by proteomics after Xoo challenged in SA0423 mutant. To improve the bacterial blight resistance, makers are developed from QTLs, eQTLs and pQTLs to pyramid the resistance genes through marker-assisted breeding in our rice breeding programs

(64(1):10-20)Development of an Efficient Fluorescent Visualization Method for the Detection of Xanthomonas oryzae pv. oryzae Infection in Rice Plants

為能即時觀察白葉枯病菌於剪葉接種後感染水稻的完整過程，本研究利用新型強效的ZsGFP 基因及pBBR1MCS 載體，構築可於白葉枯病病原菌Xanthomonas 中表現綠色螢光之重組質體pBRRZsGFP。接著，轉型至Escherichia coli DH5α 及Xanthomonas oryzae pv. oryzae XF89b。經基因型確認之轉型菌株 (DH5αZsGFP及XF89bZsGFP) 菌落，皆可持續地表現綠螢光蛋白質。進一步於最高分蘗期接種水稻「台農67號」(感病品種)植株，結果發現因葉綠素螢光的干擾，在接種3 d 後 (3 DAI)，勉強可在切口處觀察到XF89bZsGFP 的增殖；直至7 DAI，方能觀察XF89bZsGFP 自葉尖沿著葉脈向葉基入侵。為了降低葉綠素螢光的干擾，本研究改用「台農67 號」暗培養之白化苗作為接種宿主，結果發現在感染0.5 h，即可發現XF89bZsGFP 的增殖與入侵。在接種後1 h，接種葉的螢光達到最大的表現。但可能隨水稻宿主啟動免疫系統，壓制入侵XF89bZsGFP 的繁殖，使接種葉的螢光表現轉弱。然而，至7 DAI 接種葉的螢光表現轉強，並持續向病葉基部發展，顯示XF89bZsGFP可能突破水稻的免疫系統，沿著維管束繼續侵入、繁殖，向葉片基部蔓延感染。這是目前已知最早觀測到白葉枯病菌感染水稻植株的研究報告。同時，此系統為非破壞性取樣，可連續在同一病葉觀察病原菌於接種後之侵入、增生及病勢的發展。因此，本研究所建構之XF89bZsGFP，確實可作為即時檢測接種後病菌感染能力及感染過程的平台，亦能作為未來白葉枯病菌與水稻宿主間交互作用的檢測平台。 Detection of pathogen infection is an important step in pathogen-host interaction study. A Xanthomonas fluorescent expression plasmid, pRBBZsGFP, was constructed with a strong fluorescent gene ZsGFP and the pBBR1MCS vector for immediate detection of bacterial blight pathogen infection. The recombinant plasmid was transformed into Escherichia coli DH5α and Xanthomonas oryzae pv. oryzae XF89b, respectively. The transformants, DH5αZsGFP and XF89bZsGFP, were confirmed by PCR genotype analysis and the observation of continuously expression of green fluorescent proteins. After infection of ‘TNG67’ at the maximum tillering stage by XF89bZsGFP, the observation of ZsGFP was interfered with the fluorescence of chlorophyll. The results showed that the bacterial multiplication of XF89bZsGFP only could be observed at the incision site at 3 DAI (days after inoculation) and XF89bZsGFP colonized from the cutting site to leaf base at 7 DAI. Using dark-treated albino rice seedlings, the interference of chlorophyll fluorescence could be reduced and the multiplication and colonization of XF89bZsGFP could be detected in 0.5 h after inoculation and the maximum fluorescence was observed on the same leaf in 1 h after inoculation. However, the fluorescence was reduced in the following time course indicating that the multiplication and colonization of XF89bZsGFP might be suppressed by the endogenous immune system of rice. At 7 DAI, the stronger fluorescence was observed again on the same leaf and extended continuously to the leaf base, suggesting that the rice immune system was broken down by the XF89bZsGFP. To our knowledge, this is the first report on the observation of X. oryzae pv. oryzae in rice plants. Importantly, this is a non-destructive system for pathogen detection and the multiplication and colonization of pathogen can be directly observed on the inoculated leaf. Therefore, the XF89bZsGFP and system established in this study can be served as a platform for real-time observation of virulence and pathogenicity of X. oryzae, and can also be applied to the study of the interaction between pathogen and host

