利用 CRISPR/Cas9 基因編輯技術探討水稻 DEAD-box RNA 解旋酶 42 之功能

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研究生：	梁竣瑋 Zing-Wei Loong
論文名稱：	利用 CRISPR/Cas9 基因編輯技術探討水稻 DEAD-box RNA 解旋酶 42 之功能 Functional analysis of the rice DEAD-box RNA helicase 42 via CRISPR/Cas9 technology
指導教授：	陸重安 Chung-An Lu
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
論文出版年：	2022
畢業學年度：	110
語文別：	中文
論文頁數：	85
中文關鍵詞：	水稻、解旋酶、基因編輯
外文關鍵詞：	Oryza sativa, RNA helicase, CRISPR/Cas9
相關次數：	點閱：12 下載：0
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冷逆境影響植物生長發育與農作物的產量。生物處於低溫環境時，RNA分子結構難以維持正常導致無法執行應有的功能。RNA解旋酶在幫助RNA維持正常結構上扮演了重要的角色。DEAD-box RNA解旋酶是RNA解旋酶家族超級家族二的成員，這個家族成員參與了許多細胞生理過程，包括轉錄、mRNA剪接、核糖體rRNA生合成、RNA出核、轉譯等。根據先前研究，OsRH42與水稻生長發育相關且參與冷逆境相關基因之pre-mRNA剪接機制。進一步瞭解水稻OsRH42之功能，本論文利用CRISPR-Cas9技術產生OsRH42突變株，以基因編輯T0代突變株中的兩條染色體上OsRH42都有突變，分別是刪除11對鹼基對（為out-frame突變）及刪除60對鹼基對（為剪接缺失突變），而T1代所分析的成熟種子之基因型都是同型合子剪接缺失突變，且T0代成熟的種子佔熟穗種子總數之四分之一。剪接缺失突變株T2代較野生型水稻矮小且對冷逆境更加敏感。進一步分析發現，剪接缺失突變株T2代在冷處理18小時後與野生型相比，其受冷誘導表現基因之pre-mRNA有較多的内含子殘留量。這些結果顯示剔除OsRH42基因影響水稻雌雄配子發育，導致種子無法生成。OsRH42片段缺失蛋白也影響水稻小苗生長發育，並降低水稻冷逆境相關基因之内含子剪接效率，使突變株的抗冷逆境能力下降。

Low temperature adversely affects plant growth and crop production. Correct RNA structure is difficult to be maintained under low temperature conditions. RNA helicases play an important role in maintaining functional RNA structures. DEAD-box RNA helicases belong to superfamily II of the RNA helicase family, and their function are involved in many cellular processes, including transcription, RNA splicing, ribosomal RNA biosynthesis, RNA export, translation, etc. Previous study showed that the OsRH42 is involved in rice growth and cold-responsive genes pre-mRNA splicing. In this study, a OsRH42 mutant line was generated by using a CRISPR-Cas9 system. T0 generation of OsRH42 mutant occured fragment deletion mutations at coding region of OsRH42 gene at both chromosomes, one chromosome has 60 bp deletion (in-frame mutation) and another chromosome has 11 bp deletion (frameshift mutation). T0 generation mutant had 25% seed setting rate. Interestingly, T1 generation seeds all were homozygous of 60 bp deletion in-frame mutation. The T2 generation in-frame mutation mutant showed shorter plant height and reduced cold tolerance, compared with wild type. These results showed OsRH42 defect by frameshift mutation leaded to seed abortion, implied that OsRH42 is an essential gene during develpoment of male and female gametes. Besides, T2 generation in-frame mutant slightly increased the intron retention of cold stress-responsive genes after 18 h cold stress. Thus, the T2 generation in-frame mutant showed adversely affect rice growth, reduced the intron splicing efficiency of cold stress-responsive genes, and ultimately led to reduced cold tolerance in rice.

