本文選題：中黑盲蝽 + RNA-Seq　； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：中黑盲蝽(Adelphocoris suturalis Jakovlev)隸屬于半翅目盲蝽科,是中國(guó)南方一種重要的農(nóng)業(yè)害蟲,其寄主范圍廣泛,食性雜。目前,中黑盲蝽在生理、生態(tài)方面得到廣泛的研究。然而,未見有報(bào)道伴生微生物(如:病毒)的相關(guān)研究。本文利用Illumina HiSeq?2000高通量測(cè)序技術(shù),對(duì)中黑盲蝽成蟲3個(gè)樣本進(jìn)行轉(zhuǎn)錄分析,通過RNA-Seq得到約23.28 Gb質(zhì)量修剪后的數(shù)據(jù)(clean data),產(chǎn)生約134722個(gè)重疊群(contigs)其中包括107486條unigenes,序列平均長(zhǎng)度797.79 bp,N50為1568 bp。利用功能注釋得到了幾個(gè)病毒基因,主要研究結(jié)果如下:1通過RNA-seq在中黑盲蝽體內(nèi)確定了一種新的病毒,將其命名為Adelphocoris suturalis-associated virus 1(簡(jiǎn)稱ASV1)。ASV1有一個(gè)長(zhǎng)度為10845個(gè)核苷酸的單鏈RNA基因組,且包含5個(gè)開放閱讀框(ORFs)。ORF1編碼一個(gè)由2592個(gè)氨基酸構(gòu)成的多肽并包含4個(gè)功能保守域:一個(gè)病毒RNA甲基轉(zhuǎn)移酶(MTase)結(jié)構(gòu)域,一個(gè)FtsJ-like甲基轉(zhuǎn)移酶(FtsJ)結(jié)構(gòu)域,一個(gè)病毒RNA解旋酶(Hel)結(jié)構(gòu)域,一個(gè)RNA-dependent RNA聚合酶(RdRP)結(jié)構(gòu)域。ORF2,ORF3,ORF4和ORF5的多肽鏈分別由190,461,103和159個(gè)氨基酸構(gòu)成,其中只有ORF5包含一個(gè)結(jié)構(gòu)域:煙草花葉病毒超家族(CP)。前期NCBI Blastx結(jié)果表明ASV1與Paprika mild mottle virus(一種植物病毒)表現(xiàn)出較高的同源性,但在寄主植物上卻沒有發(fā)現(xiàn)ASV1,同時(shí)傳毒實(shí)驗(yàn)的結(jié)果也表明其很可能不是一種植物病毒。后期再次聚類分析發(fā)現(xiàn)ASV1與新發(fā)現(xiàn)的果蠅屬(Drosophila)Boutonnet virus同源性較高,且在采用最大似然法(maximum-likelihood)的進(jìn)化樹中相似的基因組結(jié)構(gòu)和高引導(dǎo)值(high bootstrap value)表明ASV1(可能伴隨著Boutonnet virus)可以被考慮為是一種新的昆蟲病毒分類的原型。另外,野外中黑盲蝽ASV1的感染率處于較低水平(6.60%,在212個(gè)樣本中只有14個(gè)陽性樣本)。2 Adelphocoris suturalis-associated virus 2(簡(jiǎn)稱ASV2)是在中黑盲蝽體內(nèi)發(fā)現(xiàn)的第二種新的病毒,其長(zhǎng)度為9417 bp有4個(gè)ORF,分別由2697、106、169和61個(gè)aa構(gòu)成,其中只有ORF1包含3個(gè)保守功能區(qū)域:一個(gè)MTase,一個(gè)RNA螺旋酶病毒(Hel),一個(gè)RdRP。聚類分析結(jié)果表明ASV2同ASV1一樣與果蠅屬Boutonnet virus展現(xiàn)出較高的同源性,且構(gòu)建系統(tǒng)發(fā)育樹的結(jié)果表明ASV2同ASV1一樣可以被考慮為是一種新的昆蟲病毒分類的原型。另外,野外中黑盲蝽ASV2的感染率處于較低水平(1.42%,在212個(gè)樣本中只有3個(gè)陽性樣本)。3在中黑盲蝽體內(nèi)發(fā)現(xiàn)的第三種病毒為Adelphocoris suturalis-associated virus 3(簡(jiǎn)稱ASV3)。ASV3的長(zhǎng)度為9922 bp包含6個(gè)ORF,每個(gè)ORF分別有322、233、719、1662、68和123個(gè)氨基酸,且功能保守域的總數(shù)為5個(gè)。其中ORF2有一個(gè)似小核糖核酸病毒衣殼蛋白功能域(Rhv),ORF3有一個(gè)Rhv和一個(gè)crpv衣殼蛋白類(CRPV),ORF4有一個(gè)Hel和一個(gè)RdRP。聚類分析結(jié)果表明ASV3與Sanxia water strider virus 7(Iflaviridae:iflavirus)展現(xiàn)出較高的同源性,且構(gòu)建系統(tǒng)發(fā)育樹的結(jié)果說明ASV3是一種昆蟲病毒。同樣,ASV3的感染率也較低(6.60%,在212個(gè)樣本中只有14個(gè)陽性樣本)。4綜上所述,本文利用RNA-seq在中黑盲蝽體內(nèi)發(fā)現(xiàn)了三個(gè)新病毒的完整基因組,ASV1和ASV2的聚類分析結(jié)果和發(fā)育樹圖均表明這兩種病毒的分類地位到目前為止仍處于待定的狀態(tài),而ASV3則被確定為是Iflaviridae:iflavirus的一種昆蟲病毒。
[Abstract]:Adelphocoris suturalis Jakovlev belongs to the family of Hemiptera and is an important agricultural pest in southern China. It has a wide range of host and miscellaneous food. At present, the middle black bug is widely studied in physiological and ecological aspects. However, there is no related research on the associated microbes (such as virus). This paper uses Illumina Hi. Seq? 2000 high throughput sequencing technology was used to transcriptional analysis of 3 samples of the adults of the middle black bug. About 23.28 Gb pruned data (clean data) were obtained by RNA-Seq, producing about 134722 overlapping groups (contigs) including 107486 unigenes, the average length of 797.79 BP, and N50 to 1568 bp. using several viral bases. The main results are as follows: 1 a new virus