中黑盲蝽三种新病毒的基因组结构与田间感染情况研究

发布时间：2018-05-13 03:02

本文选题：中黑盲蝽 + RNA-Seq　；参考：《华中农业大学》2017年硕士论文

【摘要】：中黑盲蝽(Adelphocoris suturalis Jakovlev)隶属于半翅目盲蝽科,是中国南方一种重要的农业害虫,其寄主范围广泛,食性杂。目前,中黑盲蝽在生理、生态方面得到广泛的研究。然而,未见有报道伴生微生物(如:病毒)的相关研究。本文利用Illumina HiSeq?2000高通量测序技术,对中黑盲蝽成虫3个样本进行转录分析,通过RNA-Seq得到约23.28 Gb质量修剪后的数据(clean data),产生约134722个重叠群(contigs)其中包括107486条unigenes,序列平均长度797.79 bp,N50为1568 bp。利用功能注释得到了几个病毒基因,主要研究结果如下:1通过RNA-seq在中黑盲蝽体内确定了一种新的病毒,将其命名为Adelphocoris suturalis-associated virus 1(简称ASV1)。ASV1有一个长度为10845个核苷酸的单链RNA基因组,且包含5个开放阅读框(ORFs)。ORF1编码一个由2592个氨基酸构成的多肽并包含4个功能保守域:一个病毒RNA甲基转移酶(MTase)结构域,一个FtsJ-like甲基转移酶(FtsJ)结构域,一个病毒RNA解旋酶(Hel)结构域,一个RNA-dependent RNA聚合酶(RdRP)结构域。ORF2,ORF3,ORF4和ORF5的多肽链分别由190,461,103和159个氨基酸构成,其中只有ORF5包含一个结构域:烟草花叶病毒超家族(CP)。前期NCBI Blastx结果表明ASV1与Paprika mild mottle virus(一种植物病毒)表现出较高的同源性,但在寄主植物上却没有发现ASV1,同时传毒实验的结果也表明其很可能不是一种植物病毒。后期再次聚类分析发现ASV1与新发现的果蝇属(Drosophila)Boutonnet virus同源性较高,且在采用最大似然法(maximum-likelihood)的进化树中相似的基因组结构和高引导值(high bootstrap value)表明ASV1(可能伴随着Boutonnet virus)可以被考虑为是一种新的昆虫病毒分类的原型。另外,野外中黑盲蝽ASV1的感染率处于较低水平(6.60%,在212个样本中只有14个阳性样本)。2 Adelphocoris suturalis-associated virus 2(简称ASV2)是在中黑盲蝽体内发现的第二种新的病毒,其长度为9417 bp有4个ORF,分别由2697、106、169和61个aa构成,其中只有ORF1包含3个保守功能区域:一个MTase,一个RNA螺旋酶病毒(Hel),一个RdRP。聚类分析结果表明ASV2同ASV1一样与果蝇属Boutonnet virus展现出较高的同源性,且构建系统发育树的结果表明ASV2同ASV1一样可以被考虑为是一种新的昆虫病毒分类的原型。另外,野外中黑盲蝽ASV2的感染率处于较低水平(1.42%,在212个样本中只有3个阳性样本)。3在中黑盲蝽体内发现的第三种病毒为Adelphocoris suturalis-associated virus 3(简称ASV3)。ASV3的长度为9922 bp包含6个ORF,每个ORF分别有322、233、719、1662、68和123个氨基酸,且功能保守域的总数为5个。其中ORF2有一个似小核糖核酸病毒衣壳蛋白功能域(Rhv),ORF3有一个Rhv和一个crpv衣壳蛋白类(CRPV),ORF4有一个Hel和一个RdRP。聚类分析结果表明ASV3与Sanxia water strider virus 7(Iflaviridae:iflavirus)展现出较高的同源性,且构建系统发育树的结果说明ASV3是一种昆虫病毒。同样,ASV3的感染率也较低(6.60%,在212个样本中只有14个阳性样本)。4综上所述,本文利用RNA-seq在中黑盲蝽体内发现了三个新病毒的完整基因组,ASV1和ASV2的聚类分析结果和发育树图均表明这两种病毒的分类地位到目前为止仍处于待定的状态,而ASV3则被确定为是Iflaviridae:iflavirus的一种昆虫病毒。
[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.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433.3

【参考文献】