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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 洪傳揚(Chwan-Yang Hong) | |
| dc.contributor.author | Ke-Chun Lin | en |
| dc.contributor.author | 林科均 | zh_TW |
| dc.date.accessioned | 2021-06-16T02:38:19Z | - |
| dc.date.available | 2020-10-12 | |
| dc.date.copyright | 2015-10-12 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-07-24 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54058 | - |
| dc.description.abstract | 第一部分：水稻是一個對鹽分相當敏感的物種，而鹽分逆境會對植物細胞的膜系造成傷害。植物的脂質運輸蛋白(Lipid transfer proteins, Ltps)是一群小分子蛋白質，普遍存在於各種植物中，具有運輸多種疏水性脂質分子之功能，在細胞膜系的修補與生合成、抵抗病蟲害與環境逆境等多種重要生理反應中扮演著重要角色。然而水稻的脂質運輸蛋白在非生物性逆境下所呈現的生理功能與分子特性仍然未知，因此本研究的目的為篩選並鑑定水稻中會受鹽分逆境誘導的Ltps，並進行分子特性分析。利用DNA微陣列法分析受鹽分逆境誘導表現的水稻LTPs基因，結果顯示有5個LTPs (OsLTPII.3、OsLTPII.5、OsLTPII.6、OsLTPV.1與OsLTPV.2) 及2個LTP-like (OsLtpL1、OsLtpL1) 基因顯著受到鹽分誘導表現。胺基酸序列比對及親緣關係分析發現這7個LTPs或LtpLs的胺基酸 N 端皆有訊息胜肽，典型的LTPs具有8個保守性半胱胺酸功能區塊，LtpLs則只有不完全的保守性半胱胺酸。分析水稻台農67號三葉齡幼苗在地上部與根部對各種非生物性逆境處理下的反應發現，除了鹽分外，這些基因亦受到缺水、低溫、H2O2、ABA與CaCl2等誘導表現。 進一步分析這些基因的啟動子區域顯示，受逆境誘導的基因多具有ABRE、MYB、MYC、ROSE與CRT/DRE等順式序列。組織專一性表現結果指出，受鹽分誘導且屬於LtpII次家族的成員(LtpII.3、LtpII.5 與LtpII.6)主要表現在花與種子；LtpIII.1則會大量累積在生殖生長期的莖部組織。分析跟DsRed螢光蛋白融合的LTP次細胞定位顯示這些基因皆表達在內質網上。以上結果可以作為未來在研究水稻脂質運輸蛋白生物性功能的新線索。 第二部分：從鹽分處理水稻Glutathione reductase 2-RNAi植株(gr2-RNAi)、Ascorbate peroxidase 8突變株(apx8)以及野生型(WT)水稻幼苗的microarray試驗中，發現OsLTP2基因只會在gr2-RNAi與apx8中受鹽分逆境的極大量誘導，但WT則不受鹽分影響。進一步以RT-PCR分析發現OsLTP2會在水稻幼苗中受到ABA與鈣離子的誘導，並專一性地表現在水稻的胚與花藥。選殖OsLTP2啟動子並分別以GUS、YFP作為報導基因轉殖到水稻中，結果顯示1 kb啟動子可賦予YFP表現出與RNA相似的表達趨勢。GUS及YFP均表現在種子的胚與花藥，外加ABA與氯化鈣處理會大量誘導GUS累積。以玉米Ubi 1啟動子驅動OsLTP2基因大量表現轉殖株相較於野生型水稻，對高溫、低溫表現出過敏感外表型，但外加ABA不影響其生長。反之以OsLTP2-RNAi抑制LTP2表現之轉殖株則有株高變矮的外表型，與WT比較，降低約24%的株高。RT-PCR分析轉殖株顯示OsNCED3以及OsNCED5基因在OsLTP2-RNAi植株中受到誘導，但不出現在WT與大量表現的LTP2植株中。同時apx8與gr2-RNAi在鹽分處理後，OsNCED3基因表現量也高於WT。這些結果說明了OsLTP2基因只會在突變株受鹽分逆境大量誘導的原因可能跟apx8與gr2-RNAi對鹽分較敏感，累積較多的ABA進而誘使OsLTP2基因表現。綜合以上結果說明了OsLTP2基因參與水稻ABA所調控的生理功能，其更確切的功能與在水稻所扮演的角色有待未來更進一步的研究。 | zh_TW |
| dc.description.abstract | Part 1. Rice is sensitive to salt. Salt stress can cause injury to plant cellular membrane. Plant lipid transfer proteins (LTPs) are abundant lipid binding proteins that are important in membrane vesicle biogenesis and trafficking; however, the biological importance of Ltps on salt-stress response in rice remains unclear. Therefore, salt-responsive rice Ltps were identified and characterized in this study. Microarray analysis showed seven genes positively regulated by salinity, including five Ltp genes (LtpII.3, LtpII.5, LtpII.6, LtpV.1, and LtpV.2) and two Ltp-like (LtpL; LtpL1 and LtpL2) genes. Amino acid alignment revealed that all these Ltp and LtpL genes contained the N-terminal signal peptides. In addition to LtpL1, all salt-inducible Ltp genes have the conserved eight cysteine residue motifs backbone. Verification of gene expression to different stimuli in rice seedlings revealed that salt-regulated Ltp