檳榔心芋可溶性澱粉合成酶基因之選殖、表現與性質分析

可溶性澱粉合成酶（soluble starch synthase, SSS）是高等植物中參與澱粉合成的重要酵素。本論文利用檳榔心芋（芋，Colocasia esculenta var. esculenta）做為材料，進行其SSSI及SSSII cDNA的選殖、表現與特性分析，以探討它們在澱粉合成中所扮演的角色。 本研究利用RT-PCR與rapid amplification of cDNA ends（RACE）的方法，自芋葉組織中選殖出SSSI cDNA。SSSI在芋基因體中是單一拷貝的基因，其cDNA長度為2340 bp，包含643個胺基酸序列之閱讀框架，其中前54個殘基為訊息胜肽（transit peptide）。而芋SSSI在Escherichia coli中所表現的重組蛋白質，具有primer-dependent及primer- independent二種澱粉合成酶的活性。另一方面，芋SSSI在葉組織表現較多，塊莖表現較少，而葉柄中則未觀察到其表現。同時，在澱粉快速積存生長期的塊莖-- 597± 37g重的塊莖，發現較多的SSSI mRNA及蛋白質表現量。在葉組織發現72 及66 kDa兩種SSSI，但在塊莖組織只有66 kDa SSSI。此外，芋SSSI有一較特殊的性質，是其只存在塊莖萃取物的可溶性部分，不像其它已發表的SSSI會同時存於可溶性與澱粉顆粒的部份。 芋SSSII cDNA亦以相同方法自芋塊莖中選殖出來。其長度為2939 bp，包含804個胺基酸序列之閱讀框架，其中前52個殘基亦為訊息胜肽。芋SSSII演譯的胺基酸序列與其他物種的SSSII，有58- 63%的相同性及63- 69%的相似性。雖然芋是單子葉植物，但比對及親緣的分析結果，均顯示其與同為單子葉來源的SSSII較不相似，反與雙子葉植物的SSSII較為相近。而SSSII在E. coli中表現出具有澱粉合成活性的重組蛋白質，更確定其為芋中的一個澱粉合成酶。南方墨漬法結果顯示，其在芋基因體可能是單一拷貝數或低拷貝數基因。另一方面，在澱粉快速積存生長期的塊莖-- 597± 37g重的塊莖，可發現較多的SSSII mRNA及蛋白質。西方墨漬法分析結果顯示，芋SSSII同時存於塊莖萃取物的可溶性與澱粉顆粒的部份，且其在老化葉中有較多的表現量。這些結果顯示芋SSSII是一種特殊的澱粉合成酶，其同時參與芋暫存性與儲藏性澱粉的合成。Soluble starch synthase (SSS) is one of the important enzymes involved in the starch synthesis of higher plants. In this thesis, we used Bin Lang Hsin Yu (taro, Colocasia esculenta var. esculenta) as material to precede the cloning, expression and characterization of SSSI and SSSII for studying their functions in starch synthesis. SSSI cDNA was isolated from taro leaves by RT-PCR and rapid amplification of cDNA ends reaction (RACE). The cDNA of this single copy gene is 2340bp and encodes a 643 amino acid protein containing a putative transit peptide of 54 residues. Recombinant SSSI protein displayed both primer-dependent and primer-independent activities of starch synthase. More SSSI transcript was expressed in taro leaves than in tubers, with no evident expression in petioles; and more transcript and protein were found in tubers of 597 37g fresh weight than in smaller or larger ones. Two forms of SSSI, i.e., 72 and 66 kDa, exist in leaves, but only the 66 kDa form is found in tubers. The taro SSSI, proposed as a novel member, was located only in the soluble fraction of tuber extract, while SSSI from other sources exist in both soluble and granule-bound forms. A novel SSSII cDNA was isolated from taro tubers by the same cloning method. This 2939 bp SSSII cDNA encodes a 804 amino acid protein with a putative transit peptide of 52 residues at the N terminus. It displays 58- 63% identity and 63- 69% similarity with SSSIIs from other sources. Alignment and phylogenetic analyses showed that taro SSSII is more closely related to dicot SSSIIs