Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30777
標題: 木瓜輪點病毒鞘蛋白轉基因木瓜抗病性狀之研究
Studies on the resistance of transgenic papaya conferred by the coat protein gene of papaya ringspot virus
作者: 包慧俊
Bau, Huey-Jiunn
關鍵字: papaya ringspot virus
木瓜輪點病毒
coat protein gene
transgenic papaya
broad-spectrum resistance
strain-specific resistance
field trial
papaya leaf-distortion mosaic virus
gene silencing
鞘蛋白基因
轉基因木瓜
廣效性抗病
系統專一抗性
田間試驗
木瓜畸葉嵌紋病毒
基因沉寂作用
出版社: 植物病理學系
摘要: 木瓜輪點病毒(papaya ringspot virus;簡稱PRSV)幾乎是世界上所有木瓜栽培區生產的最大限制因子。數種方法曾用在防治病害上,包括種植時避開蚜蟲高峰期、以銀色塑膠布覆蓋畦面忌避蚜蟲、交互保護等。然皆無法達到長期抑病的效果。根據病原誘導抗病(pathogen-derived resistance)理論的基礎,以及植物基因轉殖的手法,本實驗室以台灣典型輪點病毒品系PRSV YK的鞘蛋白基因為轉殖標的,構築了鞘蛋白轉基因木瓜,共獲得了45個品系(line)。本研究經由溫室抗病檢定,其中有16個品系不具抗輪點病毒的能力,17個表現中度抗性,10個表現高度抗性,而2個則具有免疫的抗病現象。選擇2個(line 18-1-3,18-1-4)不具抗性,2個(17-0-5, 17-7-1)中度抗性,3個(16-0-1,17-0-5,18-2-4)高度抗性,和2個(18-0-9, 19-0-1)免疫型的品系進行轉基因在細胞內表現的分析,結果發現,免疫型的2個品系在細胞質內測不到鞘蛋白轉基因轉錄體(mRNA)的存在,3個高度抗性的品系亦很低,而2個中度抗病品系則累積較高。顯示抗性的表現與轉基因轉錄體mRNA的累積成相反關係。因此推測這些轉基因木瓜品系的抗病機制是由RNA媒介的抗性(RNA-mediated resistance)所誘導。除了2個不具抗性的品系外,其餘7個品系再進行接種其他來自3個不同地域的PRSV系統(strain),HA(夏威夷系統)、TH (泰國系統) 和MX(墨西哥系統)。結果顯示,品系19-0-1仍提供對3個不同地域的PRSV系統免疫的抗性,其他6個品系則表現不同程度的系統專一抗性(strain-specific resistance)。在轉基因遺傳特性研究上發現,18-0-9具有兩套顯性基因的遺傳特性,而16-0-1,17-0-1,17-0-5,18-2-4為單一顯性基因遺傳。 為了了解轉基因木瓜品系,表現系統專一抗性的行為是否與植物的年齡有關,將上述7個品系的幼苗分別在5公分齡期與10公分齡期偵測細胞內轉基因的表現,結果發現3個高度抗性,和2個免疫型品系在10公分苗期皆測不到轉基因mRNA的存在。在分別接種YK與上述3個來自台灣以外的PRSV系統後,顯示10公分苗期的轉基因木瓜對此4個系統的PRSV表現較強的抗性。另外,構築不同病毒系統間鞘蛋白基因與3¢端非轉譯區雜合體病毒來探討系統專一抗性的表現是否與轉基因核酸序列有關,結果顯示當雜合體病毒帶有與轉基因核酸序列相似度越高的序列時,轉基因木瓜對其抗性越強。綜合以上結果,鞘蛋白轉基因木瓜所提供的抗性強弱的關鍵決定在植株本身的發育年齡和轉基因核酸序列與入侵病毒序列間相似度。 經由溫室檢定轉基因木瓜的抗病行為後,挑選了4個高度抗YK系統的轉基因品系進行田間抗病評估。於農試所隔離試驗田,分兩試區進行。在觀察18個月後,受測的轉基因木瓜品系無一表現被病毒感染的嚴重型症狀。在試驗期間雖有20-30%的轉基因木瓜表現輕微斑駁病徵,然並不影響其植株的生長與產果。尤其果實皆無病斑,亦未扭曲變形。在仔細觀察後,冬季與梅雨季表現輕微病徵的株數較多,然而多數表現輕微病徵的轉基因植株在夏季高溫時,病徵會消失。病徵一旦消失後,此植株就不再會被感染。顯示轉基因木瓜的抗性似乎與環境的溫度有關。在產量的調查上,各轉基因木瓜品系的總產果量,在第一試區比對照組有2.5-2.8倍的增加,具商品價值的果則有10.8-11.6倍的增加。在第二試區,則有3.0-3.2倍的增加,而具商品價值的果更高達54.3-56.7倍的增加。顯然大大的提高了經濟效益,極具應用價值。 轉基因木瓜在幼齡期具系統專一抗性的缺點,使得將來在田間大規模應用時,仍可能造成一潛在的限制因子。因此未雨綢繆,事先調查台灣田間PRSV的病毒相,結果意外的在台中的大里分到一與PRSV無血清學關係的DL病毒分離株。由於此分離株在奎蔾上並不會造成單斑,因此以稀釋終點法純化此病毒並以電子顯微鏡觀察。結果為長絲狀顆粒,大小約為780-820 nm,並觀察到風車狀、螺旋狀、薄層狀內含體於感染細胞內,據此DL分離株推測可能為馬鈴薯Y群病毒屬。寄主範圍試驗顯示,DL病毒分離株只感染木瓜,對數種葫蘆科瓜類皆不感染。以馬鈴薯Y屬病毒專一性引子經由反轉錄聚合酵素連結反應(RT-PCR)放大出此病毒的鞘蛋白基因與3¢端非轉譯區,再經選殖與解序,比對後發現,DL病毒分離株與最早在琉球發現的木瓜畸葉嵌紋病毒(papaya leaf distortion mosaic virus;簡稱PLDMV),在鞘蛋白核酸序列上有95.1%的相同度,而胺基酸序列上則有94.9%的相同度。顯示此DL分離株應為PLDMV之一系統。然分離自琉球的PLDMV會感染數種葫蘆科瓜類,此乃DL與分離株最大的不同。將此大里分離株接種木瓜輪點病毒鞘蛋白轉基因木瓜,結果發現無一轉基因品系能抗此病毒。此病毒在台灣之所以一直未被重視,可能是長期被PRSV所掩蓋而忽略。 綜觀以上結果顯示,由本地典型的木瓜輪點病毒YK系統所構築的鞘蛋白轉基因木瓜,對PRSV而言,其所表現的抗性不僅對當地的輪點病毒系統有很強的抗病能力,同時對來自不同地域的輪點病毒系統亦有良好的保護效果,可說是具廣效性的抗病能力。具有極佳潛力可應用於世界其他地區防治木瓜輪點病害。然而,PLDMV在台灣已被發現,一旦輪點病毒鞘蛋白轉基因木瓜被推廣大量應用於田間時,極有可能會再一次造成台灣木瓜產業的危機。因此,逕速的做好準確的調查鑑定與廣泛的監測系統,以了解其目前在本島的分佈情形,實為當務之急。
Abstract Papaya ringsopt virus (PRSV) is a major limiting factor for papaya (Carica papaya L.) production in tropical and subtropical areas throughout the world. Control measures that has been used to protect papaya plants from PRSV infection, include the selection of planting time to avoid the peak of winged aphids, the use of silver mulch to prevent aphids from visiting seedlings, and conventional cross protection. None of these control methods provided a long period of effective protection against PRSV. Based on the concept of pathogen-derived resistance and the CP-transgenic approach, the CP gene of a local mosaic type strain isolated from Taiwan, designed PRSV YK, has been sequenced, and transferred into papaya via Agrobacterium-mediated transformation. All together, 45 putative transgenic lines were obtained. When the transgenic lines were challenged with PRSV YK by mechanical inoculation, they showed different