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

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題名:	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
作者:	梁竣瑋;Loong, Zing-Wei
貢獻者:	生命科學系
關鍵詞:	水稻;解旋酶;基因編輯;Oryza sativa;RNA helicase;CRISPR/Cas9
日期:	2022-08-29
上傳時間:	2022-10-04 11:03:20 (UTC+8)
出版者:	國立中央大學
摘要:	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.
顯示於類別:	[生命科學研究所 ] 博碩士論文

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