than the monocot ones, though taro is a monocot. The identification of taro SSSII clone as starch synthase was confirmed by the expression of its enzymatic activity in Escherichia coli. Genomic DNA blot analysis revealed a single copy or low copy number of SSSII in taro. Expression profile showed that more transcript and protein were highly accumulated in tubers of 597 37 g fresh weight, at a stage of rapid starch synthesis, than tubers of other stages. By Western blot analysis, SSSII was found in both soluble and granule bound portions of tuber extracts; and more SSSII protein was found in aged leaves than leaves of other stages. These results suggested that taro SSSII is a novel starch synthase for the synthesis of both transit and storage starch.目 錄 頁次 目錄 i 縮寫表 v 摘要 中文摘要 vii ABSTRACT ix 第一章 緒論 1 第一節 澱粉顆粒之組成與結構 1 第二節 澱粉合成代謝途徑 4 第三節 澱粉合成酶之作用機制 5 第四節 可溶性澱粉合成酶之相關研究 6 第五節 芋之簡介 9 第六節 實驗緣由與目的 10 第二章 材料與方法 12 第一節 芋之來源與採樣之處理 12 第二節 Escherichia coli宿主 12 第三節 質體 14 第四節 藥品 14 第五節 儀器與設備 15 第六節 實驗方法 16 1 芋SSS之分離 16 1.1 粗酵素液之製備、硫酸銨劃分區分及膠體過濾層析分離 16 1.2 澱粉合成酶活性之測定 17 1.2.1 Primer-dependent activity 17 1.2.2 Primer-independent activity 18 1.2.3 酵素活性單位 19 1.3 電泳分析 19 1.4 蛋白質含量之測定 20 1.5 活性染色 20 2 芋SSS基因之選殖 21 2.1 芋之RNA的抽取與分析 21 2.1.1 芋之總體RNA（total RNA）的抽取 21 2.1.2 甲醛瓊脂膠體電泳分析法 23 2.1.3 mRNA之回收 24 2.2 以RT-PCR、5’及3’ cDNA尾端快速增殖法（rapid amplification of cDNA ends, RACE）選殖SSS基因 24 2.2.1 利用反轉錄酶-聚合酶鏈鎖反應（reverse transcriptase- polymerase chain reaction, RT-PCR）方法增殖部分SSS cDNA片段 25 2.2.2 以3’-RACE方法分析SSSs基因之3’端序列 26 2.2.3 以SMARTTM RACE分析SSSs基因之5’端序列 27 2.2.4 cDNA片段之回收 28 2.2.5 TA選殖（cloning） 29 2.2.6 全長SSSs基因之選殖與表現系統之構築 29 2.2.7 轉形作用 29 2.2.7.1 CaCl2方法 30 2.2.7.2 電穿孔法 30 2.2.8 質體DNA之抽取 31 2.2.9 生物資訊學之分析 32 3 SSS表現系統之構築 33 3.1 以PCR方式增殖SSSI及SSSII上具閱讀框架的cDNA片段 33 3.2 內切限制酶之剪切與DNA接合作用 34 3.3 DNA分子端之補齊（3’端位配對之補齊） 34 4 重組SSS之生產與純化 35 4.1 重組SSSI蛋白質之生產 35 4.2 細胞浸噬液的製備 35 4.3 重組SSSI蛋白質之純化 36 4.3.1 變性條件下之純化 36 4.3.2 可溶性蛋白質之純化 37 4.4 重組SSSII蛋白質之生產 38 4.5 重組SSSII蛋白質之純化 38 4.6 抗體之製備 39 5 芋中SSSI及SSSII性質之分析 40 5.1 芋之總蛋白質抽取 40 5.2 芋SSSs分佈之部位 41 5.3 二維電泳分析 42 5.4 西方墨漬法分析 42 5.5 芋基因體DNA之抽取 43 5.6 南方墨漬法 44 第三章 結果與討論 46 第一節 芋之澱粉合成酶 46 一 芋澱粉合成酶之分佈 46 二 芋塊莖中可溶性澱粉合成酶之分離 46 第二節 芋SSSI基因之研究 52 一 芋SSSI基因之選殖 52 二 芋SSSI cDNA之序列與胺基酸序列之生物資訊學分析 55 三 芋SSSI基因在Escherichia coli之表現、生產與純化 62 四 芋SSSI基因性質之分析 76 第三節 芋SSSII基因之研究 83 一 芋SSSII基因之選殖 83 二 芋SSSII cDNA之序列與胺基酸序列之生物資訊學分析 83 三 芋SSSII基因在Escherichia coli之表現、生產與純化 92 四 芋SSSII基因性質之分析 98 第四章 結論 104 第一節 芋澱粉合成酶之分佈與同功酶數目之鑑定 104 第二節 芋可溶性澱粉合酶cDNA的選殖與選殖cDNA的分析 104 第三節 芋可溶性澱粉合成酶於 Escherichia coli 表現系統中之表現、生產及純化與抗體製備 107 第四節 可溶性澱粉合成酶在芋植物中之表現 110 第五節 未來展望 111 第五章 參考文獻 113 附錄一 124 附錄二 12

