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Genetic Analysis of Recessive Resistance to Turnip mosaic virus and Development of Transgenic Plants using eIF(iso)4E in Brassica rapa

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Authors
김진희
Advisor
강병철
Major
농업생명과학대학 식물생산과학부(원예과학전공)
Issue Date
2013-08
Publisher
서울대학교 대학원
Keywords
Brassica rapaTurnip mosaic virus (TuMV)recessive resistancetranslation initiation factorhost factor
Description
학위논문 (박사)-- 서울대학교 대학원 : 식물생산과학부 원예과학 전공, 2013. 8. 강병철.
Abstract
순무 모자이크 바이러스(Turnip mosaic virus)는 주로 배추과(Brassicaceae)를 기주로 하여 심한 모자이크 병징과 더불어 기형, 위축, 왜화 등의 병징을 유도하여 배추 등 여러 작물에서의 피해가 보고되었다. 본 연구는 궁극적으로 배추에 많은 피해를 주는 것으로 보고된 TuMV가 어떻게 기주를 감염시키고 병징을 일으키고 식물은 어떠한 방법으로 저항성형질을 보일 수 있는지에 대한 이해를 넓히는 데에 그 목적이 있다. 제 1강에서는 순무 모자이크 바이러스에 대한 trs 열성 저항성의 유전 양상을 분석하고 trs 저항성 유전자와 배추의 해독 개시 인자인 eIF(iso)4E가 강한 연관이 보이는 것을 확인하였다. 세 가지 eIF(iso)4E 인자 중 Braiso4Ea의 분자표지가 저항성과 공 분리하는 것을 확인하였으며 trs의 유전자 지도를 작성하여 그 위치를 찾았다. 제 2강에서는 eIF(iso)4E Braiso4E를 이용하여 TuMV에 저항성을 갖는 배추를 개발하였다. 우선 TuMV VPg 단백질과 eIF(iso)4E와의 상관관계가 저항성 기작과 연관이 있음을 확인한 후 이병성 배추에서 eIF(iso)4E 돌연변이들의 과대발현을 유도하였다. 바이러스 이병성 및 저항성 검정 결과, eIF(iso)4E의 W95와 K150 부분의 아미노산의 돌연변이가 VPg 단백질과의 상호작용을 억제하고 광범위 저항성을 유도한다는 것을 확인하였다.
Turnip mosaic virus (TuMV) is one of the major viruses in Brassicaceae crops including Chinese cabbage. The genetic analysis of two Chinese cabbage lines that showed broad spectrum resistance toward TuMV was performed and the recessive resistance gene was named trs. An eIF(iso)4E gene was selected as a candidate for the trs gene. Among the three copies of eIF(iso)4E (Braiso4Ea, Braiso4Eb and Braiso4Ec) found in the Chinese cabbage, the Braiso4Ea marker (trsSCAR) was perfectly co-segregated with trs. But the marker polymorphism was not consistent between resistant lines and susceptible lines, indicating that Braiso4Ea is not the corresponding resistance gene. Mapping analysis indicates that the trs is located at chromosome A04, between scaffold 000104 and scaffold 040552. The trs was highly expected to be one of the Braiso4Ea homologs. So Braiso4Ea cDNA was used for the further study to confirm its relationship between TuMV resistances. The interaction between eIF4E/eIF(iso)4E and VPg is a critical step in many potyviral infections. To elucidate the key amino acids in the interaction between TuMV VPg and eIF(iso)4E, eight amino acids in the cap binding pocket were mutated. The results of yeast two-hybrid and co-immunoprecipitation analysis showed that Trp 95 and Lys 150 amino acid positions are the key amino acids for the TuMV VPg interaction. Complementation of an eIF4E knockout yeast strain by mutated eIF(iso)4E proteins showed that all eIF(iso)4E mutants were able to complement eIF4E of yeast, indicating the mutated eIF(iso)4Es retained their function. Transformant analysis was performed to find out if these mutations affect the susceptibility of Chinese cabbage. eIF(iso)4E W95L, W95L/K150E and susceptible wild type were over-expressed in susceptible Chinese cabbage. According to the TuMV screening result of T1 and T2 transformants, over-expression of the eIF(iso)4E mutants showed broad spectrum TuMV resistance. In conclusion, this research supports that the eIF(iso)4E of Brassica rapa is strongly related to TuMV recessive resistance and single amino acid mutation in the eIF(iso)4E can confer resistance to susceptible plants.
Language
English
URI
https://hdl.handle.net/10371/121047
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Plant Science (식물생산과학부)Theses (Ph.D. / Sc.D._식물생산과학부)
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