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標題: 環境因子對於南方根瘤線蟲性別分化之影響
Effects of Environmental Factors on the Sex Differentiation of Southern Root-Knot Nematode(Meloidogyne incognita)
作者: 林怡欣
Lin, Yi-Hsin
關鍵字: 雄蟲
Meloidogyne incognita
sex differentiation
suppression subtractive hybridization
出版社: 植物病理學系所
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摘要:   根瘤線蟲二齡幼蟲在寄主處於逆境(stress)的狀況下,會改變其性別分化方向而發育為雄蟲,本研究分別以三種不同氮磷鉀成份比例肥料添加於寄主、將二齡幼蟲接種於具抗性基因之寄主、於接種後將寄主施以剪枝或甲基茉莉酸(MeJA)處理以及使用五種不同酸鹼值溶液浸泡處理二齡幼蟲後接種,探討寄主養分效應以及酸鹼值等環境因子對於南方根瘤線蟲(Meloidogyne incognita)性別分化的影響。亦利用抑制性扣除雜交法(Suppression subtractive hybridization,SSH)比較線蟲體內特殊之基因表現。在線蟲動物中,C. elegans的性別分化系統(Sex differentiation system)是被研究最透徹的例子,本研究以C. elegans性別決定基因之兩組引子對分別增幅南方根瘤線蟲二齡幼蟲、雄蟲及母蟲的cDNA,結果發現此三種發育階段的線蟲體內均不表現此兩種基因。推測在南方根瘤線蟲體內並沒有與C. elegans已知性別決定相關基因相似的同源性基因,兩者的性別決定系統並不相同,南方根瘤線蟲之性別決定機制受環境因子調控之可能性較大。添加三種肥料之所有處理其雄蟲數量均與對照組無顯著差異,推測因肥料有助於植物生長,對於寄生的根瘤線蟲而言為適合生存的環境,故其族群中雄蟲的分化量並無顯著提升。抗性寄主試驗於兩次種子發芽袋之結果皆顯示,接種南方根瘤線蟲後的抗性豇豆品系CB46具有顯著較低的根瘤指數以及較低的二齡幼蟲數量,而其雄蟲則顯著較感性品系CB46 NIL null為多,推測具Rk基因的抗性植物其細胞於二齡幼蟲侵入後期即無法繼續提供養分給發育中的線蟲,此種缺乏養分的環境壓力(food stress)可能為誘導雄蟲分化的因子。將接種南方根瘤線蟲之植物施以剪枝或施用甲基茉莉酸,所有處理之雄蟲數量均較對照組為多,再次佐證雄蟲分化之提升與寄主植物細胞供給根瘤線蟲養分之能力受影響有關。將南方根瘤線蟲二齡幼蟲以不同pH值溶液浸泡處理一天後接種於寄主植物,模擬環境酸鹼值對於後續齡期性別分化之影響,在種子發芽袋試驗系統下,以pH 5和pH 7的溶液浸泡幼蟲之處理有顯著較多的雄蟲分化,但在盆缽試驗系統下則以pH 9和pH 11之處理有顯著較多的雄蟲產生。以抑制性扣除雜交法(SSH)得到pH 11溶液處理下二齡幼蟲之表現基因標幟(Expressed sequence tag,EST),於NCBI資料庫比對結果顯示,此些基因的功能在現有資料庫中沒有任何同源性。本研究亦比較南方根瘤線蟲二齡幼蟲與雄蟲體內各自特殊表現之基因差異,於NCBI資料庫比對結果顯示,以雄蟲為背景值所建立之二齡幼蟲減扣基因資料庫(Subtracted J2 library)中表現了ATP synthase、CTP synthase、Sugar transporter-like protein和Transcription termination factor等同源基因;而在Nematode Genome Sequencing Center資料庫的比對結果中,二齡幼蟲減扣基因資料庫中亦有與蟲生線蟲Steinernema feltiae乾旱逆境相關表現基因標幟(Desiccation stress related ESTs)具同源性之基因表現,推測南方根瘤線蟲二齡幼蟲時期會大量表現能量合成相關基因以及抵抗環境逆境之相關基因以利於尋找適當寄主、侵入或於無適當寄主狀況下殘存,而雄蟲階段特殊表現的基因在現有資料庫中則無發現任何同源性序列。由於現有基因資料庫無法預測此些南方根瘤線蟲雄蟲體內特殊表現基因或是鹼性環境刺激下二齡幼蟲特殊表現基因之功能,故無法確定本研究四個資料庫中新的表現基因標幟(novel ESTs)是否與後續性別分化過程有所相關。未來可繼續利用抑制性扣除雜交法製作以二齡幼蟲為背景值之雌蟲減扣基因資料庫(Subtracted female library),分析在雌蟲體內特殊表現的基因功能,而將其與本研究之雄蟲減扣基因資料庫(Subtracted male library)結果相互比較,則可更深入探討南方根瘤線蟲性別分化相關基因在雌雄蟲體內表現有無之差異或表現量之差異。
The second-stage juveniles of Meloidogyne spp. have been reported to redirect their development towards males when the host is under stress. In this study, five treatments were used to investigate the effects on the sex differentiation of M. incognita, they were applying three different N-P-K fertilizer proportions, host resistant gene, pruning, methyl-jasmonic acid (MeJA) applications and five different pH value treatments of juveniles. The males, juveniles and juveniles treated with high pH were proceeded for the suppression subtractive hybridization (SSH) to compare the specific gene expression in different nematodes. In nematodes, the sex differentiation system in Caenorhabditis elegans is the most well studied. Using