光肩星天牛及其近缘种线粒体DNA COⅠ基因遗传差异及其PCR鉴定试剂盒的研究
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摘要
光肩星天牛Anoplophora glabripennis(Motschulsky)，是我国分布广泛而且危害非常严重的蛀干害虫。1996年以来，美国相继在纽约、芝加哥和新泽西州等地发现光肩星天牛危害，由于美方认为该虫是从中国传入的，因此于1998年开始对我国输美的货物木质包装(SWPM)实行了更为严厉的检疫措施，这对我国的出口贸易产生了严重影响。鉴于光肩星天牛对中关两国检疫的重要性，经双方商谈决定合作开展“星天牛种间及光肩星天牛种群间分子生物学的研究”课题。本研究为该课题的一部分。
应用PCR直接测序法对15个不同地理来源的光肩星天牛及5个光肩星天牛近缘种共27个样品的线粒体DNA CO Ⅰ基因中一段504 bp的序列成功进行了测序。
从序列分析结果可见，不同地理来源的光肩星天牛样品间存在一定的碱基差异，并呈现出一定的规律。结果表明，美国、韩国与中国的光肩星天牛存在明显标记性差异，说明美国的光肩星天牛已形成自己独有的基因特点，而区别于中国、韩国的光肩星天牛。由此可以推断，美国所发生的光肩星天牛不是从中国传入的。
综合序列碱基差异分析和Bootstrap聚类结果，可将本研究中的光肩星天牛(含黄斑星天牛)样品分为4个种群：美国、韩国、中国北方和中国南方。中国北方种群包括：甘肃、内蒙古、河北、陕西；中国南方种群包括：安徽、江苏、浙江、湖北、河南、山东、宁夏、山西和辽宁。
在所测序列中，光肩星天牛与黄斑星天牛样品间的碱基差异较小，且它们在系统树中全部聚于一分枝中，由此可见，光肩星天牛和黄斑星天牛并未达到种级分类水平，应归为同一个种。
本研究结果显示，光肩星天牛样品与采自重庆的四川星天牛间碱基差异较大，且它们在系统树中位于不同的分枝，这一结果表明，四川星天牛应是一个独立的种。
测序结果表明，光肩星天牛mtDNA CO Ⅰ基因这一片断，在种间差异明显，而在不同地理来源的光肩星天牛(包括黄斑星天牛)间相对保守，具有高度的特异性和稳定性，可用来鉴定光肩星天牛。
根据所测mtDNA CO Ⅰ序列设计了一对光肩星天牛的特异性PCR引物用于光肩星天牛的PCR鉴定。对包括21个不同地理来源的光肩星天牛在内的39个样品进行实验，结果表明所设计的PCR引物具有较高的特异性和灵敏度。根据以上研究结果初步构建了光肩星天牛PCR鉴定试剂盒及其说明书，并获得了专利登记。
Anoplophora glabripennis (Motschulsky), which widely distribute in China, has been a great economic pest of forest trunk. Since 1996, A.glabripennis infestation has been discovered in New York City, Chicago City and New Jersey in U.S.A. in succession. U.S.A. government thought that this pest imported from China. So in 1998, U.S.A.. government implemented a series of more stern quarantine and inspection regulations to SWPM from China, that had influenced seriously on the export trade of our country and brought the enormous economic losses. As the importance of A.glabripennis to U.S.A. and China, the subject "Genetic differences and identification technology of A.glabripennis "was decided to study by co-operative of both sides after negotiations. This study is part of this subject.
A 504bp fragment of mtDNA CO I gene were sequenced successfully by sequencing directly from a total of 27 samples including 15 A.glabripennis samples from different areas , 4 A.nobilis samples from different areas and the other 4 sibling species of A.glabripennis.
From the result of analysis on the sequences, it can be seen that there are base difference in A.glabripennis samples from different areas and show definitive regulation. This show that there is visible marker difference in A.glabripennis from America, China and Korea. This indicates that A.glabripennis from America have formed unique character themselves and can differentiate from A.glabripennis from China and Korea. So we infer the A.glabripennis distributed in U.S.A. were not from China.
On the basis of the base difference and the Bootstrap tree, The A.glabripennis specimens including A.nobilis of this study can be divided into 4 populations: the population of America, the population of Korea, the population of the North of China and that of the South of China. The population of the North of China consist of A.glabripennis from the provinces of Gansu, Neimenggu, Hebei and Shananxi. The population of the South of China consist of A.glabripennis from Anhui, Jiangsu, Zhejiang, Hubei, Henan, Shandong, Ningxia, Shanxi and Liaoning.
The base difference between A.glabripennis and A.nobilis samples is very small. And they all grouped in a clade. Thus show these two sibling species should belong to the same species.
The morphological characters of are very similar. As a result, the biggest base difference between A.glabripennis samples and A.freyi from Chongqing is 6.8% and they were grouped into tow clades in the Bootstrap tree. Therefore, A.freyi can be seen a independent species in the light of the result.
The result of sequencing indicates the region of mtDNA CO I gene show distinct difference in different species but conservation in A. glabripennis samples relatively. This region
has special stability and specificity. It can be used to identify A.glabripennis by sequencing directly.