genes differentially responded to drought, cold, H2O2, abscisic acid (ABA) and CaCl2; furthermore, the expression of Ltp and LtpL genes was tissue-specifically regulated by ABA-dependent and independent pathways. In silico analysis of an 1.5-Kb 5’-upstream region of these genes identified regulatory cis-elements associated with ABA, calcium and cold/drought responses. Three LtpII subfamily genes, including LtpII.3, LtpII.5 and LtpII.6, were strictly expressed in flowers and seeds, and LtpIII.1 mRNA strongly accumulated in stem tissue. Subcellular localization analysis of LTP-DeRed fusion proteins revealed that the five LTPs and two LTPLs localized at the endoplasmic reticulum. The results provide new clues to further understand the biological functions of Ltp genes.
Part 2. According to our previous microarray experiments, we put seedlings of Glutathione reductase 2 – RNAi transgenic rice (gr2-RNAi), Ascorbate peroxidase 8 rice mutant (apx8), and wild type rice (WT) under salt treatment, discovering that OsLTP2 gene was only markedly induced in gr2-RNAi and apx8 but not WT rice. RT-PCR results showed that OsLTP2 gene could be induced by ABA and calcium ion in shoot of seedlings; furthermore, specifically expressed in embryo and anther. Cloning promoter of OsLTP2 gene and fused with GUS and YFP, which acted as reporter genes, then transformed into rice calli by agrobacterium, we found that 1.0 kb-length OsLTP2 promoter could make YFP express as similar way as intrinsic OsLTP2 gene expression. Both GUS and YFP expressed in embryo and anther; in addition, external stumili like ABA and calcium ion could aslo highly induce expression of GUS accumulation. The phenotype of over-expressed OsLTP2 gene transgenic rice, which was driven by constitutive promoter Ubi 1 from maize, under high and low temperature displayed over-sensitived phenotype; however, not affected by ABA treatment. On the contrary, plant height of OsLTP2-RNAi lines were severely inhibited about 24% compared to WT in reproductive stage. RT-PCR analysis showed that expression of OsNCED3 and OsNCED5 gene in OsLTP2-RNAi were higher than WT and OE-OsLTP2. Similarly, expression level of OsNCED3 in gr2-RNAi and apx8 was higher than WT under salt treatment. These results reveled possible reasons which made OsLTP2 gene only extremely expressed in gr2-RNAi and apx8 may due to over-accumulated ABA in plants. Therefore, OsLTP2 gene plays an important role in plant development and ABA-related regulation. The in-depth function of OsLTP2 gene need to be studied in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T02:38:19Z (GMT). No. of bitstreams: 1 ntu-104-R02623002-1.pdf: 3784867 bytes, checksum: 4de955100d73f9ebb3dddf0a55e71b10 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 目 錄 目錄…….………………………….……………………………………………………..I 圖目錄..………………………………………….………………………………………II 表目錄……….………………………………………………….