Expression of cyclodextrin glucanotransferase gene of Bacillus macerans in Bacillus subtilis

中文摘要本研究係以 B. macerans 之 late log phase 染色體 DNA 為模 板，利用聚合鏈反應（PCR）合成一段長度約為 2.1 kb ，含有 ribosome binding site 、signal sequence 之 CGTase 基因。將此酵素 基因選殖於一雙向載體 -- pHY300PLK，轉形至 E. coli 因而得到重組質 體 -- pHG，再轉形至 B. subtilis。pHY300PLK 及 CGTase 基因片段並 無明顯的 promoter 區域，而重組質體 - pHG 卻能分別在 E. coli 及 B. subtilis 中，表現 CGTase 蛋白質，因此推測 pHY300PLK 上有一段 未知的 promoter 區域。將攜帶 pHG 之 B. subtilis 培養於 2 x SG、 LB 及 2 YT 中，發現其在以 2 YT 培養基培養至 24 小時，菌液會具有 最高的 CGTase 酵素活性表現。將培養液離心所得之粗酵素液以β- CD 親合性管柱、 DEAE Sepharose CL 6B 離子交換管柱層析及 Sephacryl S-200 膠體過濾層析等步驟純化分離 CGTase，將純化之 B. subtilis CGTase 進行 N 端胺基酸序列分析得SPDTSVDNKVNF，與文獻之 B. macerans CGTase N 端胺基酸序列相同，此外由澱粉水解及 α-CD 偶合 反應測試之結果，顯示出 B. subtilis 所生產之 CGTase 與 B. macerans 之 CGTase 具有相似的催化活性。自 pQG - D*、pQG - Y* 載 體上，以剪切、置換的方式，導入 E344D、E344Y 等兩種突變酵素至此表 現系統，生產並純化突變酵素，經評估結果顯示此系統能生產足量的野生 型與突變型 CGTase，並將其分泌至胞外，以利生化特性分析的進行。因 此，本研究之系統可作為將來其他突變型 CGTase 之生產與活性分析之依 據。AbstractA 2.1 kb DNA fragment which encodes the cgt gene (with ribosome binding site and signal sequence)of Bacillus macerans was synthesized by polymerase chain reactions(PCR)and cloned into a shuttle vector -- pHY300PLK(which is not a expression vector). There was no promoter sequence in the cloned cgt gene and vector, but the recombination plasmid, pHG, could express CGTase in B. subtilis DB430. It is proposed that an unknown promoter sequence was located at the upstream of cgt gene on pHG. Higher amount of CGTase was produced as B. subtilis carrying pHG growing on 2YT medium than on 2xSG or LB. B. subtilis CGTase was purified through β-CD affinity chromatography, DEAE Sepharose CL 6B ion exchange chromatography and Sephacryl S - 200 gel filtration to apparent homogeneity. The N-terminal amino acid sequence of B. subtilis CGTase determined was SPDTSVDNKVNF,which is consistent with that of B. macerans CGTase. The B. subtilis CGTase also has the similar starch digesting and α- CD coupling activities as B. macerans CGTase. The results revealed that B. subtilis harboring pHG produces the authentic CGTase of B. macerans. E344D and E344Y were introduced onto this vector system by replacing the corresponding DNA fragment on pHG with that on pQG - D* and pQG- Y* contained the mutated site, respectively. It was determined that the amount of wild type, E344D and E344Y expressed extracellulary from B. subtilis was 55 mg /L, 25 mg / L and 20 mg / L, respectively. These amounts were fairly enough to be applied in the determination the properties of the two mutant CGTases. From above results, we proposed the system could be employed in the production of the mutant CGTases for studying of their catalytic activities in the future

