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標題: 篩選三種水稻種原庫水稻對水稻根瘤線蟲 Meloidogyne graminicola之抗性
Identification of root-kont nematode (Meloidogyne graminicola) resistance(s) in three rice germplasms
作者: 歐陽皓其
Hao-Chi Ouyang
關鍵字: 水稻;水稻根瘤線蟲;抗病篩選;抗性育種;野生稻;nematode resistance screening;resistance breeding program;rice root-knot nematode;wild rice
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水稻為重要的穀類作物,全球超過1/3人口仰賴稻米作為主要糧食。水稻根瘤線蟲為主要限制水稻生長重要的生物性因子之一。種植抗性品種為一可行的防治策略。但亞洲稻(Oryza sativa)栽培品種對水稻根瘤線蟲缺乏足夠的抗性,且台灣本土的亞洲稻栽培品種對根瘤線蟲抗感性之資料仍尚未建立,無法提供育種與防治參考資料。非洲稻、野生稻常保有對病蟲害的抗性及環境壓力耐受性,可以做為稻作改良上重要的基因提供者。此外台灣水稻基因插入突變庫(TRIM)建立了大量的水稻插入突變品系,提供了探討水稻基因功能的重要工具。本研究篩選了31種野生稻種及野生稻導入系(introgression lines);103年農委會公告之28種優良水稻推廣品種;526種台灣水稻插入突變庫(TRIM)提供的水稻T-DNA突變品系,並對其抗性機制做初步探討。31種野生稻種及野生稻導入系的初步篩選結果顯示導入系NT-3(Tainung 67 x Oryza officinalis)、NT-6(Tainung 67 x O. officinalis)以及來自非洲的NT-58(O. longistaminata)、NT62(O. punctata)、NT-66(O. glaberrima)、NT-72(O. barthii)等品系表現出對水稻根瘤線蟲良好的抗性潛力,其平均結瘤數分別為3.4、7.5、4.8、5.4、2.8與敏感品系IR64平均結瘤數24相較偏低。28種優良水稻推廣品種篩選結果中,台中秈10號與台東30號品系兩者有許多2級根瘤指數(結瘤率11~30%)的個體,與IR64皆為3、4級根瘤指數(結瘤率31~70%)相比,推測兩者應為耐病品系。其餘栽培品種結果則與敏感品系IR64差異不大,顯示台灣目前常用之水稻栽培品系普遍缺乏對根瘤線蟲抗性。利用水稻根部對水稻根瘤線蟲測試誘引率,IR64、NT3、台東30、台中秈10與Tainung 67於接種後0、12、24小時對水稻根瘤線蟲誘引率皆於12小時後始有上升趨勢,但各品種之間的誘引率在各時間點下於統計上無顯著差異,暗示其抗性應與寄主誘引無關,而是取決於線蟲入侵後植株其他生理反應有關。將具抗性同源導入系NT3、NT6與敏感品系IR64的根部進行切片觀察,皮層平均厚度IR64(308.6μm)、NT3(371.8μm)、NT6(413.9μm)三者間有明顯差異。中柱平均厚度IR64(344.0μm)、NT3(351.5μm)兩者間無明顯差異但與NT6(418.8μm) 有顯著差異。同源導入系NT3、NT6中,抗性較高的NT-3根部皮層與中柱較敏感的NT6有較厚的情形,顯示組織構造差異可能為決定抗性的因子之一。於接種水稻根瘤線蟲後0、12、24、48小時後以Evan's blue 對NT3與IR64根部進行染色,兩者無明顯差別,顯示NT3之抗性未涉及細胞凋亡。為了再進一步了解野生稻導入系NT3的抗性與植物防禦反應後關係,於接種水稻根瘤線蟲後0、12、24、36、48小時後對NT3與IR 64中Actin 1, NPR1, AOS2 三個基因的表現進行了分析,但結果無法判別NT3與IR64之間的差異,若欲進一步釐清其芬子抗病機制,還需要以定量即時聚合酶鏈鎖反應並針對更多抗病路徑相關基因的表現量加以比較。526個水稻T-DNA突變品系中,篩選出23個品系具有對根瘤線蟲抗性潛力。23個品系中的8個已有插入點位置資料, 其中5個品系與生長素基因有關。本研究結果針對野生稻、野生導入系與台灣栽培品系的篩選結果,能提供未來進行水稻育種時的參考資料,有部分品系甚至可以直接用於田間,減少田間水稻根瘤線蟲的密度。而T-DNA突變品系篩選出來的品系,也可提供後人探討生長素與水稻根瘤抗性的關係,甚至能夠了解水稻根瘤線蟲如何調控水稻生長素的相關基因。

Rice is the most important staple food for a large part of the world's population. Rice Root-knot nematode Meloidogyne graminicola is one of the important biotic factors limiting rice productivity. Growing of resistant or tolerant cultivars is certainly the most practical method to reduce root-knot nematodes in the field But no strong and reliable resistance to the rice root-knot nematode has been found in Asian rice (Oryza sativa), and rice cultivars in Taiwan are also lack information of resistance to rice root-knot nematode. Compared to Asian rice, African rice and wild rice are a valuable source of resistance genes for developing resistant cultivars. Taiwan rice insertional mutants database(TRIM) developed a large population of insertional mutant rice lines, provide a strategies for rice functional genomics research. 31 wild rice or wild rice introgression lines, 28 Taiwan commercial rice cultivars and 526 Rice insertion mutant lines (TRIM) were used to screen the resistance of rice root-knot nematode. Wild rice introgression lines, NT-3(TNG67 x O. officinalis), NT-6 (TNG67 x O. officinalis) were highly resistant to M. graminicola. Wild rice NT 24(O. glaberrima), NT-58(O. logistaminata), NT62(O. punctata), NT-66(O. glaberrima), NT-72(O. barthii) also showed resistant to Meloidogyne graminicola. Among 28 Taiwan commercial rice cultivars screened, all showed susceptibility except Taichung sen 10 and Taitung 30. The chemotaxis test results indicated the resistance trait of NT-3, Taichung sen 10 and Taitung 30 is not related to feeding deterrent. NT 3, NT 6 and IR 64 were used for histological study and results indicated that NT 3 and NT 6, with narrower cortex and stele may affect the feeding activity of the rice root-knot nematode. Roots of NT3 and IR 64 were staining with Evna's blue to analyze root cell death induction by rice root-knot nematode infection. Staining results indicated no significant difference between NT 3 and IR 64. The expression pattern of Actin 1, NPR1 and AOS2 were examined in the roots of NT 3 and IR 64 infected by RKNs and no differences were found ,Real-time Quantitative Polymerase Chain Reaction should be applied in the future for more precise results. Screening the Taiwan insertion mutant rice (TRIM) lines, 23 of 526 tested lines might have potential resistances. Among 23 resistant T-DNA insertion lines, 8 already had the position of T-DNA insertion information, among them, 5 lines showed Auxin metabolic related genes might be affected. The results of this research provides the potential resistant gene were present in wild rice germplasm, and rice species that worth investigate in the future breeding program, as well as the possible resistant mechanisms for the future research.
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