比較不同通量RNA-Seq在 P1/HC-Pro介導之基因沉默效應與開發可檢測具活性Xanthomonas perforans之技術

Comparison of different RNA-Seq throughputs of P1/HC-Pro-mediated silencing suppression, and establishing a molecular detection method for viable Xanthomonas perforans

邱燕欣(Yen-Hsin Chiu)

指導教授 ： 林詩舜

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2022年)

https://doi.org/10.6342/NTU202204026

摘要

本論文架構分三大部分，第一章節為文獻探討。第二、三章節則分別為兩大試驗主軸的論述。第二章節為通過高低通量 RNA-seq 方法探討經由P1/HC-Pro主導的基因沉默效益引起的 ABA/鈣信號傳導反應，本研究運用次世代解序技術探索馬鈴薯病毒Y屬特有的 P1/HC-Pro 病毒抑制因子抑制轉錄後基因沉默 (PTGS)作用機制，研究蕪菁嵌紋病毒 (turnip mosaic virus, TuMV) P1/HC-Pro誘導的基因調控關係，對三種轉基因植物 (P1Tu、HC-ProTu 和 P1/HCTu) 進行了轉錄組分析比較，並使用高通量 (high-througput; HTP) 和低通量 (low-throughput; LTP) RNA-Seq 策略。結果顯示P1/HC-ProTu 干擾了信號通路中內源性離層酸（ABA） 的積累，特別是對乾旱逆境、冷逆境、老化和氣孔動力學的反應，LTP 網絡分析與本研究中 HTP 網絡具備相同的關鍵基因，顯示LTP的應用性。第二部分針對番茄種要細菌性斑點病，建立可快速檢定病原微生物活性的檢定技術，本研究結果顯示用 1% NaClO 進行 1 分鐘的表面消毒可將番茄種子的發芽率提高 90%。此外，在番茄種子披覆枯草芽孢桿菌可抑制原種子上病原Xanthomonas，包括抑制發芽之微生物，並將發芽率提高 90%。利用gumD 基因作為Xanthomonas屬病原體檢測標的，並且通過使用 gumD 的核酸產物為標的，減少以DNA作為分子檢測偽陽性的缺點。

關鍵字

離層酸 ； 次世代高低通量解序分析 ； P1/HC-ProTu ； 鈣信號 ； 逆境反應 ； Xanthomonas 屬 ； 枯草芽孢桿菌 6-14 ； 反轉錄酶PCR ； 發芽 ； 種子檢疫

並列摘要

Two major topics are focus on this thesis. The first topic is focus on P1/HC-Pro viral suppressor to suppress posttranscriptional gene silencing (PTGS). To address the modulation of gene regulatory by turnip mosaic virus (TuMV) P1/HC-Pro (P1/HC-ProTu), a comparative transcriptome analysis of three types of transgenic plants (P1Tu, HC-ProTu, and P1/HC-ProTu) were conducted using both high-throughput (HTP) and low-throughput (LTP) RNA-Seq strategies. The results indicated that P1/HC-ProTu disturbed the endogenous abscisic acid (ABA) accumulation and the AGBA signaling pathway. Additionally, the integrated responses of stress-related genes, in particular to drought stress, cold stress, senescence, and stomatal dynamics, altered the expressions by the ABA/calcium signaling. Crosstalk among the ABA, jasmonic acid, and salicylic acid pathways might simultaneously modulate the stress responses triggered by P1/HC-ProTu. Furthermore, the LTP network revealed crucial genes in common with those identified by the HTP network, suggesting that the effectiveness of LTP is compareatble with the HTP profile. Our findings indicate that P1/HC-ProTu-mediated suppression in RNA silencing altered the ABA/calcium signaling and a wide range of stress responses. The second topic is focus on method for the seed quarantine. We demonstrated that surface sterilization with 1% NaClO for 1 min enhanced tomato seed germination by 90%. In addition, precoating Bacillus subtilis WG 6-14 onto tomato seeds also inhibited harmful microorganisms, including Xanthomonas, and improved germination up to 90%. We also demonstrated that the gumD gene of Xanthomonas spp. can be used for pathogen detection, and false positives were overcome by reverse transcription polymerase chain reaction (RT-PCR) detection with the gumD-specific primer set. We proposed sterilizing seeds and coating them with Bacillus subtilis WG 6-14 to inhibit harmful pathogen infection and using RT-PCR to eliminate false positives for this quarantine pathogen.

並列關鍵字

ABA signaling ； HTP-Seq ； LTP-Seq ； P1/HC-ProTu ； calcium signaling ； stress response ； Xanthomonas, ； Bacillus subtilis 6-14 ； RT-PCR ； germination ； seed quarantine

參考文獻

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