(65(4):406-419) Analysis of Seed Purity for the Bell Pepper (Capsicum annuum) Hybrid Using SSR Markers

種子純度檢定是一代雜交種子生產中品質管控的關鍵性工作之一。利用生育特性的檢測 (grow out test; GOT) 方式僅比較植物生長至成熟期的外觀差異，檢測耗時且受制於環境；故亟需建立一個快速、可靠的DNA 檢測模式。本研究目的擬開發具有專一性與共顯性的簡單序列重複 (simple sequence repeat; SSR) 標誌，應用於番椒雜交種親本與雜交種子純度之檢測。利用2 個番椒雜交種及親本，篩選374 組SSR 引子對，結果獲得12 個高訊息的SSR 標誌；又取得其中5 個關鍵性SSR 標誌，進行3 個番椒雜交種 (No. 5、No. 6、No. 7) 的種子純度之檢定，經各取120 個樣品之檢測結果顯示，3 批次的雜交種子純度分別為99.2%、98.3% 和95.8%。利用SSR-PCR (polymerase chain reaction) 分析可篩選出自交種子之汙染株，也與GOT 檢測結果吻合。試驗顯示，SSR 標誌可作為快速、有效之檢測工具，有效地應用於番椒商業雜交種子純度的檢定。 Identification of seed purity of F1 hybrids is a critical factor of quality control in the production of hybrid seeds. The grow out test (GOT) is based on the morphological characters of plants grown to maturity. However, GOT may influence by the environment and is time consuming, there is necessary to identify a rapid and reliable test like DNA based assay for this purpose. The objective of this study was to generate specific and co-dominant SSR (simple sequence repeat) markers for discrimination of parental lines and the subsequent assessment of seed purity in pepper hybrids. Genomic DNA from two hybrids and their parental lines were screened with 374 SSR primer pairs and 12 informative SSR markers were identified. Among the informative SSR markers, five key markers were selected for seed purity test for No. 5, No. 6, and No. 7 hybrids. A total of 120 hybrid individuals from each hybrid were tested using the identified markers, and results showed the seed purity rate for the three hybrid seed lots were 99.2, 98.3 and 95.8%, respectively. The contamination of inbred seeds obtained by SSR-PCR analysis was comparable to the GOT method. These results indicated that SSR markers could be used as a rapid and efficient tool in testing the seed purity of the commercial pepper hybrid

A Modified Roger's Distance Algorithm for Mixed Quantitative-Qualitative Phenotypes to Establish a Core Collection for Taiwanese Vegetable Soybeans

