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研究生:李崇正
研究生(外文):Lee, Chung-cheng
論文名稱:利用隨機增幅核酸多型性分析及聚合酵素連鎖反應技術專一性檢測露疫病菌
論文名稱(外文):Specific detection of Peronophythora litchii using Random Amplified Polymorphic DNA analysis and Polymerase Chain Reaction
指導教授:陳隆鐘
指導教授(外文):L. C. Chen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:101
中文關鍵詞:荔枝露疫菌隨機增幅核酸多型性分析聚合酵素連鎖反應檢測
外文關鍵詞:litchiPeronophythora litchiiRAPDPCRdetection
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荔枝露疫病, 又名霜霉病 ( litchi downy blight ), 由荔枝
露疫菌 (Peronophythora litchii ) 所引起,為荔枝之嚴重病害, 主
要危害近成熟的果實,此病害經由風雨傳播,其發生與氣溫及雨水有密切
關係,持續雨季將致使此病害嚴重發生,因此若能在病害發生前作有效且
快速偵測此病原菌,除有助於防治工作之實施,並能降低本病害在儲運期
造成大量感染之損失及增加荔枝外銷競爭力。荔枝露疫菌以掃瞄式電子顯
微鏡 ( SEM ) 觀察有類似露菌屬 ( Peronospora ) 之胞囊柄及有類似疫
病菌屬 ( Phytophthora ) 之明顯乳突 ( papillae ) 的檸檬狀胞囊 (
lemon-shaped sporangium )。菌絲生長最適溫為 22 ~ 28 ℃,低於 4
℃及高於 36 ℃菌絲則無法生長;光週期處理時生長較快,黑暗次之,光
照最慢; CV8蔬菜汁培養基的濃度則以 5 % 最佳;pH 4.5 ~ 8 皆有利於
菌絲的生長。刺傷或未刺傷二種不同處理的成熟果實,在病徵上無差異存
在,果表皆覆蓋一層白色的胞囊柄及胞囊,可推測荔枝露疫菌在適合發病
的田間環境下,果實勿須有機械性傷害,即可進行病害的傳播。利用逢機
引子在隨機增幅核酸多型性析反應 ( RAPD ) 中產生的核酸增幅條帶,
以作為荔枝露疫菌供檢測用之專一性核酸標幟 ( DNA marker),在篩選的
引子中有 9 個引子能對所有供試之荔枝露疫菌產生專一性核酸增幅條帶
。 而引子 OPW-02 ( 5'TCGCAGGTTC3' ) 可由測試所有供試之荔枝露疫菌
菌株增幅得一 595 bp 之專一性核酸片段。本研究進一步將此 595 bp 之
專一性核酸片段與載體 pCRRII-TOPO vector 進行核酸片段之選殖 (
cloning ),以 EcoR I 內限制酵素切出嵌入片段,經非放射性標定後製
備為核酸探針 ( DNA probe ),並命名為 pR4, 再進行南方氏漬染分析
( Southern blotting analysis ),與各供試菌株作專一性測定。 探針
pR4 與所有的 P. litchii 皆可產生專一性核酸雜合訊號( signal ),與
相對照的荔枝酸腐病菌、疫病菌、腐霉病菌菌株,則皆無訊號產生。 將
此 595 bp 之專一性核酸片段進行核甘酸定序 ( sequencing ),並由此
序列衍生設計出正向引子 ( forward primer ):R4F7、R4MF245、R4
MF258 及返向引子( reverse primer ): R4R562、 R4R592, 其中以引
子對 R4MF245 ( 5'GCTCCAAGATGTTGGTTGGGATCGG3' ) / R4R562 (
5'GATCTACAGCACAGCCCAAA GAAGG3' ),利用聚合酵素連鎖反應 ( PCR )
技術來檢測荔枝露疫菌, 可由所有供試之荔枝露疫菌菌株增幅得一 317
bp 專一性的核酸片段,而與相對照的疫病菌菌株,則皆無訊號產生。 此
專一性引子對 R4MF245 / R4R562 利用 PCR 技術來檢測露疫菌基因體核
酸 ( genomic DNA ) 時,其靈敏度可達 500 pg; 輔以 pR4 核酸探針,
進行南方氏漬染分析,其偵測的結果,更可達到 50 pg。綜合上述研究,
可利用隨機增幅核酸多型性分析之結果,以電腦軟體進行荔枝露疫菌菌株
之遺傳親緣性關係的分析探討,引子 OPW-02 可應用於荔枝露疫菌以
RAPD 技術之分生輔助鑑定,專一性引子對 R4MF245 / R4R562 可利用
PCR 及南方氏漬染分析技術來檢測荔枝露疫菌,未來更可進一步實際應用
於田間,偵測栽培田土及果實上的荔枝露疫菌,以進一步瞭解荔枝露疫菌
在田間生態、傳播及在卵菌門之遺傳演化中所扮演的角色。

Peronophythora litchii Chen ex Ko et al is a fungal plant
pathogenthat cause downy blight of litchi ( Litchi chinensis
Sonn. ). Thispathogen caused serious disease of premature
fruit during the rainyseason by the rain and wind. It might can
be controlled the developmentof!this disease and decreased the
yield loss during the storage by rapiddetection. The
