• 제목/요약/키워드: PVY CP gene

검색결과 8건 처리시간 0.02초

형질전환 연초의 복합바이러스 저항성

  • 이기원;채순용;이청호;이영기;강신웅;박성원;박은경
    • 한국연초학회지
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    • 제21권1호
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    • pp.70-76
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    • 1999
  • KF 116 was TMV resistant tobacco plant and KB 301 was PVY resistant plant transformed with TMV CP gene and PVY CP gene, respectively. These resistant plants were cross-fertilized and the 4 lines of the TMV-PVY resistant plants were selected from F1 hybrid plants. The rate of PVY-resistant plant in these hybrids was 100 percent and that of TMV-resistant plants including delay type was 90-98 percent at 4 weeks after virus inoculation. It was confirmed that the TMV and PVY CP genes were integrated into the genome of hybrid plants by genomic PCR, and Southern blot hybridization. The genome of F1 hybrid plants had one copy and 4 copies of PVY-CP gene and TMV-CP gene, respectively, and CaMV 35S promoters were not methylated, regardless of the difference symptom development to TMV.

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우리나라 감자에 발생하는 PVY의 병원학적 특성 및 외피단백질 유전자 분석 (Etiological Properties and Coat Protein Gen Analysis of Potato Virus Y Occuring in Potatoes of Korea)

  • 정승룡
    • 한국식물병리학회:학술대회논문집
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    • 한국식물병리학회 1995년도 Proceedings of special lectures on Molecular Biological Approaches to Plant Disease National Agricultural Science and Technology Institute Suwon, Korea
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    • pp.77-96
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    • 1995
  • To obtain basic informations for the improvement of seed potato production in Korea, some etiological properties of potato virus Y(PVY) distributed in the major seed potato production area(Daekwanryeong) were characterized, and the nucleotide and amino acid sequences of the coat protein gene of the PVY strains isolated were analyzed. PVY strains in Daekwonryeong, an alpine area, were identified to be two strains, PVYo and PVYN by symptoms of indicator plants, and their distribution in potato fields was similar. Major symptom on potato varieties by PVY was grouped as either mosaic alone or mosaic accompanied with veinal necrosis in the lower leaves. The symptom occurrence of the two symptoms was similar with Irish Cobbler, but Superior showed a higher rate of mosaic symptom than the other. The PVY strain which was isolated from potato cv. Superior showing typical mosaic symptoms produced symptoms of PVY-O on the indicator plants of Chenopodium amaranticolor, Nicotiana tabacum cv. Xanthi nc and Physalis floridana, but no symptom o Capsicum annum cv. Ace. Moreover, results from the enzyme-linked immunosorbent assay with monoclonal and polyclonal antibodies showed that the isolated PVY reacts strongly with PYV-O antibodies but does not react specifically with PVY-T antibodies. The purified virus particles were flexious with a size of 730$\times$11nm. On the basis of the above characteristics, the strain was identified to be a PVY-O and named as of PVY-K strain. The flight of vector aphids was observed in late May, however, the first occurrence of infected plants was in mid June with the bait plants surrounded with PVY-infected potato plants and early July with the bait plants surrounded with PVY-free potato plants. PVY infection rates by counting symptoms on bait plants (White Burley) were 1.1% with the field surrounded with PVY-free potato plants and 13.7% the fields surrounded with PVY-infected potato plants, showing the effect of infection pressure. The propagated PVY-K strain on tobacco(N. sylvestris) was purified, and the RNA of the virus was extracted by the method of phenol extraction. The size of PVY-K RNA was measured to be 9, 500 nucleotides on agarose gel electrophoresis. The double-stranded cDNAs of PVY-K coat protein(CP) gene derived by the method of polymerase chain reaction were transformed into the competent cells of E. coli JM 109, and 2 clones(pYK6 and pYK17) among 11 clones were confirmed to contain the full-length cDNA. Purified plasmids from pYK17 were cut with Sph I and Xba I were deleted with exonuclease III and were used for sequencing analysis. The PVY-K CP gene was comprised of 801 nucleotides when counted from the clevage site of CAG(Gln)-GCA(Ala) to the stop codon of TGA and encoded 267 amino acids. The molecular weight of the encoded polypeptides was calculated to be 34, 630 daltons. The base composition of the CP gene was 33.3% of adenine, 25.2% of guanine, 20.1% of cytosine and 21.4% of uracil. The polypeptide encoded by PVY-K CP gene was comprised of 22 alanines, 20 threonines, 19 glutamic acids and 18 glycines in order. The homology of nucleotide sequence of PVY-K CP gene with those of PVY-O(Japan), PVY-T(Japan), PVY-TH(Japan), PVYN(the Netherlands), and PVYN(France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. The amino acid sequence homology of the polypeptide encoded by PVY-K CP gene with those encoded by viruses was represented as 97.4%, 92.5%, 92.9%, 92.9%, and 98.5%, respectively.