Vegetable soybeans [Glycine max (L.) Merr.] have characteristics of larger seeds, less beany flavor, tender texture, and green-colored pods and seeds. Rich in nutrients, vegetable soybeans are conducive to preventing neurological disease. Due to the change of dietary habits and increasing health awareness, the demand for vegetable soybeans has increased. To conserve vegetable soybean germplasms in Taiwan, we built a core collection of vegetable soybeans, with minimum accessions, minimum redundancy, and maximum representation. Initially, a total of 213 vegetable soybean germplasms and 29 morphological traits were used to construct the core collection. After redundant accessions were removed, 200 accessions were retained as the entire collection, which was grouped into nine clusters. Here, we developed a modified Roger’s distance for mixed quantitative–qualitative phenotypes to select 30 accessions (denoted as the core collection) that had a maximum pairwise genetic distance. No significant differences were observed in all phenotypic traits (p-values > 0.05) between the entire and the core collections, except plant height. Compared to the entire collection, we found that most traits retained diversities, but seven traits were slightly lost (ranged from 2 to 9%) in the core collection. The core collection demonstrated a small percentage of significant mean difference (3.45%) and a large coincidence rate (97.70%), indicating representativeness of the entire collection. Furthermore, large values in variable rate (149.80%) and coverage (92.5%) were in line with high diversity retained in the core collection. The results suggested that phenotype-based core collection can retain diversity and genetic variability of vegetable soybeans, providing a basis for further research and breeding programs

Genetic analysis and molecular mapping of QTLs associated with resistance to bacterial blight in a rice mutant, SA0423

Bacterial blight disease, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases in rice producing areas. SA0423 is a broad-range resistance mutant selected from a popular japonica-type variety, TNG67, using sodium azide mutagenesis. Genetic analysis and QTL mapping of SA0423 were performed using the descendants obtained from crossing with Taichung Native 1, a susceptible and well-known indica variety, by challenging with a Taiwanese Xoo isolate, XF89b. Genetic analysis displayed that the resistance of SA0423 is regulated by quantitative trait loci (QTLs) with incomplete dominance. A linkage map covering 12 chromosomes and consisting of 148 SSR as well as 3 InDel markers was constructed. Three QTLs are identified on chromosomes 11, 8 and 6 and account for 21.1, 11 and 9.6 % of the observed phenotypic variance, respectively. Three QTLs are localized to 6, 7 and 14 confidence intervals, respectively. These QTLs contribute to approximately 47 % of the total phenotypic variation of the F-2 population. No epistatic effect could be detected among the three QTLs. Our results provide a suitable source of potential disease resistance genes and establish a system for improving rice bacterial blight resistance through marker-assisted selection

A proteomic study of rice cultivar TNG67 and its high aroma mutant SA0420

Fragrance is a very important economic trait for rice cultivars. To identify the aroma genes in rice, we performed a proteomics analysis of aroma-related proteins between Tainung 67 (TNG67) and its high aroma mutant SA0420. Seventeen of the differentially identified proteins were close related with the aroma phenotype of SA0420. Among them, 9 were found in leaves and 8 were found in grains. One protein (L3) was identified as the chloroplastic glyceraldehyde-3-phosphate dehydrogenase B (OsGAPDHB) which was less abundant in SA0420 than TNG67. Sequence analysis demonstrated that this protein in SA0420 carries a P425S mutation in the C-terminal extension domain, which might hinder the formation of holoenzyme, thereby changing the profile of aroma compounds. The protein profile of OsGAPDHB showed only a weak correlation to its transcription profile. This result indicated that the reduction of OsGAPDHB in SA0420 is regulated by post-translational processes and can only be analyzed by proteomics approach. Transgenic lines suppressing OsGAPDHB through RNAi harbored more fragrance than TNG67 but less than SA0420. With betaine-aldehyde dehydrogenase as the only fragrance gene identified in rice to date, OsGAPDHB may serve as the second protein known to contribute to the aroma phenotype