桃蚜傳播黃瓜花葉病毒機(jī)理的研究
發(fā)布時(shí)間：2019-05-29 22:38
【摘要】：桃蚜 Myzus persicae(Sulzer),屬半翅目Hemiptera,蚜科Aphididae,是一種幾乎遍布全球的多食性害蟲。桃蚜不僅可以吸取植物汁液,還可以作為介體傳播多種植物病毒,是常見的媒介昆蟲,給農(nóng)業(yè)生產(chǎn)帶來了嚴(yán)重的損失。桃蚜主要以非持久性的方式來傳播黃瓜花葉病毒(Cucumber mosaic virus,CMV),這種方式主要是指病毒在昆蟲體內(nèi)僅停留幾秒至幾分鐘,病毒不會隨昆蟲的繁殖而傳遞給下一代。目前,關(guān)于桃蚜傳播CMV的傳毒機(jī)制尚不清楚。本研究一方面根據(jù)文獻(xiàn)的報(bào)道,利用qPCR、酵母雙雜交、RNAi等分子生物學(xué)方法,研究桃蚜表皮蛋白基因在傳毒過程中的作用;另一方面利用RNA-seq和iTRAQ等組學(xué)研究技術(shù),進(jìn)一步挖掘與傳毒相關(guān)的潛在傳毒因子,為全面理解桃蚜的傳毒機(jī)制奠定理論基礎(chǔ),同時(shí)也為生產(chǎn)中桃蚜和病毒病的防治策略提供參考依據(jù)。在研究表皮蛋白基因的作用方面,前期篩選出用于擴(kuò)增MPCP1、MPCP2、MPCP4、MPCP5的引物,并優(yōu)化了反應(yīng)條件;用整個(gè)蟲體和口針擴(kuò)增的各表皮蛋白基因序列是一致的,故桃蚜整個(gè)蟲體可以作為后續(xù)的研究對象。在基因水平上,CMVCP的相對表達(dá)量與各表皮蛋白基因的變化趨勢相似,并且MPCP1、MPCP2和MPCP4的相對表達(dá)量明顯高于MPCP5;在酵母雙雜交系統(tǒng)里,只有基因MPCP4編碼的蛋白質(zhì)與CMVCP是相互作用的。在功能研究上,基因MPCP4的沉默可以降低桃蚜獲得病毒的效率。綜上所述,桃蚜表皮蛋白相關(guān)基因參與了傳毒過程,且基因MPCP4在其中起主要作用。在挖掘與傳毒相關(guān)的潛在傳毒因子方面,對帶毒的和不帶毒的桃蚜分別進(jìn)行了轉(zhuǎn)錄組和蛋白質(zhì)組研究。在轉(zhuǎn)錄組測序中,一共找到20550個(gè)差異表達(dá)的基因。其中,9731個(gè)差異基因是上調(diào)表達(dá)的,10818個(gè)差異基因是下調(diào)表達(dá)的。在KEGG數(shù)據(jù)庫中,差異表達(dá)的基因共注釋到501個(gè)通路,其中有32個(gè)是富集分布的(p-value≤0.05),多集中在cAMP信號通路(cAMP signaling pathway)和藥物代謝通路(drug metabolism)。在蛋白質(zhì)組測序中,共找到744個(gè)差異表達(dá)的蛋白。其中,上調(diào)表達(dá)的差異蛋白有437個(gè),下調(diào)表達(dá)的差異蛋白有307個(gè)。在KEGG數(shù)據(jù)庫中,差異表達(dá)蛋白富集(p-value≤0.05)分布在了 14個(gè)通路里,其中包括脂肪酸代謝途徑(fatty acid metabolism)、碳代謝途徑(carbon metabolism)和各種氨基酸代謝途徑等。最后,對桃蚜轉(zhuǎn)錄組和蛋白質(zhì)組進(jìn)行聯(lián)合分析,發(fā)現(xiàn)兩者存在一定的相關(guān)性,并且有282個(gè)蛋白質(zhì)在轉(zhuǎn)錄組數(shù)據(jù)中可以找到聯(lián)系,這其中表達(dá)模式(均上調(diào)表達(dá)或均下調(diào)表達(dá))一致的有207個(gè)蛋白質(zhì),包括表皮蛋白、核糖體蛋白和細(xì)胞色素P450等。
[Abstract]:Aphididae, of Hemiptera, aphids of the genus Myzus persicae (Sulzer), is a kind of polyfeed pest almost all over the world. Peach aphid can not only absorb plant juice, but also spread a variety of plant viruses as a mediator. It is a common vector insect, which has brought serious losses to agricultural production. Peach aphids mainly transmit cucumber mosaic virus (Cucumber mosaic virus,CMV in a non-persistent way, which mainly means that the virus stays in insects for only a few seconds to a few minutes, and the virus is not passed on to the next generation with the reproduction of insects. At present, the mechanism of transmission of CMV by peach aphids is not clear. On the one hand, according to the reports in the literature, the role of epidermis protein gene in the transmission process of peach aphid was studied by qPCR, yeast two-hybrid, RNAi and other molecular biological methods. On the other hand, RNA-seq and iTRAQ were used to further excavate the potential transmission factors related to virus transmission, which laid a theoretical foundation for a comprehensive understanding of the transmission mechanism of peach aphid. At the same time, it also provides a reference for the control strategy of peach aphid and virus disease in production. In the study of the role of epidermis protein gene, primers for amplification of MPCP1,MPCP2,MPCP4,MPCP5 were selected in the early stage, and the reaction conditions were optimized. The sequence of epidermis protein genes amplified by the whole insect body and oral needle is the same, so the whole aphid body can be used as the follow-up research object. At the gene level, the relative expression of CMVCP was similar to that of each epidermis protein gene, and the relative expression of MPCP1,MPCP2 and MPCP4 was significantly higher than that of MPCP5;. In yeast two-hybrid system, only the protein encoded by gene MPCP4 interacts with CMVCP. In functional research, the