基于16S rDNA序列比對分析贛南臍橙上柑橘黃龍病病原的分子特征

摘 要: 運用PCR技術以柑橘黃龍病病原亞洲種、非洲種和美洲種特異性引物對贛南臍橙上7個代表性柑橘黃龍病樣品進行擴增檢測，結合限制性內切酶XbaⅠ對贛南臍橙上的黃龍病病原16S rDNA基因RFLP分析和測定的16S rDNA基因序列同GenBank登錄的亞洲種、非洲種和美洲種的相應基因序列進行比對分析。結果表明，在贛南臍橙上檢測到黃龍病病原亞洲種，未檢測到非洲種和美洲種；贛南臍橙上黃龍病病原16S rDNA基因 XbaⅠ酶切具有亞洲種圖譜；16S rDNA基因序列比對分析表明7個贛南臍橙上的黃龍病病原間同源性為99.8%~100%，與亞洲種具有98.4%~99.9%的序列同源性，高于與非洲種/美洲種的序列同源性；與亞洲種間遺傳距離小于與非洲種/美洲種間的遺傳距離；系統聚類分析表明7個贛南臍橙上的黃龍病病原同亞洲種聚類在同一組群，具有較高的同源性和較近的親緣關系，而與非洲種和美洲種在系統發育樹中分屬不同的組群，試驗結果表明贛南臍橙上感染的黃龍病病原為亞洲種。

關鍵詞: 贛南臍橙； 柑橘黃龍病； 16S rDNA； RFLP分析； 序列比對

中圖分類號：Ｓ６６6．4 文獻標識碼：Ａ 文章編號：１００９－９９８０?穴２０11?雪０6－1099－０5

Molecular characterization of Citrus Huanglongbing Bacteria in Gannan navel orange based on 16S rDNA sequences alignment

YI Long1，2， LU Zhan-jun1，2， ZHONG Ba-lian2，LAI Xiao-hua2， CHEN Ci-xiang2

（1College of Life and Environmental Sciences， Gannan Normal University， Ganzhou，Jiangxi 341000 China； 2Jiangxi Navel Orange Engineering and Technology Research Center， Ganzhou，Jiangxi 341000 China）

Abstract: Seven Citrus Huanglongbing (HLB) bacteria of Gannan navel orange in Jiangxi Province were detected by PCR with primers specific to Asian， African and American liberibacters. The XbaⅠrestriction endonucleases were used to digest the HLB 16S rDNA fragment of Gannan navel orange， and the sequences of 16S rDNA were sequenced and compared with those corresponding sequences of Asian， African and American liberibacters from GenBank by BioEidt and MEGA software. The results showed that the HLB pathogen of Gannan navel orange could be detected by the specific primers of Candiatus L. asiaticus. RFLP analysis demonstrated that the digested XbaⅠprofiles of 16S rDNA of the HLB isolates from Gannan navel orange had similar profiles with Ca. L. asiaticus. The 16S rDNA sequences of the 7 HLB isolates have identity ranging from 99.8% to 100%， and 98.4% to 99.9% with Asian liberibacters were higher identity than those with African liberibacters or American liberibacters. The same results were found by analyzing the sequences average nucleotide distance of HLB isolates of Gannan navel orange， Asian， African and American liberibacters. Phylogenetic analysis revealed that the 7 HLB isolates of Gannan navel orange were located in different phylogenetic clusters with African and American liberibacters， and classified into one cluster with Asian liberibacters， and shared higher homology and closer relationships. The results indicated that the HLB isolates of Gannan navel orange were Ca L. asiaticus.

Key words: Gannan navel orange； Citrus Huanglongbing； 16S rDNA； RFLP analysis； Sequence alignment

柑橘黃龍病（Citrus Huanglongbing， HLB）是柑橘產區毀滅性的病害，廣泛分布于亞洲、非洲、美洲等柑橘主產區，由一種迄今尚不能人工培養的韌皮部限制性細菌（Candiatus Liberibacter）引起[1-2]。根據病原物的熱敏感性、發生區域和蟲媒類型，柑橘黃龍病病原存在亞洲種（Ca. L. asiaticus）、非洲種（Ca. L. africanus）[3]和美洲種（Ca. L. amercanus）[4]。受全球氣候持續變暖的影響，黃龍病傳播媒介柑橘木虱活動范圍的擴大和患病苗木和果品不經檢疫、無證調運導致該病目前存在不斷擴增蔓延的趨勢。……