【作者】 张红梅；

【导师】 仵均祥；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2004， 硕士

【摘要】 实蝇作为一种有重要经济意义的有害生物类群，不仅对水果生产构成直接的危害，而且对水果进出口贸易也造成严重的影响。目前，世界上危害果蔬类的实蝇达 150 余种，且分布在不同的国家和地区，各国政府均采取了严密的检疫措施，以防止检疫性实蝇的传播扩散。目前，实蝇的分类鉴定主要是以成虫的外部形态特征作为依据，但在口岸实际检疫工作中，截获的往往是幼虫或蛹，通常的做法是对其进行室内饲养待获得成虫后再进行鉴定，这样不仅延长了进出口物品的通关时间，而且增加了检疫人员的工作强度。因此，对实蝇进行快速、准确的鉴定，无论是在口岸检疫上还是在防治上都具有重要意义。本文利用 RAPD 技术和 PCR 方法在国内首次对实蝇的分子鉴定进行了研究，旨在找到一种能准确、快速鉴定实蝇的方法，主要结果如下： 1、利用目前国内提取干标本 DNA 的多种方法，如 CTAB 法、SDS 法等试验结果表明，常规保存条件下，保存一年左右的干标本很难进行 DNA 的提取，不管采用何种方法，大多数标本均不能提取到 DNA；仅少数保存比较好的干标本，可以提取出 DNA并进行 PCR 扩增。而利用 CTAB 法、SDS 法等方法对绝大多数保存在超低温冰箱中的干标本都能提取到足量的 DNA。 2、在多次筛选试验的基础上，优化了实蝇的 RAPD 体系，建立了一套稳定的实蝇鉴定 RAPD 反应体系，为今后采用分子生物学技术进行实蝇鉴定奠定了一定的基础。 3、从 69 条引物中筛选出 S361、S67 和 S252 可以对南瓜实蝇、具条实蝇和三点棍腹实蝇进行区别鉴定；S112 引物可以对具条实蝇、桔大实蝇和三点棍腹实蝇进行区别鉴定；S126 和 S134 可以对具条实蝇和三点棍腹实蝇进行区别鉴定，S83 可以对南瓜实蝇的雌性和雄性进行鉴定。 4、利用 3 条扩增结果稳定、清晰的引物，每种实蝇用 7 个个体对 4 种实蝇的种内稳定性进行了研究，结果表明南瓜实蝇、具条实蝇、桔大实蝇和三点棍腹实蝇的种内平均相似系数分别为 0.551、0.538、0.373、0.778，从相似系数可以看出三点棍腹实蝇种内最稳定，其次为南瓜实蝇和具条实蝇，桔大实蝇种内分化最大。 5、用 S61、S83、S107、S264、S265、S266、S271、S274、S275、S276、S1142、S 合引等 12 条引物，每种实蝇用 1 个个体，对 4 种实蝇进行扩增，根据 DICE 遗传距离聚类结果表明南瓜实蝇和具条实蝇先聚在一起，然后与三点棍腹实蝇聚在一起，最后再与桔大实蝇聚在一起。用 S61、S107、S126、S275、S1142 等 5 条引物，每种实蝇用3 个个体，对 4 种实蝇进行扩增，用 DICE 和 NEI 遗传距离聚类结果表明南瓜实蝇和具条实蝇先聚在一起，再与桔大实蝇聚在一起，最后它们 3 个与三点棍腹实蝇聚在一起。这与传统分类所提出的亲缘关系完全一致。 6、通过扩增 ITS1 片段，成功的对 4 种实蝇进行了鉴定，其中南瓜实蝇扩增出的条<WP=6>带大小为 650bp 左右，具条实蝇扩增出的条带大小为 640bp 左右，桔大实蝇扩增出的条带大小为 1010bp 左右，三点棍腹实蝇扩增出的条带大小为 773bp 左右，该方法比 RAPD方法用的时间更短，整个过程可以在 30 个小时内完成，重复性好，可以在不同实验室，不同 PCR 仪上进行重复。本论文在国内首次利用两种分子标记对实蝇进行了鉴定，利用这两种方法对实蝇进行鉴定快速、准确，从理论上来讲，不受虫态的影响，可以用于实蝇幼虫和蛹的鉴定，而且在其他实蝇的鉴定中也具有一定的参考价值。更多还原

【Abstract】 Fruit flies, a kind of the important pest insects economically, make directly a seriousdamage and have a significant effects on the production and trade of import and export ofmany fruits. There are more than 150 species of fruit flies which damage fruits andvegetables in different country. In order to avoid the introduction of fruit flies, almost eachgovernment adopts a series of serious quarantine methods. The identification of fruit flies wasmainly based on their externally morphological characters of adults in past many years, butmore larvae and ova rather than adults are found in trade of import and export of fruits.Generally, larvae and ova are very difficult to identify precisely and they have to be raised formore a long time so as to obtain their adults in laboratory. Both time of pass-customs isdelayed and more work is added. Therefore, it is important to identify fruit flies accuratelyand quickly in the pest quarantine and control. By means of RAPD and PCR, studies on themolecular identification of fruit flies were carried out. The results are as follows: 1. Genomic DNA in the fruit flies of dried specimen (stored generally more than a year)was extracted by CTAB and SDS etc. The results showed that genomic DNA of driedspecimen of most fruit flies could not be extracted, only a few can be extracted and beprocessed PCR. But the genomic DNA could well be extracted from dried specimen that werestored in super-low temperature refrigerator. 2. A reaction system with more stability and repeatability was established by many timesof screening in this paper. It could also be used in studies of other fruit flies. 3. In tested 69 primers, S361, S67 and S252 could be used to distinguish B. scutellaris,B. tau and D. trimacula. S112 could be used to distinguish B. tau, B. scutellaris and B. citri.S126 and S134 could be used to distinguish B. scutellaris and D. trimacula. S83 could beused to distinguish female and male of B. tau. 4. Genetic stability of 4 species of fruit flies was studied by means of 3 selected primerswhich amplify to get the stable and clear band. Average genetic similarity indexes of B. tau ,B. scutellaris, B. citri and D. trimacula are 0.551, 0.538, 0.373, 0.778 respectively. On thebasis of the genetic similarity indexes, genetic variation of D. trimacula is the lest, B. tautakes second place, the third is B. scutellaris, B. citri had the biggest genetic variation. 5. The genetic polymorphism of 4 species was studied by 12 primers and 5 primersrespectively. An individual was assessed for each species when 12 primers were used. Thephylogenetic tree of 4 species was constructed by UPGMA. The results showed that B. tauand B. scutellaris were clustered firstly, they and D. trimacula were clustered. Finally threespecies and B. tau were clustered. 3 individual was assessed for each species when 5 primers<WP=8>were used. Phylogenetic tree constructed by Dice and Nei distance was different with theabove. B. tau and B. scutellaris were clustered firstly, they and B. citri were clustered. Finallythree species and D. trimacula were clustered. The latter was consistent with the conventionalphylogenetic relationship among 4 species. 6. 4 species were distinguished by amplified ITS1 fragment. Amplified special band of B.tau, B. scutellaris, B. citri and D. trimacula was 650bp, 640bp, 1010bp, 773bp respectively.ITS1 amplification needed less time than RAPD. Generally ITS1 amplification only needed30h or so and had better repeatability. It is the first time that the fruit flies was identified by two kinds of the molecular markermethods in China. Two molecular markers were more accurate and quicker than conventionalmethod based on morphological characters. In theory, they were not affected by insectdevelopment stage and could also be used to identify larvae and pupae of 4 species. At thesame time, these methods could be consulted in the identification of other fruit flies.更多还原