silencing of gene MPCP4 can reduce the efficiency of peach aphid in obtaining virus. In conclusion, the epidermis protein related genes of peach aphid are involved in the transmission process, and the gene MPCP4 plays a major role in it. In the aspect of excavating the potential transmission factors related to virus transmission, the transcripts and proteome of peach aphids with and without virus were studied respectively. A total of 20550 differentially expressed genes were found in the transcriptional group. Among them, 9731 differentially expressed genes and 10818 differentially expressed genes were down-regulated. In the KEGG database, the differentially expressed genes were commented to a total of 501 pathways, 32 of which were enriched and distributed (p-value 鈮，
本文編號：2488280
[Abstract]:Aphididae, of Hemiptera, aphids of the genus Myzus persicae (Sulzer), is a kind of polyfeed pest almost all over the world. Peach aphid can not only absorb plant juice, but also spread a variety of plant viruses as a mediator. It is a common vector insect, which has brought serious losses to agricultural production. Peach aphids mainly transmit cucumber mosaic virus (Cucumber mosaic virus,CMV in a non-persistent way, which mainly means that the virus stays in insects for only a few seconds to a few minutes, and the virus is not passed on to the next generation with the reproduction of insects. At present, the mechanism of transmission of CMV by peach aphids is not clear. On the one hand, according to the reports in the literature, the role of epidermis protein gene in the transmission process of peach aphid was studied by qPCR, yeast two-hybrid, RNAi and other molecular biological methods. On the other hand, RNA-seq and iTRAQ were used to further excavate the potential transmission factors related to virus transmission, which laid a theoretical foundation for a comprehensive understanding of the transmission mechanism of peach aphid. At the same time, it also provides a reference for the control strategy of peach aphid and virus disease in production. In the study of the role of epidermis protein gene, primers for amplification of MPCP1,MPCP2,MPCP4,MPCP5 were selected in the early stage, and the reaction conditions were optimized. The sequence of epidermis protein genes amplified by the whole insect body and oral needle is the same, so the whole aphid body can be used as the follow-up research object. At the gene level, the relative expression of CMVCP was similar to that of each epidermis protein gene, and the relative expression of MPCP1,MPCP2 and MPCP4 was significantly higher than that of MPCP5;. In yeast two-hybrid system, only the protein encoded by gene MPCP4 interacts with CMVCP. In functional research, the silencing of gene MPCP4 can reduce the efficiency of peach aphid in obtaining virus. In conclusion, the epidermis protein related genes of peach aphid are involved in the transmission process, and the gene MPCP4 plays a major role in it. In the aspect of excavating the potential transmission factors related to virus transmission, the transcripts and proteome of peach aphids with and without virus were studied respectively. A total of 20550 differentially expressed genes were found in the transcriptional group. Among them, 9731 differentially expressed genes and 10818 differentially expressed genes were down-regulated. In the KEGG database, the differentially expressed genes were commented to a total of 501 pathways, 32 of which were enriched and distributed (p-value 鈮，
本文編號：2488280
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