白背飞虱基因组测序及其受南方水稻黑条矮缩病毒侵染的转录组分析

发布时间：2018-03-05 12:10

本文选题：白背飞虱　切入点：南方水稻黑条矮缩病毒　出处：《中国科学技术大学》2017年博士论文　论文类型：学位论文

【摘要】：白背飞虱是一种重要的农业害虫,主要通过取食植物韧皮部汁液和传播病毒两种方式危害水稻。白背飞虱是目前已知传播南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)的特异介体昆虫,SRBSDV在其体内以持久增殖方式扩增。由于缺少白背飞虱的基因组等信息,白背飞虱的相关生物学研究进展缓慢,包括白背飞虱与SRBSDV互作机制的研究。本课题针对这种研究现状,首先对白背飞虱的基因组测序,获得了高质量的基因组序列;系统鉴定了白背飞虱的免疫相关基因;对感染SRBSDV的白背飞虱转录组进行详细的比较分析。结果如下:我们首先对每代白背飞虱进行同胞配对传代,获得基因组杂合度不断降低的近交系。提取F6代个体的基因组DNA,构建一系列插入片段180 bp～40 kb的DNA文库进行高通量测序,获得了总量达到241.3 Gb测序深度为330X的原始序列。经组装和拼接获得约720 Mb的白背飞虱基因组序列,其Contig N50为70.7 kb,Scaffold N50为1.18 Mb。生物信息学分析白背飞虱基因组含有21,254个蛋白编码基因。利用64个白背飞虱F1代和F2代个体,构建了23个RAD-seq文库,鉴定发现2,386个遗传标记,构建了15个遗传连锁群。这些连锁群包括529个Scaffolds,占基因组总长度的71%;含13,626个基因,占基因总数的64%。基于白背飞虱基因组和转录组信息,我们通过同源比对分析鉴定了348个免疫相关基因,其中231个基因可以归类至28个免疫基因家族和4个功能组中。为了研究免疫基因在白背飞虱抗SRBSDV过程中的作用以及SRBSDV侵染对白背飞虱产生的影响,我们分析了SRBSDV侵染的白背飞虱转录本。传毒后不同个体SRBSDV S9-2表达水平在第12天和15天有10∧6级别的差异。按照白背飞虱个体SRBSDV表达水平分组,提取高病毒滴度组(High viral titers,HVT)、中病毒滴度组(Median viral titers,MVT)和非侵染组(Non-viruliferous,NVF)的白背飞虱RNA进行转录组测序。分析发现SRBSDV侵染对于介体白背飞虱基因表达具有广泛影响。和健康对照组相比,我们在HVT,MVT和NVF组鉴定了278个共同上调表达基因和406个共同下调表达基因(差异倍数=2),这些基因参与了很多种生物学过程,在初级代谢通路和氧化还原反应中有一定的富集。鉴于NVF,MVT,HVT组白背飞虱含有SRBSDV渐次增加,分析发现1,906个单向递增基因(Monotonically increasing exprssion genes,MIEGs)和1,467个单向递减基因(Monotonically decreasing exprssion genes,MDEGs),这些基因表现出一定程度的病毒滴度依赖。RNAi通路基因在MIEGs中显著富集,表明RNAi是白背飞虱抗SRBSDV病毒的一种主要免疫反应。对小RNA分析同样证明白背飞虱RNAi通路在细胞和成虫水平上参与抗SRBSDV反应。综上所述,通过本课题的研究,获得了高质量的白背飞虱基因组序列,明确了白背飞虱的免疫系统组成,认识了白背飞虱在感染SRBSDV后复杂的基因差异表达情况。这些数据和发现为深入研究SRBSDV-白背飞虱-水稻三者之间的互作机制提供了研究基础。
[Abstract]:The white backed planthopper is one of the most important agricultural pests, harm of rice mainly through feeding plant phloem sap and the spread of the virus in two ways. Sogatellafurcifera is now known to spread southern rice black streaked dwarf virus (Southern rice black-streaked dwarf virus, SRBSDV) the specific insect, SRBSDV in the body with persistent proliferation due to the lack of amplification. Sogatellafurcifera genome information, research progress of related biological sogatellafurcifera slow, including sogatellafurcifera and SRBSDV to study the interaction mechanism. This paper based on this situation, the first genome sequencing of WBPH, obtained the genome sequences of high quality; system identification of immune related genes in white backedplanthopper; detailed comparative analysis on sogatellafurcifera transcriptome of SRBSDV infection. The results are as follows: firstly we each generation of WBPH were sib pairs were obtained Inbred to genomic heterozygosity decreased. The genomic DNA of individual extraction F6, construct a series of insert 180 BP ~ 40 KB DNA library for high-throughput sequencing, the total reached 241.3 Gb sequencing depth of the original sequence of 330X. By assembling and splicing sogatellafurcifera genomic sequences obtained about 720 Mb the Contig N50 70.7 KB Scaffold N50 1.18 Mb. bioinformatics analysis of sogatellafurcifera genome contains 21254 protein encoding gene. Using 64 sogatellafurcifera F1 and F2 generation individuals, 23 RAD-seq libraries were constructed, identified 2386 genetic markers, 15 linkage groups were constructed these linkage groups including 529 Scaffolds, accounting for 71% of the total length of the genome; containing 13626 genes, accounting for 64%. of the total gene sogatellafurcifera genome and transcriptome information based on the homologous analysis we identified 348 immune related Genes, including 231 genes can be classified into 28 immune gene family and 4 functional groups. In order to study the influence of immune genes in sogatellafurcifera anti SRBSDV process and the effect of SRBSDV infection on WBPH, we analyzed SRBSDV infection sogatellafurcifera transcripts. Expression of different individual SRBSDV S9-2 virus after the level difference at twelfth and 15 days, 10 a 6 level. In accordance with sogatellafurcifera individual expression of SRBSDV group, extraction of high virus titer group (High viral, titers, HVT), virus titer group (Median viral titers, MVT) and non infection group (Non-viruliferous, NVF) of sogatellafurcifera the RNA transcriptome sequencing. The result showed that SRBSDV infection has broad implications for the expression of mediator sogatellafurcifera gene. Compared with healthy control group, we at HVT, MVT and NVF group identified 278 common up-regulated genes and 406 down regulated table As gene (fold difference =2), these genes are involved in many biological processes in the primary metabolic pathways and redox reactions in certain concentration. In view of NVF, MVT, HVT group of sogatellafurcifera containing SRBSDV gradually increase, the analysis found that 1906 single gene (Monotonically increasing exprssion genes by MIEGs, and 1467) one gene (Monotonically decreasing exprssion genes., MDEGs), these genes showed a certain degree of virus titer dependent.RNAi pathway genes were significantly enriched in MIEGs, indicating that RNAi is a kind of white back lice primary immune responses against SRBSDV virus. The RNA analysis also showed that the small white backed planthopper RNAi pathway is involved in anti SRBSDV reaction in cells and the adult level. To sum up, through the research, obtained the WBPH genome sequence of high quality, the components of the immune system white back lice, We have known the complex gene expression of white backed planthopper after infection with SRBSDV. These data and findings provide a basis for further research on the interaction mechanism between SRBSDV-, the three white planthopper rice.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S435.11

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