Glycine soja 에서 분리 동정된 Cucumber mosaic virus 의 분자 생물학적 특성 구명

학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2014. 8. 김국형.기주적응은 바이러스의 진화에 있어 바이러스 게놈서열의 수렴과 변이에 주요한 요인 중 하나이다. 이 연구에서 우리는 Glycine soja (wild soybean) 로부터 CMV-209라 명명한 CMV strain을 분리하였으며 전체 게놈의 감염 가능한cDNA clone을 제작함으로써 분자적, 생물학적 특징을 규명하였다. CMV-209의 전체 게놈 서열을 이용한 계통유연학적 분석은 CMV-209 strain이 CMV의 subgroup I에 속한다는 것을 보여주었으나, 기존 subgroup IA와 subgroup IB에 속하는 CMV strain들과는 서열상 89-93%의 상동성을 보이는 것으로 보아 IA 또는 IB에도 속하지 않은 것으로 나타났다. 또한 CMV-209게놈의 각각의 segment와 각각의 바이러스 단백질에 대한 계통학적 분석은 CMV-209가 subgroup IA와 IB와는 다른 계통에 위치한다는 것을 나타냈으며 CMV-209의 기주범위에 대한 조사는 CMV-209가 기주 선택적 strain이라는 것도 나타냈다. CMV는 Vigna sinensis에서는국부 병반을야기하고 Pisum sativum과 Glycine soja에서는전신감염하였으나 실험에 사용된 여타 콩과 다른 품종들 대부분은 감염하지 못했다. CMV-209는 전형적인 CMVstrain인 CMV-Fny가 콩과 식물을 감염시키지 못한다는 것을 고려했을 때 콩과 식물을 감염시킬 수 있도록 적응해 온것으로 보여진다.한편 본 연구에서는 CMV-Fny와의 비교를 통하여 CMV-209의 Nicotiana benthamiana에서의 병징에 대한 분석을 수행하였으며 CMV-Fny가 Nicotiana benthamiana에감염하여 모자이크, 왜화, 잎의 기형화 등의 전형적인 병징을 보이는데 반해 CMV-209는 눈에 뛰는 병징을 보이지 않으면서 감염하는 잠복감염의 특성을 보인다는 것을 알게 되었다. CMV-Fny와 CMV-209의 각 RNA 절편들의pseudorecombinant들을 이용한 감염실험 결과는 CMV의 RNA2가 병징의 발생에 기여한다는 것을 증명하였다. 예를 들면 Fny-RNA2는 모자이크, 왜화또는 잎의 기형화를 나타내는데 반해 209-RNA2는 잠복 감염의 특성을 보인다.또한 우리는 구별된 strain간의 pseudorecombination이 더욱 강한 감염성과 심각한 피해를 야기할 수 있다는 점을 통해 strain간의 RNA 절편들이 서로 호환 가능하다는 것을 관찰하였다. 209-RNA1과 Fny-RNA2를 지닌 pseudorecombinant는 Fny-RNA1과 Fny-RNA2의 그것에 비해 더욱 심한 잎의 기형화 및 왜화 증상을 나타내었다.요약하면 본 연구를 통하여 CMV-209는 Cucumovirus속 CMVsubgroup I에 보다 가까운 새로운 strain이라는 것을 보여준 것이다.Host adaption is one of the major factors for sequence convergence or divergence in viral evolution. In this study, a Cucumber mosaic virus (CMV) isolate from Glycine soja (wild soybean), named as CMV-209, was characterized molecular and biological properties by generating its infectious full-genome cDNA clones. Sequence analysis of full-length genome of CMV-209 showed CMV-209 belonged to subgroup I of CMV, but neither subgroup IA nor subgroup IB since the CMV-209 only shared overall 89-93% sequence identity in their genomes. Phylogenetic analysis of both each CMV-209 genomic segment and each viral protein also exhibited that CMV-209 was located in a new branch separated from subgroups IA and IB. The investigation of CMV-209 host ranges suggested that CMV-209 is a host-selective strain. CMV did not infect most of the tested legume cultivars except causing local lesion on Vigna sinensis (cowpea) and systemic infection on Pisum sativum (pea) and Glycine soja. CMV-209 seems to have adapted itself to infect pea because other typical CMV strain such as CMV-Fny could not infect pea. In the other hand, the symptom expression of CMV-209 in Nicotiana benthamiana was further analyzed by comparing with that of CMV-Fny, which is a representative strain of CMV. CMV-209 induced latent symptom in Nicotiana benthamiana whereas CMV-Fny caused typical symptoms including mosaic, stunting, or distortion of leaves. Pseudorecombinant results demonstrated that RNA2 of CMV was responsible for symptom development, e.g. Fny-RNA2 was accountable for mosaic, stunting or leaf distortion and 209-RNA2 for latent symptom. Furthermore, the compatibility among CMV genomic RNA segments also played a role in symptom expression because pseudorecombination between distinct strains of a species can result in emergence of virus progenies carrying higher infectivity or causing severe damages. The pseudorecombinants containing 209-RNA1 and Fny-RNA2 induced more severe leaf distortion and stunting than those consisting of Fny-RNA1 and Fny-RNA2. In summary, results of our study indicate that CMV-209 is a novel strain and belongs to subgroup I of CMV in the genus Cucumovirus.CONTENTS page ABSTRACT .......................................................................................... i CONTENTS .......................................................................................... iv LIST OF TABLES ............................................................................. vii LIST OF FIGURES ............................................................................. viii page GENERAL INTRODUCTION ............................................................ 1 LITERATURE CITED ...................................................................... 10 CHAPTER 1. Molecular and biological characterization of an isolate of Cucumber mosaic virus from Glycine soja by generating its infectious full-genome cDNA clones page ABSTRACT ........................................................................................ 17 INTRODUCTION .............................................................................. 18 MATERIALS AND METHODS I. Virus source and construction of the full-length cDNA clones of CMV-209 .............................................................................................. 21 II. Infectivity test and screening host range for CMV -209 ................... 24 III. Sequencing, comparative, and phylogenetic analyses .................... 25 page RESULTS AND DISCUSSIONS I. Infectivity and host range of CMV -209 ............................................ 29 II. Molecular characteristics of CMV-209 ............................................ 33 III. Comparative analysis of the CMV -209 genome sequence ............. 37 IV. Phylogenetic analysis ...................................................................... 42 LITERATURE CITED ...................................................................... 46 CHAPTER 2. Pseudorecombination between two distinct strains of Cucumber mosaic virus results in enhancement of symptom severity page ABSTRACT ........................................................................................ 53 INTRODUCTION .............................................................................. 54 MATERIALS AND METHODS I. Virus source and construction of the full-length cDNA clones of CMV -209 and CMV -Fny ...................................................................... 57 II. Construction and characterization of pseudorecombinants of CMV -209 and CMV -Fny ................................................................................ 60 page RESULTS I. CMV RNA 2 contains a symptom determinant (s) causing stunting and leaf distortion ................................................................................. 62 II. The compatibility among CMV genomic RNA segements plays a role in symptom expression ................................................................ 67 DISCUSSION ...................................................................................... 70 LITERATURE CITED ...................................................................... 75 ABSTRACT IN KOREAN................................................................. 83Docto