levels of resistance ranging from delay of symptom development to complete immunity. Molecular analysis of nine selected lines that exhibited different levels of resistance revealed that the expression level of the transgene is negatively correlated with the degrees of resistance, suggesting that the resistance is manifested by a RNA-mediated mechanism. The segregation analysis showed that the transgene in the immune line 18-0-9 has an inheritance of two dominant loci and the other four highly resistant lines a single dominant locus. Seven selected lines were further tested for resistance to three PRSV heterologous strains that adapted from Hawaii, Thailand and Mexico. Six of the seven lines showed varying degrees of resistance to the heterologous strains, and one line 19-0-1 was immune not only to the homologous YK strain but also to the three heterologous strains. Our results also indicated that some CP-transgenic papaya lines displayed a strain-specificity of resistance and may be correlated with plant development. To further investigate this phenomenon, test plants of transgenic line 18-2-4 were tested at different developmental stages, the first stage of 5-cm height and the second stage of 10-cm height. Northern blot analysis revealed that the steady state levels of the CP transgene mRNA in the plants of two resistant and three highly resistant lines at the stage of 5-cm height were detectable, with higher levels in the two resistant lines. However, when plants further grew to the 10-cm stage, the CP transgene mRNA decreased to low levels in plants of the two resistant lines and declined to undetectable in plants of the three highly resistant lines, suggesting that the posttranscriptional gene silencing (PTGS) triggered in transgenic papaya cells is more conspicuous at the later developmental stage. When the transgenic seedlings were inoculated with four different geographic strains of PRSV YK, HA, TH and MX, the results showed that the phenomenon of strain-specific resistance is more conspicuous in plants at the 5-cm stage than at the 10-cm stage. Sequence comparison of the 3 terminal regions including the CP gene and the 3noncoding region, of the four strains of PRSV exhibited diversity within 10%. Hybrid viruses constructed by exchanging a segment covering the CP transgene were used to examine whether sequence homology is essential for strain-specific resistance. The results showed that the expression of strain-specific resistance in transgenic papaya seedlings is correlated with the sequence identity between the transgene and the challenging virus. On the basis of these observations it was concluded that the strain-specific resistance in transgenic papaya plants is affected by plant development and dependent on transgene homology. After the evaluation of resistance under greenhouse conditions, four highly resistant transgenic papaya lines 16-0-1, 17-0-1, 17-0-5 and 18-2-4 were further evaluated under field conditions for their reactions to infection by PRSV and for fruit production. Test plants were exposed to natural inoculation by aphids in two different locations and planting periods. The first trial started from September 1996 and the second from November 1996. In the first trial, the control plants were 100 % infected with PRSV five months after planting, while all plants of test lines 16-0-1, 17-0-1 and 17-0-5 displayed a high level of resistance and were free from PRSV infection 18 months after planting. In the second trial, which was under a severe challenge pressure, the controls were completely infected with PRSV three months after planting, while the transgenic lines 16-0-1, 17-0-5 and 18-2-4 showed a similar result as the first