………………………III 縮寫字對照表…………………………………………………………………….……IV 第一部分 題目：受鹽分誘導表現水稻非專一性脂質運輸蛋白基因的分子特性分析……......1 中文摘要……………………………………………………………………………...…2 英文摘要………………………………………………………………………………...3 壹、 前人文獻回顧………………………………………………………….…………..4 貳、 研究目的……………………………………………………………………….…..8 參、 材料方法……………………………………………………………….………..…9 肆、 結果…………………………………………………………………………….…21 伍、 討論……………………………………………………………….………………24 陸、 參考文獻………………………………………………………………….………27 第二部分 題目：受鈣離子與離層酸誘導的水稻非專一性脂質運輸蛋白基因OsLTP2之分子特性與功能性分析………………………...…………………………………..42 中文摘要…………………………………………………..…………………………...43 英文摘要…………………………………………………………..…………………...44 壹、 前人文獻回顧………………………………………………………….…………45 貳、 研究目的……………………………………………………………………….…50 參、 材料方法……………………………………………………………….…………51 肆、 結果…………………………………………………………………………….…56 伍、 討論………………………………………………………………….……………59 陸、 參考文獻…………………………………………………………………….……63 圖目錄 第一部分：受鹽分誘導表現水稻非專一性脂質運輸蛋白基因的分子特性分析 圖1. 水稻非專一性脂質運輸蛋白LTP與LtpL基因之親緣關係分析圖……….......34 圖2. 水稻受鹽分逆境誘導的LTP與LtpL基因之胺基酸序列比對圖。….………....35 圖3. 受鹽分逆境誘導的Ltp與LtpL基因在不同處裡下的表現情況。...…………....36 圖4. 受鹽分逆境誘導的Ltp與LtpL基因其啟動子區域之in silico預測分析。.…....37 圖5. 受鹽分逆境誘導的Ltp與LtpL基因之組織專一性分析。………………...…....38 圖6. 受鹽分逆境誘導的Ltp與LtpL基因之次細胞定位分析。…...………………....39 第二部分：受鈣離子與離層酸誘導的水稻非專一性脂質運輸蛋白基因OsLTP2之分子特性與功能性分析 圖1. 微陣列分析TNG67、apx8與gr2-RNAi於鹽處理6 hr後之基因表現變化。…..69 圖2. 水稻OsLTP2基因之非生物性逆境誘導性與組織專一性分析。……...……....70 圖3. 使用於本研究中基因轉殖實驗之質體構築。……………………………..…... 71 圖4. 穩定性轉殖水稻P(1.0k)-OsLTP2-GUS之GUS染色組織專一性分析。……....72 圖5. 轉殖水稻P(0.5k) , (1.0k)-OsLTP2-GUS的calli受逆境處理之GUS誘導性分析。..73 圖6. 轉殖水稻P(1.0k)-OsLTP2-GUS於ABA與氯化鈣處理之GUS染色分析。…....74 圖7. 穩定性轉殖水稻P(1.0k)-OsLTP2-YFP之組織專一性螢光分析。………….........75 圖8. OsLTP2基因之次細胞定位分析。………………………………………….…....76 圖9. 大量表現OsLTP2基因與RNAi功能弱化之穩定性轉殖水稻的分子檢測。.....77 圖10. TNG67、OE-OsLTP2與OsLTP2-RNAi轉殖株之農藝性狀分析。……….……78 圖11. 大量表現OsLTP2基因之轉殖株於非生物性逆境處理下的功能分析。…….79 圖12. TNG67與轉殖株之ABA生合成、反應相關基因之RT-PCR分析。…………..80 圖13. OsLTP2之潛在功能預測模型。………………………………………...……....81 表目錄 第一部分：受鹽分誘導表現水稻非專一性脂質運輸蛋白基因的分子特性分析 表1. 微陣列分析TNG67、apx8與gr2-RNAi於鹽處理6 hr後之基因表現變化。......40 表2. 本研究中Ltp與LtpL基因命名方式之前人文獻與Locus_ID。…………..…....41 附表1. 本實驗中用於RT-PCR、次細胞定位與質體構築之引子列表。……..……....82 附表2. 水稻基因轉殖用培養基列表。………………………………………..……....83 附表3. 木村氏水耕液配方。…………………………………………………..……....85 | |
| dc.language.iso | zh-TW | |
| dc.title | 水稻非專一性脂質運輸蛋白基因對非生物性逆境的反應及OsLTP2的功能分析 | zh_TW |
| dc.title | Response of rice non-specific lipid transfer protein genes to abiotic stress and functional analysis of OsLTP2 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 賀端華(Tuan-Hua David Ho),葉國楨(Kuo-Chen Yeh),張孟基(Men-Chi Chang),蔡育彰(Yu-Chang Tsai) | |
| dc.subject.keyword | 第一部分：水稻,nsLTPs,LtpLs,鹽分逆境,第二部分：水稻,OsLTP2,ABA,鹽分逆境, | zh_TW |
| dc.subject.keyword | Part 1. Lipid transfer proteins,LTP-like proteins,Rice,Salinity,Abiotic stress Part 2. OsLTP2,Rice,Salinity,Abiotic stress,ABA, | en |
| dc.relation.page | 85 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-07-24 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農業化學研究所 | zh_TW |
| 顯示於系所單位： | 農業化學系 | |
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