番茄抗青枯病品系Hawaii7996之解序與其抗第一演化型青枯病菌基因座Bwr12之研究

Genome draft of bacterial wilt resistant tomato Hawaii7996 and study of the resistance locus Bwr12 against Ralstonia solanacearum phylotype I strains

林露(Lu Lin)

指導教授 ： 鄭秋萍

國立臺灣大學/生命科學院/植物科學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10705

摘要

由細菌Ralstonia solanacearum (Rs) 引起的土壤傳播性青枯病 (bacterial wilt，簡稱 BW) 可感染全球兩百多種植物，其中包含許多重要經濟作物，且是番茄前五大病害之一。番茄品系Hawaii7996 (H7996)是目前最為穩定的青枯病抗病植物，且已發現數個抗病數量性狀基因座(quantitative trait loci，簡稱QTL)，然而，其抗病分子機制與關鍵抗病基因卻尚未知。為深入了解 H7996遺傳性質，採用全基因組解序策略並與 Heniz 1706 (H1706) 基因組進行序列比對後，結果發現核苷酸具有多型態變異並且普遍存在於各條染色體之基因片段與非基因片段。本研究針對位在 H7996第12條染色體上之抗第一演化型青枯病菌 QTL (命名為Bwr12) 進行基因搜尋與功能研究。在Bwr12區段預估的67個基因中，包含12個leucine-rich repeat (LRR)-receptor like kinase (RLKs)與LRR-receptor like protein (RLPs)，序列分析結果顯示其中多個RLKs基因的序列在H7996與H1706中具明顯差異。病毒誘導基因靜默 (virus-induced gene silencing，簡稱 VIGS) 分析所得結果發現其中數個 RLKs 被個別或共同靜默後會造成 H7996之抗性明顯降低，且病毒誘導基因表現 (virus-mediated gene over-expression，簡稱 VMGO) 分析結果更進一步顯示12g520與12g550之表現可使不抗病番茄品系 WVa700之抗性增加，顯示12g520與12g550是 H7996抗青枯病之關鍵正調控者。 Pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) 被認為參與在抗病數量性狀位點中，根據基因靜默試驗結果得知12g520與12g550參與青枯病 PTI 反應之癒傷葡聚醣累積、 H2O2累積及 PTI 基因PTI5生合成，具有抵抗Pseudomonas syringae pv. tomato DC3000 hrcC- mutant 與Rs Pss4 hrpG- mutant 的能力。此外，蛋白質定位結果顯示12g520坐落在細胞膜。以上研究結果不但提供了番茄抗青枯病關鍵基因的重要訊息，預期亦可有助於發展基因標記，以提高抗病育種的篩選精準度與效率。

關鍵字

青枯病 ； 青枯病菌 ； 數量性狀位點 ； H7996 ； LRR ； RLK ； PTI

並列摘要

Bacterial wilt (BW), caused by Ralstonia solanacearum,is a highly complex and serious disease affecting a wide range of economically important crops worldwide, including tomato. Breeding for durable resistance in crops for this disease is important and urgent. Thus, comprehensive information on plant defense mechanisms would be highly desirable and necessary, but are still limited. Tomato cultivar Hawaii 7996 (H7996) is currently the most durable resistance source against BW. Despite various BW-resistance associated quantitative trait loci(QTLs) have been mapped on H7996 chromosomes, the involved molecular mechanism and gene identity remain undetermined. To gain insight into the genetic nature of H7996, we have determined its genome draft. Genome-wide sequence comparison analyses showed high sequence variations on several chromosomes between H7996 and the announced BW-susceptible cultivar Heniz 1706. In addition, we also carried out functional study on 12 putative leucine-rich repeat (LRR)-receptor like kinases (RLKs) and LRR-receptor like proteins (RLPs), which are located on one major QTL (Bwr12) associated with the H7996 resistance against Rs phylotype I strains. Comparative sequence analysis in H7996 and the Heniz1706 revealed significant sequence diversity in most of these genes. Virus-induced gene silencing (VIGS) assays suggested the involvement of a few RLKs in H7996 BW-resistance, and virus-mediated gene silencing (VMGO) assays further confirmed a positive role of 12g520 and 12g550 in H7996 BW-resistance. Pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) is suggested to be involved in disease-resistance associated quantitative traits. Furthermore, transient gene silencing assays suggested play an important role in PTI responses including callose deposition, H2O2accumulation and marker gene PTI5 expression against Pseudomonas syringaepv. tomato DC3000 hrcC- mutant and Rs Pss4 hrpG- mutant. In addition, GFP-12g520 is localized at the plasma membrane. These results together not only provide important information on BW-resistance genes involved in H7996 defense, as well as pave the way to develop gene-based markers useful for improving efficacy and efficiency of tomato breeding programs in industry and public sectors.

並列關鍵字

Bacterial wilt ； Ralstonia solanacearum ； QTL ； Hawaii7996 ； LRR ； RLK ； PTI

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