杏褪绿卷叶植原体新疆分离物16S rDNA基因克隆与序列分析  

Cloning and sequencing of 16S rDNA gene of the phytoplasma of apricot chlorotic leaf roll in Xinjiang

作　　者：艾克热木·买买提 韩剑[1,2] 罗明 唐章虎[3] AIKEREMU·Maimaiti;HAN Jian;LUO Ming;TANG Zhanghu(Agronomy Faculty, Xinjiang Agricultural University, Urumqi 830052, China;Key Laboratory of Harmful Biological Monitoring and Safety Prevention of Xinjiang Autonomous Region Forestry Universities, Urumqi 830052, China;Luntai National Pomology Germplasm Garden, Academy of Agricultural Science of Xinjiang, Luntai 841600, China)

机构地区：[1]新疆农业大学农学院,乌鲁木齐830052 [2]新疆维吾尔自治区高校农林有害生物监测与安全防控重点实验室,乌鲁木齐830052 [3]新疆农科院轮台国家果树资源圃,轮台841600

出　　处：《植物保护》2020年第2期186-192,共7页Plant Protection

基　　金：国家自然科学基金(31600011);新疆维吾尔自治区研究生教育创新计划科研创新项目(XJGRI2017-072)。

摘　　要：利用植原体16S rDNA基因通用引物对新疆轮台县疑似杏褪绿卷叶病植株总DNA进行巢氏PCR检测,扩增出大小约1.2 kb的特异性条带。对扩增产物克隆和测序,确定特异片段大小为1248 bp。序列同源性比较和系统进化分析表明,新疆杏褪绿卷叶植原体不同分离株16S rDNA基因序列同源性极高,达到99.8%~100%。与16SrⅤ组成员的同源性达到98.2%以上,其中与16SrⅤ-B亚组的枣疯病植原体山东宝山分离株,甜樱桃绿化植原体山东分离株同源性最高,达到99.4%~99.6%。进一步虚拟RFLP分析,结果表明该植原体属于榆树黄化组(16SrⅤ)的一个新的亚组,与其相似性最高的是16SrⅤ-B亚组,相似系数为0.94。本研究首次报道了新疆杏褪绿卷叶植原体16S rDNA的序列,确定了其分类地位,为杏褪绿卷叶病的早期诊断和检测提供了基础。By using Nested-PCR,the 16S rDNA of phytoplasma of apricot chlorotic leaf roll(ACLR)in diseased sample from Luntai region in Xinjiang was amplified by the universal primer pairs,then a phytoplasma-specific 1.2 kb fragment were amplified by nested-PCR.The 16S rDNA gene was cloned by polymerase chain reaction from samples of ACLR,they were then sequenced.The result of sequencing showed that the ACLR strains from Xinjiang consists of 1248 bp in 16S rDNA gene.The similarity and phylogenetic analysis showed that the six ACLR isolates sequenced from Luntai region are closely related to each other,displaying 99.8%-100%nucleotide sequence similarity,and the six isolates shared approximately 98.2%identity with 16SrⅤgroup,the results of blast showed that the genetic distance of six isolates was closely to the 16SrⅤ-B subgroup,which shared the highest similarity(99.4%-99.6%)with the strains of prunus avium virescence phytoplasma(MF848965)from Shandong and jujube witches’-broom phytoplasma(EU999737)from Shandong.Furthermore,virtual RFLP analysis showed that the phytoplasma belonged to a new subgroup within the elm yellows group(16SrⅤ),the most similar was the reference pattern of the 16Sr groupⅤ,subgroup B(GenBank accession:AB05287),with a similarity coefficient of 0.94.This study reports 16S rDNA sequences of ACLR phytoplasmas and classified phytoplasma of ACLR in Xinjiang for the first time,it provides the base for early diagnosis and detection.

关 键 词：杏 杏褪绿卷叶病 植原体 16S RDNA 序列分析 

分 类 号：S436.6[农业科学—农业昆虫与害虫防治]