探討受仙人掌X病毒與紅龍果X病毒感染影響之寄主基因

Investigation of host genes affected by Cactus virus X and Pitaya virus X infection

李克(Ke Lee)

指導教授 ： 張雅君

共同指導教授 ： 林詩舜(Shih-Shun Lin)

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202003267

摘要

仙人掌科三角柱屬(Hylocereus spp.)的紅龍果是熱帶地區常見的水果作物。根據報告，目前台灣感染紅龍果的病毒有仙人掌X病毒(CVX)、紅龍果X病毒(PiVX)和蟹爪蘭X病毒(ZyVX)三種。田間調查顯示，在台灣主要紅龍果種植區中紅龍果植株均至少感染這三種Potexvirus屬病毒中的一種，且常見複合感染。因這些紅龍果病毒的分子感染機制仍待探討，本論文採用轉錄體與文獻搜尋的方法研究紅龍果與這些病毒之間的交互作用。熱休克蛋白70家族(Hsp70s)和葉綠體磷酸甘油酸激酶被已被報導為其他potexvirus的相關的寄主基因，以這些植物蛋白為對象，分析被CVX和PiVX感染的植物。半定量反轉錄聚合酶連鎖反應的結果顯示，Hsp70c-4的表現量在PiVX感染的圓葉菸草中被向下調控。至於初步的轉錄體分析，分別以對照組、感染CVX和PiVX的紅龍果(H. undatus)總RNA進行次世代定序，利用de-novo 組裝法，總共獲得60,510個片段重疊組(contig)；之後使用阿拉伯芥(TAIR)和歐洲分子生物學實驗室(EMBL)的編碼序列(cds)資料庫，進行註解和開放閱讀框(ORF)預測。透過轉錄體表現量與文獻搜索，自差異表現基因中挑出11個主要研究目標。為驗證轉錄體資料的可信度，從中找出HSPRO2與BMY3作為參考基因，並以定量RT-PCR分析幾個差異表現基因的表現量，但結果與轉錄體資料不符。為了取得更有說服力的結果與資料，對另外兩批紅龍果RNA樣品進行定序，並結合之前的定序資料重新建構轉錄體。根據不同處理之間的基因表現量的變化和皮爾森相關係數，建構出基因網絡圖。PiVX感染網路圖的初步分析揭露數個與離層酸相關的基因被負調控，而參與在吉貝素訊息傳導路徑的基因也發現被調控；暗示PiVX感染可能會影響吉貝素訊息傳導路徑，並抑制離層酸訊息傳導路徑。CVX感染網路圖所包含的基因數量遠高於PiVX，其中幾個已被報導的potexvirus寄主因子與主要目標基因也在其中，但他們在CVX感染中扮演的角色仍待探討。CVX和PiVX感染的紅龍果網絡共享38個基因，其中heat shock transcription factor A2 (HSFA2.2)、outer membrane tryptophan-rich sensory protein-related gene (TSPO)和phytochrome interacting factor 3 (PIF3)的基因表現量皆顯著低於對照組，但這些基因對CVX與PiVX感染的貢獻仍屬未知。總而言之，本研究為我們解析CVX和PiVX感染如何影響其寄主紅龍果，提供了基礎資料與未來研究方向。

關鍵字

紅龍果 ； 寄主因子 ； 仙人掌X病毒 ； 紅龍果X病毒 ； 轉錄體分析

並列摘要

Pitaya (Hylocereus spp.), in the family Cactaceae, is a common fruit crop in tropical regions. Three potexviruses, Cactus virus X (CVX), Pitaya virus X (PiVX), and Zygocactus virus X (ZyVX), are reported to infect pitayas in Taiwan. Field surveys revealed that all pitayas at the major planting area of Taiwan are infected by at least one of the three potexviruses and mixed infections are commonly found. Because the molecular infection mechanisms of these potexviruses have not yet been revealed, in this study transcriptome approach and paper mining were applied to investigate the interaction between pitaya and its viruses. Among previously reported host genes related to potexviruses, heat shock protein 70 family and chloroplast phosphoglycerate kinase were selected and used to analyze the CVX and PiVX-infected plants. Semiquantitative RT-PCR revealed that expression level of Hsp70c-4 was down-regulated in PiVX-infected Nicotiana benthamiana. As for preliminary transcriptome analysis, purified total RNAs of mock, CVX- and PiVX-infected pitaya (H. undatus) plants were collected for next-generation sequencing. A total of 60,510 contigs were assembled using de novo assembly method followed by annotation and open reading frame prediction with TAIR and EMBL cds databases. We eventually selected 11 primary targets from differentially expressed genes (DEGs) based on their expression levels in transcriptome and literature review. To confirm transcriptome results, the expression levels of several DEGs were verified with quantitative RT-PCR using HSPRO2 and BMY3 as reference genes which were identified from the transcriptome data. The results of quantitative RT-PCR and transcriptome were different from each other. To obtained a more convincing result, another two sets of pitaya RNA samples were sequenced to construct a transcriptome combined with previous sequencing data. Gene-to-gene networks were generated based on fold changes between genes in each treatment and their calculated Pearson correlation coefficient value. Early analysis of PiVX infection network disclosed that several ABA-associated genes were down-regulated, while genes involved in GA signaling pathway were also found to be regulated, suggesting that PiVX infection may affect GA signaling pathway and influence ABA signaling pathway. Infection network of CVX contained a lot more genes than that of PiVX, and few reported potexvirus host genes and primary targets were found in the network, but their roles in the infection of CVX required more study. Network of CVX- and PiVX-infected pitayas shared 38 genes. Among them, the expression levels of heat shock transcription factor A2 (HSFA2.2), outer membrane tryptophan-rich sensory protein-related gene (TSPO.2) and phytochrome interacting factor 3 (PIF3) in CVX- and PiVX-infected treatments were significantly lower than mock treatment. Nevertheless, how these genes contribute to the infections of CVX and PiVX remain unclear. In short, this study improves our understanding of how CVX and PiVX infection affect their pitaya host, and provides the basic information and future research direction.

並列關鍵字

pitaya ； host factor ； Cactus virus X ； Pitaya virus X ； transcriptome analysis

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