六种豆象的线粒体Co1基因的检测及鉴定

发布时间：2019-01-10 15:01

【摘要】：四纹豆象与花生豆象、菜豆象、鹰嘴豆象、绿豆象、咖啡豆象形态特征相似,菜豆象、四纹豆象、鹰嘴豆象、花生豆象、咖啡豆象已被国家列为禁止进境的检疫性有害生物,绿豆象是中俄、中南植检植保双边协定中规定的检疫性害虫,六种豆象的经济意义均十分重要,口岸检验检疫部门主要以成虫的外部形态特征作为主要鉴定依据。另外,口岸截获的不单是成虫,经常还有些是卵、幼虫、蛹等未成熟虫态,寻找一种正确、快速的鉴定方法在口岸检验检疫中的应用具有十分重要的指导意义。本研究对象为四纹豆象,首先提取四纹豆象的线粒体基因,通过设计引物,对四纹豆象等六种豆象的线粒体特异性基因Co1进行提取,测序,通过构建系统发育树,完成检疫性豆象类的分子鉴定,然后进一步对四纹豆象进行线粒体基因组全序列的测定与分析。将有利于建立更稳健的系统发育关系。实验结果如下:(1)试验方法的优化:由于常规检验豆象的方法不能够准确快速地鉴定出豆象的种类的限制,对临沂出入境检验检疫局截获的豆象以及泰安粮食市场沉积储藏物(豆类)中的豆象,用PCR级柱式动物线粒体DNAout试剂盒处理,对其线粒体DNA中特异性基因Co1进行提取鉴定测序。(2)Co1基因全长420bp,Blast对比分析六种豆象Co1基因的差异。(3)以六种实验材料豆象特异性基因Co1序列为基础,结合GenBank上查找的不同豆象的特异性Co1序列构建系统发育树,加以分析,了解不同豆象的亲缘差异,尝试构建参照体系,方便对截获的豆象及幼虫卵等通过分子测序确定其所属种。(4)验证:通过截获检疫性豆象卵若成虫等个体进行分子水平的鉴定及测序代入系统发育树,经比对确认所属种,完成测序。
[Abstract]:The four-striped bean elephant is similar to the peanut bean elephant, the chickpea elephant, the mung bean elephant, the coffee bean elephant. The bean elephant, the four-striped bean elephant, the chickpea, the peanut bean elephant and the coffee bean elephant have been listed as quarantine pests banned by the state. The economic significance of the six species of legume is very important. The port inspection and quarantine departments mainly take the external morphological characteristics of adults as the main basis for identification. In addition, not only adults but also eggs, larvae, pupae and other immature insects are intercepted at ports. It is of great significance to find a correct and rapid identification method in port inspection and quarantine. Firstly, the mitochondrial gene of tetracarpus was extracted. The mitochondrial specific gene (Co1) was extracted from six species of legume by designing primers, and sequenced, and the phylogenetic tree was constructed. The molecular identification of quarantine legumes was completed, and then the complete mitochondrial genome sequence was determined and analyzed. It will help to establish a more stable phylogenetic relationship. The experimental results are as follows: (1) the optimization of the test method: because of the limitation of the conventional method to identify the species of the elephant accurately and quickly, The beans intercepted by the Entry-Exit Inspection and Quarantine Bureau of Linyi and the legumes in the depositional storage products (legumes) of the Tai'an grain market were treated with PCR grade column animal mitochondrial DNAout kit. The specific gene Co1 in mitochondrial DNA was extracted and sequenced. (2) the total length of Co1 gene was 420 BP blast. (3) based on the Co1 sequence of six experimental materials, the difference of Co1 gene of six soybean elephants was compared. The phylogenetic tree was constructed by combining the specific Co1 sequences of different bean elephants found on GenBank. The phylogenetic differences of different legumes were analyzed and the reference system was constructed. It is convenient to identify the species of the intercepted bean elephants and larval eggs by molecular sequencing. (4) Verification: through the molecular level identification and sequencing of individuals such as the quarantine elephant eggs if the adult worm, and so on, the phylogenetic tree can be identified by comparison and identification of the species to which they belong. Complete the sequencing.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433.5

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