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RISS 인기검색어
- 검색키워드
- 주제어 : CMV-resistant (검색결과 6 건)
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고추 Cucumber mosaic virus(CMV)에 대한 저항성 재료 평가
Translocation of CMV on Nicotiana tabaccum, 'Xanthi' plants was tested by immuno-strip and RT-PCR technique. Three species of Nicotiana and 3 species of plants were evaluated for inoculum multiplication host and for local lesion host, respectively. Pepper accessions selected on the basis of field observation and on record as sources of resistance to CMV or other viral diseases occurring on pepper were evaluated for resistance by mechanical inoculation at seedling stage and subsequent observation on plants transplanted to pots and field. It was confirmed that CMV administered into plant leaf by inoculation moved to the actively growing leaves on the apex. Nicotiana tabaccum, 'Xanthi', and Chenopodium amaranticolor were selected for CMV- multiplication host and for local lesion host, respectively. KC1151, KC1212, LCA463, KC205, KC1237, KC1263, and 'Dokyacheong' and 'PR Manita' among commercial hybrid cultivars were the no or less diseased of 28 accessions tested for resistance to CMV.
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Transgene Stability and Karyotype Analysis of CMV-resistant Transgenic Hot Peppers
Many genetically modified crops were developed but the genetic instability of the introduced gene came to be a big problem to be solved for application of the transgenic plants. The existence and expression stability of transgene in CMVP0-CP introduced transgenic Hot peppers was investigated with two lines, H15 and B20. Genomic DNAs, total RNAs and total proteins extracted from leaf, flower and fruit tissues of non-transgenic and transgenic plants of T4 generation were analyzed by PCR, southern blot, RT-PCR, and western blot techniques. The transgenes were confirmed to exist stably during the generation, and transcription and translation of the gene was detected constantly in transgenic plant of two lines. It seems that the existence and expression stability of the introduced gene in CMVP0-CP transgenic Hot peppers keeps up during the generation. Karyotypes of transgenic Hot peppers were compared with those of non-transgenic plants using mitotic metaphase chromosomes. The somatic chromosome compositions of both transgenic and non-transgenic plants were diploids of 2n=24 and no significant difference in mitotic karyotypes considering chromosome length, type, and satellites etc., was observed between transgenic plants and non-transgenic plants. 최근 여러 목적으로 형질전환체 식물이 다양하게 개발되고있다. 그러나, 형질전환체 식물에서 도입 유전자의 불활성화 현상이 종종 일어나 형질전환작물 개발의 문제점로 대두되고 있다. 따라서, 본 연구는 CMV 저항성 형질전환 고추 2 계통의 핵형분석과 도입유전자의 발현 안정성 분석을 실시하였다. CMV 저항성 형질전환 고추 2 계통 H15와 B20의 잎, 꽃 그리고 열매를 이용하여 DNA, RNA 그리고 protein을 추출하여 PCR, southern blot, RT-PCR, western blot의 분자생물학적 방법을 통한 도입유전자의 존재 확인 및 발현안정성을 분석하였다. PCR 결과, 2계통 모두 1054 bp의 35S promoter 와 CMVP0-CP, 654 bp의 CMVP0-CP, 443 bp의 NPTⅡ band가 조사한 모든 식물체 부위에서 검출되었다. CMVP0-CP probe와 NPTⅡ probe를 사용하여 southern blot을 수행한 결과 형질전환 2 계통 모두에서 single band를 나타내어 single copy임을 확인 할 수 있었다. 또한. RT-PCR을 이용하여 CMVP0-CP의 654 bp와 NPTⅡ의 443 bp의 band를 2계통 모두에서 확인하여 mRNA로의 전사를 확인하였으며, western blot에 의해 형질전환 2계통 모두에서 약 27kDa의 band가 확인되어 도입유전자가 단백질로 발현됨을 확인할 수 있었다. 핵형분석 결과, 염색체 수는 H15와 B20 모두 2n = 24로 나타났으며, 이는 각각의 야생형 모본과 일치함을 확인하였다. 특히, H15와 야생형 모본, B20과 야생형 모본에서 이차협착부위로 인한 satellite의 위치와 수가 대조적인 차이를 보였다. H15와 야생형 모본은 2쌍(chromosome 11, 12)의 satellite chromosome이 관찰된 반면, B20과 야생형 모본은 1쌍의 satellite chromosome이 관찰되었다. 위 결과를 통해 CMV 저항성 형질전환체 고추의 분자생물학적 분석과 핵형분석으로 H15, B20에서 도입유전자가 안정되게 존재하며 발현되고 있음을 확인하였다.
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Nonsynonymous Evolution in RNA1 Enabled Cucumber Mosaic Virus to Overcome Resistance in Pepper
Plant RNA viruses are one of the most prevalent pathogens that cause significant damage in crops. The most efficient method to prevent viral diseases is to utilize resistance sources in breeding. Cucumber mosaic virus (CMV) belonging to plant RNA virus is the most destructive virus in pepper. Especially, CMV pathotypes P0 and P1 have occurred continuously in pepper fields in Korea and caused significant damage in pepper production. Currently, some pepper cultivars resistant to CMV pathotypes P0 and P1 have developed and grown in the fields. However, a new resistance-breaking variant of CMV (named CMV-GTN) capable of infecting peppers resistant to CMV pathotypes P0 and P1 was emerged in 2013. In this study, we examined the biological and molecular characteristics of CMV-GTN to understand evolutionary mechanisms underlying emergence of resistance-breaking variants of CMV. We constructed full-length cDNA clones of CMV-GTN (named pCMV-GTN) and demonstrated the virulence of pCMV-GTN based on Agrobacterium-mediated infiltration (agroinfiltration) in Nicotiana benthamiana and various pepper cultivars. To identify genetic resources for resistance against CMV-GTN, 34 commercial peppers were screened by inoculating with pCMV-GTN. However, all tested pepper cultivars were susceptible to CMV-GTN, while a few cultivars, including Baerota, were resistant to CMV-Fny (pathotype P0) and CMV-P1 (pathotype P1). Analysis of pathogenicity of pseudo-recombinants between CMV-P1 and CMV-GTN showed that the CMV 1a protein encoded in RNA1 acts as the pathogenic determinant causing resistance-breaking in pepper. Interestingly, sequence analysis revealed that there were only two amino acid differences at positions 253 and 553 in 1a between CMV-P1 and GTN and these amino acid substitutions were caused by nonsynonymous mutations at nucleotide positions 852 and 1752 in RNA1, respectively. Further analysis of amino acid substitution mutants carrying mutations at position 253 or 553 in 1a demonstrated that either amino acid mutation could cause resistance-breaking in pepper. Therefore, it appears that nonsynonymous mutations in RNA1 are responsible for the emergence of resistance-breaking variants in pepper. Our findings increase the understanding of evolutionary mechanisms of resistance-breaking in the CMV-pepper pathosystem.
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