發(fā)布時間：2018-05-29 09:47

  本文選題：小菜蛾 + 間隙連接蛋白��； 參考：《福建農(nóng)林大學(xué)》2016年碩士論文

【摘要】：小菜蛾P(guān)lutella xylostella (L.),是世界性十字花科植物的重要害蟲,每年造成巨大的經(jīng)濟損失,且已經(jīng)幾乎對所有農(nóng)藥都產(chǎn)生了抗藥性,亟待尋求更加有效的防治措施。間隙連接是細(xì)胞間通過細(xì)胞質(zhì)膜相互聯(lián)系,維持組織結(jié)構(gòu)協(xié)調(diào)和完整性的聯(lián)系結(jié)構(gòu),若阻斷此連接,可以阻止細(xì)胞間的交流,影響生物體的生長發(fā)育,為制定有效的小菜蛾防治策略提供新思路。以小菜蛾基因組數(shù)據(jù)立庫,與已知的innexin基因進行比對,篩選出了9個候選innexin基因,分別為Px009151、Px009152、Px009153、Px009154、Px009155、 Px009208、Px013282、Px013283和Px016319,長度分別為1080,1257,1086,1112,315,1161,1020,978,1113 bp；設(shè)計引物,經(jīng)PCR擴增,克隆并測序獲得預(yù)測的9個innexin基因CDS序列；將小菜蛾innexin蛋白家族與其他已知5種昆蟲的innexin蛋白家族成員以最大似然法構(gòu)建進化樹,分析小菜蛾此蛋白家族成員的進化關(guān)系,結(jié)果表明Px016319蛋白序列與昆蟲保守的zpg蛋白序列高度相似；9個innexin基因在不同發(fā)育階段和組織的表達模式：Px016319、Px009151以及Px009208較其它innexin基因的相對表達量高；Px016319在卵和雌性成蟲中大量表達,且在卵巢相對高表達,與果蠅中同屬innexin基因家族的zpg具有相類似的表達模式；Px009151與Px009208在所有發(fā)育階段均有表達,在卵、蛹和成蟲中相對高表達。表達模式分析的結(jié)果表明小菜蛾P(guān)x016319可能參與生殖細(xì)胞的分化與形成,參與雌雄成蟲的繁殖行為；進一步明確Px016319與其它昆蟲zpg在功能上的一致性與分化,對其在小菜蛾中的表達進行抑制,探索并建立了利用CRISPR/Cas9系統(tǒng)在小菜蛾細(xì)胞系中敲除Px016319的方法,結(jié)果顯示敲除后該基因的表達量顯著降低；同時在小菜蛾雌蛹和雄蛹中對Px016319進行RNAi,干擾后該基因的表達量降低。綜上所述,推測在小菜蛾中Px016319可能具有和zpg相似的功能,可作為防控小菜蛾的干預(yù)靶基因；結(jié)合RNAi技術(shù)和基因敲除技術(shù)抑制該基因在小菜蛾中表達,研究結(jié)果可為開發(fā)小菜蛾的遺傳防治新策略提供科學(xué)依據(jù)。
[Abstract]:Plutella xylostella Plutella xylostella L.is an important pest of cruciferous plants in the world. It causes huge economic losses every year and has become resistant to almost all pesticides. It is urgent to seek more effective control measures. Intercellular junction is the connection between cells through the cytoplasmic membrane to maintain the coordination and integrity of the tissue structure. If this connection is blocked, it can prevent the communication between cells and affect the growth and development of organisms. To provide a new idea for the effective control strategy of Plutella xylostella. By comparing the genomic data of Plutella xylostella with known innexin genes, nine candidate innexin genes were screened out, namely Px009151Px009152Px009153Px009155, Px009208Px013282Px013283 and Px016319. the length of the primer was 108611123151061101109781313 BP, respectively. The primers were amplified by PCR. The predicted CDS sequences of 9 innexin genes were cloned and sequenced, and the phylogenetic relationship between the innexin family of Plutella xylostella and the innexin family members of other known five species of insects was constructed by maximum likelihood method, and the phylogenetic relationship of the members of the innexin protein family of Plutella xylostella was analyzed. The results showed that the sequence of Px016319 protein was highly similar to that of zpg protein conserved by insects, the expression patterns of 9 innexin genes in different developmental stages and tissues were: Px016319Px009151, and the relative expression of Px009208 was higher than that of other innexin genes. Px016319 was highly expressed in eggs and female adults. The expression patterns of Px009151 and Px009208 were similar to those of innexin gene family in Drosophila melanogaster. Px009151 and Px009208 were highly expressed in eggs, pupae and adults at all developmental stages. The results of expression pattern analysis showed that Px016319 might be involved in the differentiation and formation of germ cells and the reproductive behavior of female and male adults, and the functional consistency and differentiation between Px016319 and other insect zpg were further clarified. The method of knockout Px016319 using CRISPR/Cas9 system in Plutella xylostella cell line was explored and established. The results showed that the expression of the gene decreased significantly after knockout. At the same time, the expression of Px016319 in female and male pupae of Plutella xylostella was decreased after interference. In conclusion, Px016319 may have similar function to zpg in diamondback moth and can be used as an intervention target gene for controlling Plutella xylostella, and RNAi and gene knockout technology can inhibit the gene expression in Plutella xylostella. The results can provide scientific basis for developing new genetic control strategies of Plutella xylostella.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S433.4

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1 林喜建;小菜蛾innexin基因家族鑒定與innexin 2表達抑制[D];福建農(nóng)林大學(xué);2016年

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本文編號：1950421