| Biological sciences & biotechnology |
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| Genetic variation analysis of an outer membrane protein gene of Candidatus Liberibacter asiaticus. |
| Lu Lianming1, Du Danchao1, Cheng Baoping2, Hu Xiurong1, Zhang Liping1, Chen Guoqing1* |
| (1. Citrus Research Institute of Zhejiang Province, Taizhou, Zhejiang 318026, China; 2. Research Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China) |
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Abstract Huanglongbing (HLB) is a destructive disease that represents a major threat to the world citrus industry. It was first reported from Chaoshan area of Guangdong Province in the early 20th century, and is now known to occur in next to 40 different Asian, African, Oceanian, South and North American countries and areas. Its pathogen is a phloemlimited, noncultured, Gramnegative bacterium which belongs to the genus Candidatus Liberibacter. Three species, i.e., Ca. L. asiaticus, Ca. L. americanus, and Ca. L. africanus with Ca. L. africanus subsp. capensis are currently known. So far, the pathogen of HLB disease discovered in China is only Ca. L. asiaticus, which distributes widely in almost all major citrus growing areas such as Guangdong, Guangxi, Fujian, Yunnan, Zhejiang and so on. This pathogen can be transmitted by grafting and Diaphorina citri and infect almost all fruit trees of Citrus, Fortunella and Poncirus, causing different characteristic symptoms including asymmetric blotchy mottle, vein yellowing or nutrition deficiency on leaves, and “red nose”, “greening” or deformed shape on fruits. While the infected Murraya exotica usually show no symptoms. Based on the above mentioned facts, it is referred that genetic diversity might exist in populations of Ca. L. asiaticus. The gene sequences of 16S rDNA, 16S-23S rDNA ITS and 23S rDNA of Ca. L. asiaticus have been confirmed to have a high degree of conservation and are not suitable for genetic diversity analysis. The omp gene, an outer membrane protein gene of Gramnegative bacterium, has been demonstrated as a potential candidate gene used for research on the genetic diversity. In this study, genetic variations of omp genes of 18 Ca. L. asiaticus isolates from China and other countries were analyzed aiming to understand the genetic diversity in populations of Ca. L. asiaticus and the effect of geographical source and host species. First, omp genes of Ca. L. asiaticus isolates from seven different geographical regions and 11 different host species in Taizhou of Zhejiang were amplified with special primers OMP5 (5′-GATGATAGGTGCATAAAAGTACAGAAG-3′) and OMP3 (5′-AATACCCTTATGGGATACAAAAA-3′). Then, the PCR products were subjected to digestion with restriction enzymes AluⅠ, ApoⅠ, HinfⅠ, RsaⅠ, SspⅠand TaqⅠrespectively, and were separated using agarose gel electrophoresis. To further characterize the omp gene of Ca. L. asiaticus, the PCR products were cloned and sequenced. The sequences of omp genes were submitted to GenBank (JQ928882-JQ928889), and were aligned with software DNAMAN and NCBI Blast. Finally, the phylogenetic tree of Ca. L. asiaticus was constructed by neighbor-joining method using MEGA 5.05. The results showed that the omp genes of Ca. L. asiaticus isolates from different geographical regions yielded different DNA fingerprints, but those from different host species in Taizhou area didn’t. Digestion with RsaⅠ yielded two distinct RFLP patterns, in which the pattern of isolate from Guangdong was different from those of others. Two distinct RFLP patterns were also obtained with HinfⅠdigestion, and the isolate from Jiangxi had a unique pattern. However, AluⅠ, ApoⅠ, SspⅠand TaqⅠdidn’t reveal genetic variation of these eight isolates. Sequence analysis revealed that the omp gene sequences of Ca. L. asiaticus isolates from different host species in Taizhou area were completely identical, while molecular variations existed within both nucleotide sequences and amino acid sequences of those from different geographical regions. Nucleotide sequences exhibited more than 99.9% identity among these Ca. L. asiaticus isolates, about 73% identity with those of Ca. L. africanus isolates, and nearly 69% identity with those of Ca. L. solanacearum isolates. Amino acid sequences of Ca. L. asiaticus isolates had over 99.8% identity with each other, about 59% identity with those of Ca. L. africanus isolates, and about 55% identity with those of Ca. L. solanacearum isolates. All Ca. L. asiaticus isolates exhibited extremely close genetic relationship and were clustered into one group in the phylogenetic tree, while Ca. L. africanus isolate and Ca. L. solanacearum isolate were clustered into other groups respectively. In conclusion, genetic diversity exists in populations of Ca. L. asiaticus, which are mainly affected by geographical origins, while not or little affected by host species. Thoroughly understanding the genetic diversity has an important significance to promote the research on strain differentiation and pathogenic variation, make clear the evolutionary trend of HLB pathogen and epidemic characteristics of HLB disease, and provide guidance to establish more scientific and effective control and prevention measures in future.
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Published: 20 March 2014
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柑橘黄龙病菌亚洲种外膜蛋白基因的遗传变异分析
为了解柑橘黄龙病菌亚洲种（Candidatus Liberibacter asiaticus）的遗传多样性，对来源于国内外7个不同地区和浙江台州地区11个不同寄主品种的Ca. L. asiaticus的外膜蛋白（outer membrane protein, OMP）基因进行聚合酶链反应限制性片段长度多态性分析(polymerase chain reaction-restriction fragment length polymorphism，PCR-RFLP).PCR产物经克隆测序后，利用NCBI Blast和DNAMAN软件对omp基因序列进行多重比对，并用MEGA软件构建系统发育树.结果显示：在浙江台州地区不同寄主品种上Ca. L. asiaticus各分离物的omp基因的酶切片段长度不具有多态性，其基因序列也完全一致；而不同地理来源的Ca. L. asiaticus的omp基因经限制性内切酶消化后可产生不同的RFLP指纹图谱，其核酸序列和氨基酸序列均存在变异，表明Ca. L. asiaticus存在种内遗传多样性.在系统发育树上，所有的Ca. L. asiaticus分离物聚为1个分支，而Ca. L. africanus和Ca. L. solanacearum分别处于不同的分支上.
关键词：
柑橘黄龙病菌亚洲种,
外膜蛋白,
限制性片段长度多态性分析,
遗传变异
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