野生型祕魯番茄L. peruvianum LA2157之耐熱型抗線蟲基因之選殖及功能性分析研究

Molecular cloning and functional analysis of heat-tolerant nematode resistance genes from wild tomato (L. peruvianum LA2157)

何湘泠(Hsiang-Ling Ho)

指導教授 ： 葉開溫

國立臺灣大學/生命科學院/植物科學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01379

摘要

根瘤線蟲為一種重要的植物內寄生型害蟲，當其感染番茄後，會造成果實低產量、生長發育不良、萎凋及容易感染其他病原菌等，造成農業經濟的巨大損失。在前人的研究中已知野生型番茄L. peruvianum LA2157在土壤溫度高達32℃時仍然具有抗線蟲能力，該抗線蟲基因被命名為Mi-9，目前其基因尚未被選殖出來。本實驗首先利用蛋白質二維電泳技術建立番茄根部受到根瘤線蟲感染後不同時間的基因表現情形。結果顯示番茄根於感染後24小時在蛋白質有數量最多及最大的表現量，故以感染12小時後的番茄根作為tester扣掉感染0小時的driver進行抑制性扣除雜合（SSH）實驗。從二維電泳系統中挑選出具差異性表現之蛋白質點進行MALDI/MS的分析，其中比對到系統素前趨物（prosystemin）及auxin response factor為前人發表過與受傷訊息傳導及線蟲感染誘導有關之蛋白質，因此進一步設計退化性引子（degenerate primers）經由RT-PCR選殖出其cDNA片段。而較特別的是系統素前趨物具有組織差異性，在根無法選殖的到，在感染後的葉子卻可以。從SSH實驗所得之384個cDNA片段經定序後，得到361個有效之ESTs，其中以催化酶(catalase isozyme2)、熱逆境轉錄因子30(heat stress transcription factors 30)、轉錄因子JERF1(transcription factor JERF1)、半胱胺酸蛋白酶抑制劑(cysteine proteinase inhibitor)、類奇異果蛋白(thaumatin-like protein)作為目標基因進行進一步功能性的探討。北方墨漬轉印法顯示這些目標基因在感染線蟲0、4、8、12、及24小時後具有差異性表現，且大量表現的時間亦不盡相同，可見篩選到的目標基因可能在抗線蟲途徑中分別扮演階段性任務。最後以病毒誘導之基因靜默法（VIGS）研究基因功能，以觀察植物內生型目標基因被knockdown後，是否影響植株對線蟲感染之抗性。

關鍵字

根瘤線蟲 ； 番茄 ； 抗線蟲基因 ； 病毒誘導型基因靜默

並列摘要

Root-knot nematodes (Meloidogyne spp.) are important endoparasitic pests of numerous crop species. The feedings result in damages to tomato plants, including poor fruit yield, stunted growth, wilting and susceptibility to other pathogens, cause billions of dollars in agricultural losses each year. Tomato asscession Lycopersicon peruvianum LA2157 possesses heat-stable nematode resistance gene Mi-9, which is highly resistant to Mi-1-aviruent root-knot nematodes at 32 °C, was identified by Milligan et al.. However, it has not been cloned. In this experiment firstly, a 2-D gel analysis of protein patterns from infected roots was established timely. The most abundant protein spots were found at 24 hr after inoculation. Five differentially expressed proteins were picked up and identified by MALDI/MS, and two interesting proteins i.e. prosystemin and putative auxin response factor, were investigated further. Secondly, subtractive EST clones were produced by subtracting ESTs of control from those of 12-hr post-infecting roots. Total 384 ESTs were chosen for sequencing and analyzed. Among 361 valid ESTs, catalase isozyme2, heat stress transcription factors 30, transcription factor JERF1, cysteine proteinase inhibitor, thaumatin-like protein were further investigated. Northern blot analysis showed that all of these candidate genes were differentially expressed at 0, 4, 8, 12, and 24 hour after inoculation, suggesting that these genes work in different time points and in resistant pathway. Finally, virus-induced gene silencing method was used for functional analysis. The physiological functions in nematode-resistant molecular mechanism and relationship between Mi-9 of these candidate genes will be discussed.

並列關鍵字

root-knot nematode ； tomato ； nematode resistence gene ； virus-induced gene silencing

參考文獻

Holtzmann, O.V. (1965). Effects of soil temperature on resistance of tomato to root-knot nematode (Meloidogyne incognita). Phytpathology 55, 990–992.

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