trial. In both trials, 20-30 % plants of each transgenic line were found infected with PRSV and they exhibited mild symptoms of confined mottling or chlorotic spots on leaves, however, no apparent adverse effects on fruit production and fruit quality were noticed. The numbers of plants with the mild symptoms fluctuated according to season and weather conditions, with a tendency to increase in winter or in rainy season and decrease in summer. In the first trial, total fruit yields of each line harvested for nine months showed a 2.5 to 2.8-fold increase as compared to controls and the commercially valuable fruits a 10.8 to 11.6-fold increase. Total fruit yields in the second trial of each line harvested for six months showed a 3.0 to 3.2-fold increase, and the commercially valuable fruits a 54.3 to 56.7-fold increase. These results indicated that the CP transgenic papaya lines have a great potential for control of PRSV in Taiwan. In order to avoid strain specific resistance as a limiting factor when the CP-transgenic papaya lines are widely used in fields. The variability of papaya ringspot virus (PRSV) was investigated in Taiwan. A virus isolate was collected from an open papaya orchard located at DaLee, Taichung county, in the central area of Taiwan. This virus, designated as DL isoalte, did not react with the antiserum of PRSV CP in ELISA. Papaya plants infected with the DL isolate displayed severe distortion on fully expanded leaves, shoestringing on newly emerged leaves, stunting in apex, and water-soaking on petioles and stems. Because no local-lesion hosts were available, the pure line of the isolate was obtained by limiting dilution. Electron microscopy analysis revealed that filamentous particles of 780-820 nm and cytoplasmic inclusions including pinwheels, scrolls, and laminated aggregates were present in infected cells. In the host range tests, the virus only infected Carica papaya L., but did not infect other 18 plant species inoculated. Using RT-PCR with the primers specific to potyviruses, a DNA fragment of 2.0 kb which contained the 3 terminal region of this isolate was amplified, cloned, and sequenced. A long open reading frame (ORF) encoding a polypeptide of 572 amino acids was found present in the amplified fragment of 1927 nucleotides. The determined 1927-nucleotide fragment reflected the C-terminal part of the NIb gene, the complete CP gene, and the 3noncoding regions of a potyvirus. The results of sequence analyses showed that the DL isolate shares 94.9% amino acid identity in the CP gene and 96.2% nucleotide identity in the 3noncoding region with those of papaya leaf distortion mosaic potyvirus. Since the DL isolate did not infect cucurbits, it was concluded that the DL isolate is a new pathotype of PLDMV. The DL isolate was further proved to be serological unrelated to PRSV by ELISA with reciprocal tests using antisera produced against each CP of the two viruses. When PRSV CP-transgenic papaya lines were challenged with the DL isolate, it was found that they provide no resistance. From the results of above investigations, we concluded that PRSV CP-transgenic papaya lines conferred a broad-spectrum resistance to PRSV, and have a great potential to be applied in Taiwan and other areas of the world to control the disease caused by PRSV. However, PLDMV may cause another crisis of papaya production when PRSV CP-transgenic papaya plants are widely used to control PRSV. Therefore, the establishment of a broad and precise surveillance system to realize the importance and the distribution of PLDMV in Taiwan is currently crucial.
URI: http://hdl.handle.net/11455/30777
Appears in Collections:植物病理學系

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