sporangiophore of Peronophythora litchii is similar tothe same
structure of Peronospora Cordax, and the lemon-shaped
sporagiumwith papillae of this fungus is similar to the
structure of Phytophthorade Bary which were observed by scanning
electron microscopy. The optimaltemperatures for the mycelial
growth of the fungus are between 22 ~ 28℃, and the limits were
below 4 ℃ and above 36 ℃. The suitable termsof the mycelial
growth are photoperoid, pH 4.5 ~ 8, and five percentageof CV8
juice medium. The symptoms of inoculated fruits with wound
andnon-wound which were both covered with whit
sporangia andsporangiophores were no differences. It was
supposed that the diseasecan developed in the field without
mechanical injury. Selected primerswere used to find specific
DNA markers of P. Litchii by using randomamplified
polymorphic DNA ( RAPD ). There were nine primers
couldamplify the specific patterning bands by RAPD. The
primer OPW-02 (5'TCGCAGGTTC3' ) amplified a distinct
fragment of 595 bp that wasspecific to P. litchii, and
cloned by pCRRII-TOPO vector. The insertedDNA fragment was
digested by EcoR I. The digested DNA fragment waslabeled
with non- isotope and named probe pR4, to be identified
inSouthern blotting analysis. There was specific signal to P.
litchii andthe same signal was not shown in Phytophthora spp.,
Pythium spp., andGeotrichum candidum. Five primers were
designed and synthesized by thesequence of the inserted
fragment: R4F7, R4MF245, R4MF258 are forwardprimers, and R4
R562, R4R592 are reverse primers. The primer pairsR4MF245
( 5'GCTCCAAGATGTTGGTTGGGATCGG3' ) and R4R562 (
5'GATCTACAGCACAGCCCAAAGAAGG3' ) could amplify a distinct
patterning band of 317bp that was specific to P. litchii by
polymerase chain reaction ( PCR ).The minimum amount of DNA from
P. litchii that could be detected by PCRwas 500 pg. And the
sensitivity can be raised to 50 pg of DNA from P.litchii by
using Southern blotting analysis with probe pR4. In summary,the
RAPD could be a very potential technique in using the specific
RAPDbands as genetic markers for identification of P.
litchii. Specificprimer pairs-R4MF245 and R4R562 could
detect the P. litchii by thetechnique of PCR and Southern
blotting analysis. We hope thesetechniques can detect the
disease of fruit and the soil in the fielddirectly in the
future. In advance, to understand the role of P. litchiiin
ecology, disease development, and genesis from oomyces.


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