利用半胱胺酸蛋白酶抑制因子及幾丁質酶發展抗根瘤線蟲的轉殖番茄

Abstract

根瘤線蟲是植物主要寄生性病蟲害之一，種類繁多且宿主範圍廣泛，往往造成作物損失慘重。為了防治線蟲的侵襲，本研究採用農桿菌轉殖技術，將攻擊線蟲的蛋白質大量表現在耐熱蕃茄CLN2468D中。所選用的基因分別是來自Paecilomyces javanicus的幾丁質酶基因，PjCHI-1，以及來自芋頭的半胱胺酸蛋白酶抑制劑基因，CeCPI。大量表達CeCPI的轉殖蕃茄感染線蟲後產生的根瘤及卵塊數均少於非轉植株，而且母蟲的比率也大幅降低。因此，大量表達CeCPI的轉殖蕃茄不僅抑制根瘤形成，也干擾線蟲的性別轉換而降低線蟲的子代數量。同樣的，具有高量幾丁質酶活性的轉殖植物感染線蟲後產生的卵及卵塊數量也都少於非轉殖株。採自非轉殖株的線蟲卵，其卵殼的幾丁質含量高於來自轉殖株的線蟲卵，而幾丁質酶的活性則是低於採自轉殖株的卵塊。大約96 %來自非轉殖株的線蟲胚胎能夠正常的發育，但只有約30 %來自轉殖株的胚胎能夠達到同樣的發育階段。因此，將PjCHI-1大量表達於植物中，具有降低線蟲子代數量之效果，並且抑制線蟲胚胎的發育。本研究證實大量表達這兩個基因的轉殖植物能夠有效的破壞線蟲的繁殖能力，進而抑制線蟲族群，降低根瘤線蟲對作物的危害，從而達到植物保護的效果。同時，這些研究結果也提供了農藥防治以外的另一個抗線蟲策略。

Root-knot nematodes (RKN, Meloidogyne spp,) are a major pest of many plant species and cause global economic loss. Paecilomyces javanicus chitinase gene (PjCHI-1) and Colocasia esculenta cysteine protease inhibitor gene (CeCPI), driven by a CaMV35S promoter were delivered into a heat-tolerant tomato inbreeding line, CLN2468D, respectively. When infected with RKN, transgenic T1 lines overexpressing CeCPI displayed a pronounced reduction in the numbers of galls and egg mass. Additionally, a much lower proportion of female nematodes was observed in transgenic plants than it in control plants. The study demonstrated that CeCPI has inhibitory functions in the early RKN infection stage as well as in the production of offspring, which may result from intervention in sex determination. Similarly, transgenic T1 plants with high endochitinase activity exhibited a reduction in the number of eggs and egg masses when infected with RKN. The eggs found in transgenic tomato showed low chitin component in eggshell compared to the eggs collected from control plants. Egg mass in transgenic plants exhibited higher chitinase activity than in control. Furthermore, only one-third of eggs in transgenic plants developed into multi-cell/J1 stage. Our work demonstrated that PjCHI-1 can effectively reduce the production of eggs and repress the embryogenesis of M. incognita. The present endeavor hence, implicates the feasibility of a novel agro-biotech strategy for preventing crop damages by RKN.