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Molecular cloning and functional analysis of Papaya ringspot virus resistance genes in Cucumis metuliferus
Papaya ringspot virus
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|摘要:||許多瓜類在熱帶及亞熱帶地區容易受到木瓜輪點病毒(Papaya ring spot virus, PRSV)侵害，造成植株生長受阻以及產量及品質下降，因此了解植物抗病毒機制與培育抗病之栽培品種為當前重要的工作。目前對於木瓜輪點病毒的防治策略，包括:交互保護方法和利用病毒誘導基因靜默方法來達到木瓜輪點病的防治。然而上述的方法雖然可以達到有效的成果，但是交互保護需要高度勞力，且病毒基因發生突變時，病毒誘導基因靜默的抗病方法就無法達到有效抑制病毒的效果。因此本研究的目地為探討木瓜輪點病毒與其寄主植物刺角瓜之間抗病反應的交互作用，並且從中選殖與PRSV抗病反應相關的候選基因，最後希望未來將該抗病基因應用在其它改善植物的病毒抗病育種。本實驗共分成三個部分，首先為了之後分析刺角瓜候選基因的功能，必須建立刺角瓜的再生與農桿菌轉殖系統。該系統主要是使用刺角瓜的子葉作為再生與農桿菌轉殖的培殖體，結合植物生長素(BA和NAA)的使用，最後只要6~8個星期的時間就可以成功得到刺角瓜轉殖株，目前轉殖系統的成功效率有2.5%。另外，為了探討刺角瓜在PRSV感染後，其基因的差異表現，本研究利用cDNA-AFLP方法針對兩個刺角瓜品系(分別為抗病品系PI 292190和感病品系Acc. 2459)進行多型性片段的分析。經由比較抗病品系PI 292190和感病品系Acc. 2459接種PRSV，以及對照組處理後，將差異表現的片段進行選殖。最後有139個候選片段可能與PRSV抗病的反應有關係，之後利用reverse northern blot和northern blot方法篩選出25個可能與PRSV抗病反應有關的候選基因。其中CmPI 1基因的表現分析在northern blot結果顯示，抗病品系PI 292190接種病毒後48小時，該基因的表現量快速提升並達到最高;反之感病品系Acc. 2459的基因表現需要到接種後21天才出現。經由序列比對CmPI 1基因可以得知其編碼出來的蛋白與potato I type蛋白酶抑制子有很高的相似。另外，PRSV屬於potyvirus家族，該家族的病毒須要利用自身的蛋白酶進行病毒的複製和感染反應。由這些證據顯示，CmPI 1基因的蛋白產物可能與病毒的蛋白酶有交互作用，進而干擾蛋白酶的功能。本研究因此將CmPI 1基因片段構築在可以產生siRNA的RNAi載體pEPJ86i，並利用農桿菌轉殖法進行刺角瓜抗病品系PI 292190轉殖，最後也獲得CmPI 1基因靜默的轉殖株。將CmPI 1基因靜默的轉殖株接種PRSV可以發現，轉殖株在接種後21天有病徵出現，證實抗病品系PI 292190的CmPI 1基因表現被抑制時，刺角瓜對PRSV的抗性會被破壞並且導致感病。此外，CmPI 1過量表現試驗的結果，證實感病品系Acc. 2459在持續表現CmPI 1基因的情形下，轉基因Acc. 2459可以抵抗PRSV感染。綜合上述結果，可以確認刺角瓜的CmPI 1基因參與PRSV抗病反應，並且可以提供刺角瓜抗PRSV的能力。本論文的結果有助於了解抗病相關的候選基因在植物與病毒抗病反應之間可能扮演的角色，另外利用植物蛋白酶抑制子提供植物在potyvirus屬病毒的抗病能力，也將是未來病毒防治上另一個可行的策略。|
Papaya ringspot virus (PRSV) is Potyvirus genus and causes severe damages of crops especially papaya and cucurbits. It is a top priority to understand mechanisms of plant virus resistance to assist the breeding programs for developing resistant cultivars. Until now, the control of PRSV includes cross-protection and virus-induced gene silencing (VIGS). However, labor-intensive work and resistance broken have limited the application of these strategies. This study aims to investigate the interaction between Cucumis metuliferus and PRSV, and identify defense-related candidate genes. On top of that, the candidate genes could be used in the improvement of plant resistance breeding. This study contains three major parts of contribution. First, the regeneration and Agrobacterium-mediated transformation system of C. metuliferus were developed to assist functional characterization of candidate genes. The cotyledon of C. metuliferus was used as explants in the regeneration and Agrobacterium-mediated transformation experiments. By means of benzyl adenine (BA) (1 mg/l) and naphthaleneacetic acid (NAA) (0.02 mg/l), six to eight weeks are acquired to obtain transgenic C. metuliferus plants and the transformation efficiency was 2.5%. Second, the cDNA-AFLP method was used to analyze the differential displayed genes of C. metuliferus treated with or without PRSV-inoculation. The polymorphic fragments were identified by comparing the difference of fragments occurring in resistant line PI 292190 and susceptible line Acc. 2459 inoculated with PRSV or treated with mock. A total of 139 candidates were cloned and 25 candidates of them were selected based on further results of reverse northern blot and northern blot analysis. One of these candidates, CmPI 1 has shown the most possible candidate correlated with PRSV resistance. As the result of northern blot, the gene expression of CmPI 1 has been shown to increase quickly in resistant line PI 292190 at 48 hour-post inoculation (hpi), in contrast, express not until 21 day-post inoculation (dpi) in susceptible line Acc. 2459. CmPI 1 encodes a protein with homology to potato I type proteinase inhibitor by sequencing analysis. In addition, PRSV needs virus proteinases to complete its replication and infection cycle. This implicated that CmPI 1 could impede the function of virus proteinase by interacting with virus proteinases. To estimate this hypothesis, the fragment of CmPI 1 was constructed onto an RNAi vector, pEPJ86i. The resistant line PI 292190 was transferred with this RNAi construction using Agrobacterium-mediated transformation method. The transgenic plants were shown infectious and symptoms were observed after 21 dpi. In the over-expression CmPI 1, a resistant allele derived from resistant line PI 292190 was transformed into susceptible line Acc. 2459 to test the resistance ability of cloned CmPI 1 gene against PRSV. According to the above results, CmPI 1 were able to participate in PRSV resistance and contribute the resistance ability in C. metuliferus. Overall, the results in this dissertation provide a better understanding the roles of defense-related genes in host-virus interaction. The application of plant proteinase inhibitors in inducing resistance against potyviruses would also be another possible strategy for virus control.
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