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標題: | 腸炎弧菌小型核糖核酸 RyhB 的調控目標 Regulatory targets of small RNA RyhB in Vibrio parahaemolyticus |
作者: | Jia-Chi Gu 古家齊 |
指導教授: | 李佳音(Chia-Yin Lee) |
關鍵字: | 腸炎弧菌,小型核糖核酸,次世代定序,鹼基配對, Vibrio parahaemolyticus,sRNA,RyhB,RNA-seq,base-pairing, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 小型核糖核酸在許多生理功能中扮演重要角色,其中之一的 RyhB 被廣泛研究,可透過與目標 mRNA 進行鹼基配對而調控基因表現,使細菌能快速適應周遭環境的變化,目前有關腸炎弧菌 RyhB 僅有少數的報導。本研究以臺灣分離之 Vibrio parahaemolytius no.93 作為實驗菌株,建構腸炎弧菌 ryhB 突變株與補償株,探討 ryhB 所參與調控之基因。弧菌屬與大腸桿菌 ryhB 序列比對顯示 RyhB 內部具高度廣泛保守區域以及弧菌屬保守區域。以 qRT-PCR 比較刪除 RyhB 部分保守區域之 ∆ryhB1 突變株與刪除 5’ 端之 ∆ryhB2 中 vp0106 和 sodB 基因表現,發現 RyhB cis- 調控目標之vp0106 在 ∆ryhB1 中大量表現,∆ryhB2 則相對野生株有相似或較低的表現量;RyhB trans- 調控目標 sodB 則在兩株 ryhB 突變株中皆大量表現。由 qRT-PCR 與 RNA-seq 分析基因轉錄表現,鐵離子相關的超氧歧化酶 sodB (VP2118)、琥珀酸去氫酶 sdhC (VP0843),毒性相關的 T3SS1 調節子 toxS (VP0819) 受 RyhB 負調控而抑制其表現;RNA 聚合酶 σ 因子 rpoS (VP2553) 與未知功能基因 vp0106 受 RyhB 正調控而促進其表現。RNA-seq 結果顯示,野生株與 ∆ryhB2 突變株在缺鐵環境下會造成 864 個基因具 1.5 倍以上之差異表現,其中被 RyhB 抑制之基因為 648 個,GOG 分類主要為胺基酸轉運與代謝 (9.1%)、轉譯與核糖體結構與生合成 (6.6%)、轉錄 (5.3%)、無機離子轉運與代謝 (4.6%)、能量生成與轉換 (4.2%)、後轉譯修飾、蛋白質更新、伴護蛋白 (3.8%)、細胞壁 / 細胞膜 / 套膜生合成 (3.4%);被 RyhB 促進 216 個基因,COG 分類主要為醣類轉運與代謝 (3.3%) 以及訊號傳遞機制 (3.1%)。RNAhybrid 線上程式的預測顯示 RyhB 保守區與目標 mRNA 的 5’ 非轉譯區具形成雙鏈體之潛在鹼基配對區。透過後轉錄螢光報導測試分析,顯示 RyhB 藉由保守區與 vp0106 進行鹼基配對而促進 vp0106 表現。本研究證實腸炎弧菌 RyhB 其高度廣泛保守區對於目標 mRNA 鹼基配對能力有最大影響。 Small RNA (sRNA) plays an important role in many physiological functions. One of the sRNA RyhB has been extensively studied and it could regulate gene expression by base pairing with the target mRNAs, allowing bacteria to rapidly adapt to changes in the surrounding environment. However, Vibrio parahaemolyticus RyhB has only a few reports. In this study, Vibrio parahaemolyticus no.93 isolated from Taiwan was used as the experimental strain to construct the mutant and the complemented strain to investigate ryhB involved in target genes regulation. The alignment of vibrios and E. coli ryhB sequences showed that RyhB had a highly-conserved region and a conserved region of vibrios. The expression of vp0106 and sodB in the ΔryhB1 mutant which RyhB partially conserved region was deleted and in the ΔryhB2 which 5' end of RyhB was deleted were observed by qRT-PCR. It was found that the RyhB cis-regulatory target vp0106 was numerously expressed in ΔryhB1, but in ΔryhB2 was relatively similar or lower than the wild-type strain. The RyhB trans-regulated target sodB was highly expressed in both ryhB mutants. The qRT-PCR and RNA-seq analysis demonstrated that iron-related superoxide dismutase sodB (VP2118), succinate dehydrogenase sdhC (VP0843), and the virulence-related T3SS1 regulator toxS (VP0819) were negatively regulated by RyhB. RNA polymerase σ factor rpoS (VP2553) and the unknown function gene vp0106 were positively regulated by RyhB. The RNA-seq results identified that wild-type strain and ΔryhB2 mutant would have 864 genes which were more than 1.5-fold differential expression in the iron-deficiency environment. Among these genes, 648 genes were inhibited by RyhB, and the GOG classification was mainly amino acid transport and metabolism (9.1%), translation, ribosomal structure and biosynthesis (6.6%), transcription (5.3%), inorganic ion transport and metabolism (4.6%), energy production and conversion (4.2%), post-translational modification, protein turnover, chaperones (3.8%), and cell wall/membrane/envelope biogenesis (3.4%). RyhB promoted the other 216 genes, and the COG classification was mainly carbohydrate transport and metabolism (3.3%) and signal transduction mechanisms (3.1%). The prediction of the RNAhybrid online program indicated that the RyhB conserved region and the 5’ untranslated region of the target mRNAs form a potential base-pairing region required for the formation of sRNA-mRNA duplex. Through the post-transcriptional fluorescent reporter assay displayed RyhB base-paired with vp0106 by a conserved region and promoted vp0106 expression. This study confirmed that the highly-conserved region of Vibrio parahaemolyticus RyhB had the greatest effect on the ability of base pairing with target mRNAs. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7657 |
DOI: | 10.6342/NTU201704126 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 2027-08-20 |
顯示於系所單位: | 農業化學系 |
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