was identified by RNA-seq in the middle black bug, named Adelphocoris suturalis-associated virus 1 (ASV1).ASV1, which has a single strand RNA genome of 10845 nucleotides and 5 open reading frames (ORFs).ORF1 coding one consists of 2592 amino acids. The polypeptide contains 4 functional Conservatives: a viral RNA methyltransferase (MTase) domain, a FtsJ-like methyltransferase (FtsJ) domain, a viral RNA helicase (Hel) domain, a RNA-dependent RNA polymerase (RdRP) domain.ORF2, ORF3, ORF4, and polypeptide chains are composed of 190461103 and 159 amino acids, respectively. Only ORF5 contains a domain: the tobacco mosaic virus superfamily (CP). Earlier NCBI Blastx results showed that ASV1 and Paprika mild mottle virus (a plant virus) showed higher homology, but no ASV1 was found on the host plant, and the fruit of the drug transfer experiment indicated that it was probably not a plant virus. The secondary cluster analysis found that ASV1 was highly homologous to the newly discovered Boutonnet virus of the genus Drosophila (Drosophila), and the similar genome structure and high guidance value (high bootstrap value) in the evolutionary tree of the maximum likelihood (maximum-likelihood) showed that ASV1 (possibly accompanied by Boutonnet virus) could be considered as a new insect disease. In addition, the infection rate of ASV1 in the field was at a lower level (6.60%, only 14 positive samples in 212 samples).2 Adelphocoris suturalis-associated virus 2 (ASV2) were second new viruses found in the middle black bug, with the length of 9417 BP with 4 ORF, respectively, 2697106169 and 61 AA, respectively. Only ORF1 contains 3 conserved functional regions: a MTase, a RNA spiral enzyme virus (Hel). A RdRP. cluster analysis shows that ASV2 has a higher homology with the Boutonnet virus of the Drosophila, and the results of the phylogenetic tree show that ASV2 and ASV1 can be considered as a new insect disease. In addition, the infection rate of ASV2 in the field was at a lower level (1.42%, only 3 positive samples in 212 samples) and third viruses found in the middle black bug were Adelphocoris suturalis-associated virus 3 (abbreviated ASV3).ASV3 with the length of 9922 BP containing 6 ORF, and each ORF had 322233719,16. 62,68 and 123 amino acids, and the total number of functional conservatives are 5, of which ORF2 has a small ribonucleic virus capsid protein function domain (Rhv), ORF3 has a Rhv and a CRPV capsid protein class (CRPV). ORF4 has a Hel and a RdRP. cluster analysis that shows ASV3 and Sanxia. The results of high homology and the construction of phylogenetic tree indicate that ASV3 is a kind of insect virus. Similarly, the infection rate of ASV3 is also low (6.60%, only 14 positive samples in 212 samples).4, this article uses RNA-seq to find the complete genome of the new virus in the middle black bug, and the cluster analysis of ASV1 and ASV2. And the phylogenetic tree shows that the classification of these two viruses is still in a state of undetermined status so far, while ASV3 is identified as an insect virus of Iflaviridae:iflavirus.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S433.3

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