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  本文關(guān)鍵詞： 小食心蟲 DNA條形碼 COI ITS2 分子鑒定 系統(tǒng)發(fā)育　出處：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：小食心蟲屬(鱗翅目：卷蛾科)的一些物種是我國北方果樹的重要害蟲,其幼蟲蛀果危害,嚴(yán)重影響植物的生長和結(jié)實,常給農(nóng)林生產(chǎn)造成很大的損失。為了更好的防治這類害蟲,準(zhǔn)確的分類鑒定尤為必要。目前,經(jīng)典的形態(tài)學(xué)鑒定是最常用的方法,但需要由專業(yè)人員鑒定,且近緣種的鑒定較難掌握,因此需要尋求一種可以對物種進行簡單、快速、準(zhǔn)確的輔助鑒定方法。隨著分子生物學(xué)技術(shù)在昆蟲分類中的廣泛應(yīng)用,DNABarcoding為快速準(zhǔn)確地鑒定小食心蟲提供了新思路。本論文以小食心蟲屬部分種類作為研究對象,以線粒體COI基因片段作為主要分子標(biāo)記,核糖體內(nèi)轉(zhuǎn)錄間隔區(qū)ITS2序列為輔助分子標(biāo)記,對不同性別、不同蟲態(tài)、不同地域的梨小食心蟲、李小食心蟲、麻小食心蟲和沙果小食心蟲線粒體COI基因片段進行了測序,結(jié)合GenBank[中已遞交的小食心蟲序列,對主要小食心蟲進行系統(tǒng)發(fā)育分析；對梨小食心蟲、李小食心蟲、麻小食心蟲和沙果小食心蟲部分個體的ITS2部分序列進行了初步研究,結(jié)果如下：1.四種小食心蟲經(jīng)過傳統(tǒng)形態(tài)學(xué)鑒定,擴增不同地理種群的COI和ITS2序列情況如下：梨小食心蟲COI序列35條,有5個單倍體基因型,ITS2序列13條;沙果小食心蟲COI序列1條,ITS2序列1條；李小食心蟲COI序列4條,有4個單倍體基因型,ITS2序列4條；麻小食心蟲COI序列14條,有3個單倍體基因型,ITS2序列4條;共獲得小食心蟲屬COI序列54條,ITS2序列22條。進一步豐富了小食心蟲屬DNA條形碼序列信息庫。2.對不同性別,不同蟲態(tài),不同地域,不同物種小食心蟲COI基因序列片段分析發(fā)現(xiàn)：COI基因序列無性別、蟲態(tài)差異,也無明顯地理差異(李小食心蟲除外),不同物種有固定的核酸位點,表明COI基因片段作為分子標(biāo)記,對種內(nèi)不同地理種群、不同性別、不同蟲態(tài)的識別效力有限。3.對四種小食心蟲屬昆蟲與外群蘋果蠹蛾大量樣本線粒體部分COI基因序列研究發(fā)現(xiàn),考慮到地理因素和近緣種因素后,結(jié)合種間-種內(nèi)遺傳距離分析,并運用鄰接法(NJ)和最大簡約法(MP)法建樹分析,發(fā)現(xiàn)小食心蟲屬內(nèi)麻小食心蟲與外群蘋果蠹蛾的親緣關(guān)系較與屬內(nèi)昆蟲的親緣關(guān)系更近,表明COI序列單獨作為小食心蟲分類DNA條形碼標(biāo)記基因只具有一定的識別效力。4.對小食心蟲屬(四種)大量樣本核基因ITS2研究發(fā)現(xiàn),考慮到地理因素和近緣種因素后,并結(jié)合種間-種內(nèi)遺傳距離分析和NJ、MP法建樹分析,表明ITS2序列適合作為鱗翅目理想的輔助分子標(biāo)記。5.利用COI和ITS2基因探討小食心蟲的親緣關(guān)系：梨小食心蟲與沙果小食心蟲親緣關(guān)系最近,其次是和李小食心蟲,和麻小食心蟲的親緣關(guān)系最遠(yuǎn)。
[Abstract]:Some species of the genus Lepidoptera (Lepidoptera: Crixidae) are important pests of fruit trees in northern China, and their larvae are harmful to fruit borer, which seriously affects the growth and fruiting of plants. It often causes great losses to agricultural and forestry production. In order to better control such pests, accurate classification and identification is particularly necessary. At present, classical morphological identification is the most commonly used method, but it needs to be identified by professionals. And the identification of relative species is difficult to master, so we need to find a simple and fast way to identify the species. With the wide application of molecular biological techniques in insect taxonomy, DNA Barcoding provides a new idea for rapid and accurate identification of minnow. The mitochondrial COI gene fragment was used as the main molecular marker, and the ITS2 sequence of ribosomal intertranscription spacer was used as the auxiliary molecular marker. The mitochondrial COI gene fragments were sequenced and phylogenetic analysis was carried out for the main minisculids in combination with the previously submitted GenBank sequences. The ITS2 partial sequences of some individuals were studied. The results were as follows: 1. The four species were identified by traditional morphology. The COI and ITS2 sequences of different geographic populations were amplified as follows: 35 COI sequences, 13 haploid genotypes, 1 COI sequence, 1 ITS2 sequence and 4 COI sequences, respectively. There were 4 haploid genotypes, 4 ITS2 sequences, 14 COI sequences and 4 haploid genotypes. A total of 54 COI sequences and 22 ITS2 sequences were obtained, which further enriched the information base of DNA barcode sequences. 2. The sequence analysis of COI gene in different species showed that there was no sex, no difference in insect status, and no significant geographical difference (except for C. prunculus, different species had fixed nucleic acid loci, indicating that COI gene fragment was used as a molecular marker. For different geographical populations, different genders and different insect species, the recognition efficiency of different species was limited. 3. The mitochondrial partial COI gene sequences of a large number of samples of four species of the genus Coccidioctonia and the outer coterworm moth were studied, and the results were as follows: 1. Taking into account geographical factors and relative species, combined with the analysis of interspecific and intra-specific genetic distance, and using the method of NJ) and the maximum minimization method (MPP) to construct the tree analysis, It was found that the phylogenetic relationship between the inner eater and the outer coda was closer to that of the intra genus. The results showed that the COI sequence alone as the DNA barcode marker gene in the classification of hearth only had a certain recognition effect. 4. For a large number of nuclear gene ITS2 of the genus (four species), it was found that, considering geographical factors and relative species factors, Based on the analysis of genetic distance between species and intraspecies and NJMP method, The results showed that the ITS2 sequence was suitable as an ideal auxiliary molecular marker for Lepidoptera. 5. Using COI and ITS2 genes to explore the genetic relationship of minnow, the relationship between Pear fruit-eater and fruit-eater was the most recent, followed by Phellodendron prickly. It is the furthest related to the orifices.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S433

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