the primers of C. elegans sex determination-related genes to amplify the M. incognita second-stage juveniles, males and females resulted no products, indicating no C. elegans sex determination-related homology genes are in M. incognita. The sex determination mechanism in M. incognita is probably affected by the environment factors. When three N-P-K proportions were applied on the plants, the number of males from these treatments did not differ significantly. The three fertilizers might be helpful for plant growth, so the male differentiation did not increase under the favorable growth condition. The cowpea cultivar CB46 has a resistant Rk gene and CB46 NIL null is the near-isogenic line. The galling index and the number of second-stage juveniles on CB46 were relatively low, and the number of males was significantly higher than that on the CB46 NIL null. The interruption of the nutrition-support in resistant plant cells after invasion may trigger the male differentiation. The number of males of pruned hosts and all the MeJA application treatments were significantly higher than the control, again showing that the male differentiation may be induced by the host nutrition effect. The pH 5 and pH 7-treated juveniles when inoculated on the water spinach in the pouch, resulted in the most abundant males compared to other six pH treatments. However, in the pot tests, the pH 9 and pH 11-treated juveniles resulted in the most abundant males. The expressed sequence tags (ESTs) from the pH 11-treated juveniles were obtained using SSH, and these sequences had no homology in the NCBI database. Besides, two subtracted cDNA libraries of second-stage juveniles (J2) and adult males were constructed. Compared to the NCBI database, the ESTs in the subtracted J2 library were related to ATP synthase, CTP synthase, sugar transporter-like protein and transcription termination factor. When comparing subtracted J2 library to the Nematode Genome Sequencing Center, two of the ESTs matched the desiccation stress related ESTs in the entomopathogenic (insecticidal) nematode Steinernema feltiae. The EST results indicated that the second-stage juveniles of M. incognita highly express the energy synthesis-related genes and the survival-related genes to overcome the adverse environment, and search for suitable hosts. The ESTs from the males had no homology in the database. So the functions of ESTs were currently unknown. A subtracted female library could be constructed to analyze the genes specifically expressed in females in the future. By comparing that with the subtracted male library in this study, we would be able to know if the sex differentiation is controlled by the quality or quantity of the specific genes.
其他識別: U0005-0608201215100000
Appears in Collections:植物病理學系



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