A pair of specific PCR primers was designed according to the sequences of mtDNA CO I gene had sequenced. These primers can be used to identify A.glabripennis. The PCR system and condition of specific PCR of identifying A.glabripennis were optimized and defined the optimum PCR system and condition. A total of 39 specimens were tested including 21 A.glabripennis specimens of different geographical sources. The result show that the primers designed have high specificity and sensitivity. The identification kit for A.glabripennis and its working protocol were developed primarily based on the PCR and have obtained the register patent.
应用PCR直接测序法对15个不同地理来源的光肩星天牛及5个光肩星天牛近缘种共27个样品的线粒体DNA CO Ⅰ基因中一段504 bp的序列成功进行了测序。
从序列分析结果可见，不同地理来源的光肩星天牛样品间存在一定的碱基差异，并呈现出一定的规律。结果表明，美国、韩国与中国的光肩星天牛存在明显标记性差异，说明美国的光肩星天牛已形成自己独有的基因特点，而区别于中国、韩国的光肩星天牛。由此可以推断，美国所发生的光肩星天牛不是从中国传入的。
综合序列碱基差异分析和Bootstrap聚类结果，可将本研究中的光肩星天牛(含黄斑星天牛)样品分为4个种群：美国、韩国、中国北方和中国南方。中国北方种群包括：甘肃、内蒙古、河北、陕西；中国南方种群包括：安徽、江苏、浙江、湖北、河南、山东、宁夏、山西和辽宁。
在所测序列中，光肩星天牛与黄斑星天牛样品间的碱基差异较小，且它们在系统树中全部聚于一分枝中，由此可见，光肩星天牛和黄斑星天牛并未达到种级分类水平，应归为同一个种。
本研究结果显示，光肩星天牛样品与采自重庆的四川星天牛间碱基差异较大，且它们在系统树中位于不同的分枝，这一结果表明，四川星天牛应是一个独立的种。
测序结果表明，光肩星天牛mtDNA CO Ⅰ基因这一片断，在种间差异明显，而在不同地理来源的光肩星天牛(包括黄斑星天牛)间相对保守，具有高度的特异性和稳定性，可用来鉴定光肩星天牛。
根据所测mtDNA CO Ⅰ序列设计了一对光肩星天牛的特异性PCR引物用于光肩星天牛的PCR鉴定。对包括21个不同地理来源的光肩星天牛在内的39个样品进行实验，结果表明所设计的PCR引物具有较高的特异性和灵敏度。根据以上研究结果初步构建了光肩星天牛PCR鉴定试剂盒及其说明书，并获得了专利登记。
Anoplophora glabripennis (Motschulsky), which widely distribute in China, has been a great economic pest of forest trunk. Since 1996, A.glabripennis infestation has been discovered in New York City, Chicago City and New Jersey in U.S.A. in succession. U.S.A. government thought that this pest imported from China. So in 1998, U.S.A.. government implemented a series of more stern quarantine and inspection regulations to SWPM from China, that had influenced seriously on the export trade of our country and brought the enormous economic losses. As the importance of A.glabripennis to U.S.A. and China, the subject "Genetic differences and identification technology of A.glabripennis "was decided to study by co-operative of both sides after negotiations. This study is part of this subject.
A 504bp fragment of mtDNA CO I gene were sequenced successfully by sequencing directly from a total of 27 samples including 15 A.glabripennis samples from different areas , 4 A.nobilis samples from different areas and the other 4 sibling species of A.glabripennis.
From the result of analysis on the sequences, it can be seen that there are base difference in A.glabripennis samples from different areas and show definitive regulation. This show that there is visible marker difference in A.glabripennis from America, China and Korea. This indicates that A.glabripennis from America have formed unique character themselves and can differentiate from A.glabripennis from China and Korea. So we infer the A.glabripennis distributed in U.S.A. were not from China.
On the basis of the base difference and the Bootstrap tree, The A.glabripennis specimens including A.nobilis of this study can be divided into 4 populations: the population of America, the population of Korea, the population of the North of China and that of the South of China. The population of the North of China consist of A.glabripennis from the provinces of Gansu, Neimenggu, Hebei and Shananxi. The population of the South of China consist of A.glabripennis from Anhui, Jiangsu, Zhejiang, Hubei, Henan, Shandong, Ningxia, Shanxi and Liaoning.
The base difference between A.glabripennis and A.nobilis samples is very small. And they all grouped in a clade. Thus show these two sibling species should belong to the same species.
The morphological characters of are very similar. As a result, the biggest base difference between A.glabripennis samples and A.freyi from Chongqing is 6.8% and they were grouped into tow clades in the Bootstrap tree. Therefore, A.freyi can be seen a independent species in the light of the result.
The result of sequencing indicates the region of mtDNA CO I gene show distinct difference in different species but conservation in A. glabripennis samples relatively. This region
has special stability and specificity. It can be used to identify A.glabripennis by sequencing directly.
A pair of specific PCR primers was designed according to the sequences of mtDNA CO I gene had sequenced. These primers can be used to identify A.glabripennis. The PCR system and condition of specific PCR of identifying A.glabripennis were optimized and defined the optimum PCR system and condition. A total of 39 specimens were tested including 21 A.glabripennis specimens of different geographical sources. The result show that the primers designed have high specificity and sensitivity. The identification kit for A.glabripennis and its working protocol were developed primarily based on the PCR and have obtained the register patent.
引文
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