荔枝露疫病菌檢測用分子標記之建立

Establishment of molecular markers for detection and diagnosis of Peronophthora litchii

陳隆鐘(L. C. Chen) ； 李崇正(C. C. Lee) ； 鍾依紋(Y. W. Chung) ； 安寶貞(P. J. Ann) ； 葉瑩(Y. Yeh)

《植物病理學會刊》 7卷4期 (1998/12) Pp. 189-200

https://doi.org/10.6649/PPB.199812_7(4).0004

摘要

利用隨機增幅核酸多型性分析，篩選荔枝露疫病菌(Peronophythora litchii)專一性核酸標識，由供試引子中選拔出引子OPW-02 (5'-TCGCAGGTTC-3')，可將供試之荔枝露疫菌菌株基因體核酸增幅出595 bp之專一性核酸片段。利用載體pCR®II-TOPO進行此595 bp專一性核酸片段之選殖，嵌入片段經非放射性標定後製備為核酸探針pR4，經南方氏雜合反應結果顯示pR4與荔枝露疫病菌29株菌株皆可產生專一性核酸雜合訊號，而與對照組荔枝酸腐病菌(Geotrichum ludwigii)、7種疫病菌(Phytophthora spp.)、4種腐霉病菌(Pythium spp.)菌株，則皆無訊號產生。將此專一性核酸片段進行核甘酸定序，並由此序列衍生設計出正向引子R4F7、R4MF245、R4MF258及返向引子R4R562、 R4R592，其中以引子對R4MF245(5'-GCTCCAAGATGTTGGTTGGGATCGG-3')/R4R562(5'- GATCTACAGCACAGCCCAAAGAAGG-3')利用聚合酵素連鎖反應(PCR)偵測荔枝露疫病菌，可由供試之荔枝露疫病菌菌株之基因體核酸增幅得一317 bp專一性的核酸片段，而對照組疫病菌菌株，則皆無訊號產生。利用此專一性引子對R4MF245/R4R562 PCR於荔枝露疫病菌基因體核酸之偵測時，其靈敏度可達500 pg；若輔以pR4核酸探針進行南方氏雜合反應，其靈敏度可達50 pg。

關鍵字

荔枝露疫病菌 ； 隨機增幅核酸多型性分析 ； 聚合酵素連鎖反應

並列摘要

Twelve primers were used for random amplified polymorphic DNA (RAPD) analysis to find specific DNA markers of P e ronophthora litchii. There were nine primers which could amplify the specific bands patterns by RAPD. The primer OPW-02 (5'- TCGCAGGTTC-3') amplified a distinct fragment, subsequnetly cloned in pCRÒI I - TOPO vector, that was specific to P. litchii. The cloned DNA fragment labeled with nonisotope biotin, named pR4, was used as a probe in Southern blotting analysis. There was specific signal to P. l i t c h i i and the same signal was not shown in 7 species of P h y t o p h t h o r a, 4 species of P y t h i u m, and Geotrichum candidum in Southern blotting analysis. Five primers were designed and synthesized according to the sequence of 595-bp DNA fragment: R4F7, R4MF245, R4MF258 are forward primers, and R4R562, R4R592 are reverse primers. The primer pairs R4MF245 (5'- GCTCCAAGAT G T T G G T T G G G AT C G G - 3 ' ) and R4R562 (5'-GAT C TACAGCACA GCCCAAAGAAGG-3') could amplify a specific 317-bp fragment from genomic DNA of P. litchii by polymerase chain reaction (PCR). As little as 500 pg of genomic DNA of P. litchii could be detected by the developed PCR conditions. The sensitivity can be even increased up to 50 pg of DNA from P. litchii by incorporation of Southern blotting analysis with probe pR4. We hope to use these techniques for detecting P. litchii on the fruit and in the soil directly from the field to understand the ecological role, disease development and the genetic diversity from oomyces in advance.