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Comparative Transcriptome Analysis of Rice stripe virus-viruliferous and Non-viruliferous Laodelphax striatellus

DC Field Value Language
dc.contributor.advisor제연호-
dc.contributor.author안샛별-
dc.date.accessioned2017-07-14T06:43:10Z-
dc.date.available2017-07-14T06:43:10Z-
dc.date.issued2014-08-
dc.identifier.other000000021910-
dc.identifier.urihttps://hdl.handle.net/10371/125878-
dc.description학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2014. 8. 제연호.-
dc.description.abstractRice stripe virus (RSV) is one of the serious plant pathogenic viruses for rice transmitted by small brown planthopper, SBPH, Laodalphax striatellus. So far, the studies have been mainly focused on the interaction between the host plant and the virus. In this study, for better comprehension of the interactions among Rice stripe virus, rice and small brown planthopper, the transcriptomes of the RSV-viruliferous (RVLS) and non-viruliferous L. striatellus (NVLS) were comparatively analyzed. For this, NVLS were collected from non-infected rice fields were fed with RSV-infected rice for 5 days. With the RNAs extracted from the RVLS and the NVLS, we conducted Illumina RNA sequencing (Hiseq 2000) and as a result two transcriptome databases were constructed from the each sample, respectively. The transcriptome of RVLS and NVLS were compared to figure out how the gene expression of the insects was affected by Rice Stripe Virus. The RSV-dependently regulated genes analyzed from this study may have important functions in the transmission and replication of RSV.
RNA interference (RNAi) is an universal gene-knockdown mechanism in eukaryotic organisms which includes insects, and has been considered as an alternative strategy to control insect pests. Hence, we applied this technique to interfere the translation of target RNA genes to knockdown the virus gene on RVLS. Three out of seven RSV genes, RdRp, NS3, and NCP were used as target genes and each dsRNA targeting the viral genes were delivered to the insects indirectly through the rice leaves by irrigation. As a result, not only the relative expression level of target genes decreased but also those of non-target genes and the replication of RSV genome as well. In summary, leaf-mediated dsRNA feeding methods would be useful in the knockdown of target genes on piercing-sucking insects. The genes used in this experiment can be utilized for the development of pest-resistant transgenic plants based on RNAi.
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dc.description.tableofcontentsTABLE OF CONTENTS

ABSTRACT-----------------------------------------------------------------------------------------i
TABLE OF CONTENTS----------------------------------------------------------------------iii
LIST OF TABLES------------------------------------------------------------------------------ vi
LIST OF FIGURES---------------------------------------------------------------------------- vii
LITERATURE REVIEW----------------------------------------------------------------------1
1. Laodelphax striatellus---------------------------------------------------------------------1
2. Rice stripe virus----------------------------------------------------------------------------3
3. RNA-Sequencing---------------------------------------------------------------------------5
4. RNA interference---------------------------------------------------------------------------6

CHAPTER 1.
Comparative transcriptome analysis of Rice Stripe Virus Viruliferous and Non-Viruliferous Small Brown Planthopper, Laodelphax striatellus
1. Introduction----------------------------------------------------------------------------10
2. Materials and methods----------------------------------------------------------------12
2.1 L. striatellus rearing----------------------------------------------------------------------12
2.2 Inoculation of RSV to non-viruliferous L. striatellus--------------------------------12
2.3 Total RNA extraction and RT-PCR----------------------------------------------------12
2.4 RNA-sequencing and EST library construction--------------------------------------13
2.5 Gene annotation and Gene ontology analysis ----------------------------------------13
2.6 Validation of sequencing data by Real-time PCR -----------------------------------15
3. Results -------------------------------------------------------------------------------------------16
3.1 Confirmation of RSV transmission to the L. striatellus ----------------------------16
3.2 RNA-sequencing data of combined transcriptomes from RVLS and NVLS-----16
3.3 Gene annotation--------------------------------------------------------------------------19
3.4 Comparative transcriptome analysis of RVLS and NVLS--------------------------22
3.5 Validation of sequencing data by qPCR-----------------------------------------------25
3.6 Expression profiling of RSV genes in RVLS-----------------------------------------26
4. Discussion--------------------------------------------------------------------------------29

CHAPTER 2.
RNA silencing by virus-derived small interfering RNAs
1. Introduction------------------------------------------------------------------------------------32
2. Materials and methods---------------------------------------------------------------------35
2.1 L. striatellus rearing---------------------------------------------------------------------35
2.2 Target gene design from RSV genes---------------------------------------------------35
2.3 Total RNA extraction and target gene cloning---------------------------------------35
2.4 dsRNA synthesis and quantification---------------------------------------------------38
2.5 dsRNA delivery via dsRNA-permeated O. sativa leaves---------------------------38
2.6 Detection of dsRNA delivery in O. sativa leaves------------------------------------39
2.7 Detection of gene knockdown----------------------------------------------------------39
3. Results-------------------------------------------------------------------------------------------41
3.1 Confirmation of dsRNA delivery to the O. sativa leaves---------------------------41
3.2 Detection of gene knockdown by feeding dsRNA-----------------------------------42
4. Discussion---------------------------------------------------------------------------------------45


LITERATURE CITED------------------------------------------------------------------------47
ABSTRACT IN KOREAN-------------------------------------------------------------------55
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dc.formatapplication/pdf-
dc.format.extent1115624 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectRice stripe virus-
dc.subjectLaodelphax striatellus-
dc.subjectTranscriptome-
dc.subjectRNA interference-
dc.subjectfeeding RNAi-
dc.subject.ddc630-
dc.titleComparative Transcriptome Analysis of Rice stripe virus-viruliferous and Non-viruliferous Laodelphax striatellus-
dc.typeThesis-
dc.contributor.AlternativeAuthorSaes-Byeol An-
dc.description.degreeMaster-
dc.citation.pagesⅶ, 59-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
dc.date.awarded2014-08-
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