目錄

誌謝    I
中文摘要    II
Abstract    III
目錄    IV
序論    1
1.    水稻基因體研究之重要性    1
2.    DEAD-box RNA helicase簡介及其參與之細胞生理功能    1
3.    低溫逆境及水稻對於低溫的反應    3
4.    細胞核，splicing speckles及pre-mRNA剪接簡介    4
5.    水稻DEAD-box RNA 解旋酶42之簡介與研究動機    5
壹、    實驗材料及方法    8
1.    植物材料及生長條件（Plant materials and growth conditions）    8
1-1水稻培養    8
1-2 水稻冷逆境處理    8
1-3 水稻雌激素處理    8
2.    引子（Primers）    8
3.    質體建構（Plasmid construction）    8
4.    細菌質體之轉形方法及DNA萃取    10
4-1 熱刺激轉形方法    10
4-2 電穿孔轉形方法    10
4-3 萃取小量質體DNA方法    11
4-4 萃取大量質體DNA方法    12
5.    膠體回收及PCR產物純化    12
5-1 膠體回收所需之DNA片段    12
5-2 PCR產物純化    13
6.    質體DNA之連接反應    13
6-1 末端黏滯端（cohesive end）DNA片段之連接反應    13
6-2 線性載體DNA 5’ 端的去磷酸化反應    13
7.    聚合酶連鎖反應及反轉錄聚合酶連鎖反應（PCR and RT-PCR）    14
7-1 PCR with Taq DNA Polymerase    14
7-2 PCR with Phusion® High-Fidelity DNA Polymerase    14
7-3 RT-PCR    14
8.    Genomic DNA之萃取    14
9.    RNA之萃取    15
10.    cDNA之合成（cDNA synthesis）    15
11.    水稻癒傷組織誘導及轉殖    16
11-1 水稻癒傷組織之誘導及培養    16
11-2 水稻癒傷組織利用農桿菌轉殖之方法    16
12.    農桿菌之轉殖及檢測    17
12-1 農桿菌轉殖方法    17
12-2 農桿菌之檢測    17
13.    蛋白質萃取    17
13-1 CCLR萃取液萃取蛋白質之方法    18
13-2 Lysis/RIPA萃取液萃取蛋白質之方法    18
14.    SDS聚丙烯醯胺凝膠電泳（SDS-PAGE）    18
15.    Coomassie blue staining及西方墨點法（Western blot）    19
15-1 Coomassie blue staining    19
15-2 西方墨點法（Western blot）    19
16.    原生質體萃取    20
貳、    實驗結果    21
1.    水稻OsRH42突變株的產生及突變位置分析    21
2.    T0代OsRH42突變株之脫靶效應檢測    21
3.    OsRH42突變株T1代genomic DNA分析及種子發育情形    22
4.    水稻OsRH42轉殖株及突變株種子發育的外表型分析    23
5.    水稻OsRH42轉殖株及突變株生長發育的外表型分析    23
6.    水稻OsRH42轉殖株及突變株處理長時間之冷逆境（4℃）分析    24
7.    利用XVE雌激素誘導表現系統調控OsRH42之表現    24
8．    OsRH42-GFP6之蛋白質分析    25
9.     TurboID鄰近標記蛋白技術之質體建構    26
參、    討論    28
1.    OsRH42突變株之分析    28
2.    T2代OsRH42剪接缺失突變株的生長發育情形分析    29
3.    OsRH42是參與水稻抗冷逆境基因之pre-mRNA剪接的重要蛋白    30
4.    XVE誘導表現系統利用    31
5.    OsRH42之pull down assay分析    32
肆、    參考文獻    34
表目錄
表1. 比較wild type（WT）、OsRH42 knock down（RNAi）、OsRH42 overexpression（OX）轉殖株及OsRH42剪接缺失突變株T2代（Cas9-T2）之特性        39
表2. OsRH42親緣關係蛋白質相關資訊    39
圖目錄
圖1. Cas9-OsRH42質體建構及T0代OsRH42突變株genomic DNA檢測    40
圖2. OsRH42突變株之脫靶效應檢測及定序分析    41
圖3. OsRH42突變株T1代對種子發育的影響    42
圖4. OsRH42轉殖株及突變株種子發育的表現型比較    43
圖5. OsRH42轉殖株及突變株的生長發育表現型比較    44
圖6. OsRH42轉殖株及突變株之冷逆境（4℃）處理結果    45
圖7. XVE誘導表現質體建構及轉殖株檢測    46
圖8. OsRH42-GFP6質體建構，轉殖株轉基因檢測及pull down assay結果    48
圖9. TurboID-OsRH42質體建構及轉殖株轉基因檢測    50
附錄
附錄I 本論文所使用的primers及其用途    51
附錄II wild-type與OsRH42突變株序列比對    53
附錄III 培養基及藥品配方    57
附錄IV 水稻處理各種非生物逆境下及各個組織器官OsRH42之表現量    61
附錄V wild-type植株冷處理（4℃）不同天數之存活率    61
附錄VI wild-type植株、OsRH42-OX及RNAi轉殖株冷處理18 h後CBP-like及MR7之IR    61
附錄VII wild-type植株、OsRH42-OX及RNAi轉殖株pre-mRNA剪接異常之RNA-seq結果    62
附錄VIII OsRH42同源蛋白比較    62
附錄IX wild-type植株、OsRH42-OX及RNAi轉殖株OsRH42之表現量    62
附錄X wild-type植株、OsRH42-OX及RNAi轉殖株小苗期及成熟期之株高    63
附錄XI OsRH42表現在splicing speckles，與U2 snRNA及OsSF3b1有交互作用    63
附錄XII 質體建構    1


                                

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