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Variation of Potato virus Y Isolated from Potato, Tobacco, Pea and Weeds in Korea on the C-terminal Region of Coat Protein Gene and 3'Non-translated Region

  • Yun, W.S.;Jung, H.W.;Oh, M.H.;Hahm, Y.I.;Kim, K.H.
    • The Plant Pathology Journal
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    • 제18권3호
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    • pp.130-137
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    • 2002
  • Potato virus Y (PVY) is one of the most important viruses in many field crops in Korea. In this study, 31 PVY isolates were isolated from infected potato (Solanum tuberosum), tobacco (Nicotiana tabacum), pea (Pisum sativum), and weeds (Veronica persica, Lamium amplexicause and Capsella bursa-pastoris) showing different mosaic symptoms in Jeonbuk, Chungnam, Gangwon, and Gyeongbuk areas in Korea. The 640 nucleotide region containing the C-terminal portion of coat protein (CP) gene and 3'non-translated region (NTR) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using PVY-specific oligonucleotide primers. Sequence analyses of the amplified DNA fragments showed that the C-terminal portion of CP gene was not significantly different from that of previously reported PVY strains from potato (PVY-OK and -T) and tobacco (PVY-VN) in Korea. Homologies of the deduced CP amino acid sequences were 93.3-99.0% to corresponding regions of the other PVY strains including PV $Y^{N}$, PV $Y^{o}$ , PV $Y^{OK}$ , PV $Y^{T}$ , and PV $Y^{VN}$ . In contrast the sequences located at the 3'-NTR showed more diverse sequence homologies (76.4-99.7%). These results indicate that the C-terminal portion of the CP gene was relatively conserved while sequences at the 3'NTR were more diverse and variable over the host species and the regions where they were isolated.e isolated.

감자 바이러스 Y 비전이성 외피단백질 cDNA의 형질전환에 의한 바이러스 저항성 연초품종 개발 (Development of Potato Virus Y Resistant Tobacco Plant by Transformation of the Untranslatable Viral Coat Protein Encoding cDNA)

  • 이청호;이영기;강신웅;박성원;김상석;박은경
    • 한국연초학회지
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    • 제19권2호
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    • pp.117-123
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    • 1997
  • Viral coat protein (CP) encoding cDNA with artificial start and stop codons was synthesized by reverse-transcriptase polymerase chain reaction (RT-PCR) from the Korean isolate of potato virus Y-vein nectrosis strain (pVY-VN). To make PVY CP cDNA to untranslatable form, three stop codons were inserted near the start codon by "megaprimer-PCR" method. The untranslatable CP cDNA was subcloned to plant expression vector and transferred to N. tabacum cv. NC82 by Agrobacterium-mediated transformation. Highly resistant plants to PVY infection were screened, based on symptom development after mechanical virus inoculation. By genomic PCR and Southern blot analysis, one or more copies of the untranslatable CP gene were found in all transformants. From northern blot analysis, highly resistant transgenic lines had very low level of CP transcript but susceptible lines had high level, suggesting resistance to PVY infection should be related to RNA-mediated mechanism.mechanism.

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Molecular Screening and Characterization of Antiviral Potatoes

  • Tripathi, Giriraj;Li, Hongxain;Park, Jae-Kyun;Park, Yoon-Kyung;Cheong, Hyeon-Sook
    • Biotechnology and Bioprocess Engineering:BBE
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    • 제11권2호
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    • pp.89-95
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    • 2006
  • Potato plants carrying the Ry gene are extremely resistance to a number of potyviruses, but it is not known which variety expressed the resistance. In this investigation, combined classical and molecular techniques were used to identify virus resistance potatoes. Mechanical inoculation of 32 varieties of Korean potato cultivars, with potato virus Y (PVY), induced various symptoms, such as mosaic, yellowing, necrosis, mottle, vein clearing and vein bending. Different virus spreading patterns were observed, such as highly sensitive, moderate and resistant to $PVY^o$ inoculated leaves in different cultivars. From the results of double antibody sandwich-enzyme links immunosorbant assays (DAS-ELISA), coupled with reverse transcription polymerase chain reaction (RT-PCR), Winter valley and Golden valley were found to be highly susceptible and resistant cultivars to $PVY^o$ respectively. TEM was used as a complementary method to conform the localization of the virus in leaf tissues. TEM detect virus particles in Golden valley, where, ELISA and RT-PCR were unable to detect the CP gene. However, the interior part of the tissues was severely deformed in $PVY^o$ infected Winter valley, than Golden valley The Ry gene is involved in an induced response in $PVY^o$ infected Golden valley plants. The methods described in this study could be applied for the screening and development of antiviral potatoes.

감자 바이러스 Y 저항성 형질전환 감자 개발 (Development of Potato Virus Y-Resistant Transgenic Potato)

  • PARK, Young Doo;RONIS D.H.;DUYSEN M.E.;CHENG Z.M.;LORENZEN J.H.
    • 식물조직배양학회지
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    • 제24권5호
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    • pp.313-317
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    • 1997
  • ND860-2, Norchip, Russet Norkotah, Goldrush 및 Norgueen Russet 등 5개 품종의 감자 잎 조직을 감자 바이러스 Y(PVY)의 외피단백질 (CP) 유전자를 운반하는 Agrobacterium tumefaciens을 이용하여 형질전환시켰다. ND860-2와 Norchip품종과 같은 흰색 감자에서 재분화율과 형질전환율이 높게 나타났다. 형질전환체를 선발한 후 CP의 생성을 western blot으로 확인한 결과 30 kD의 밴드가 나타났다. 온실에서 재배된 형질전환체는 한 개체를 제외하고는 표현형에 있어서는 기존의 품종과 다르지 않았다. 인위적으로 감자 바이러스 Y를 감염시킨 15일 후의 검사결과 control구는 80%의 감염율을 보인 반면 형질전환체들은 38-58%의 감염율을 보였다. 30일 후의 검사 결과는 모든 5품종의 control구에서 100%의 감염을 보였으나 형질전환체는 7개체가 저항성을 보였다. 마지막 85일 후의 검사 결과 4개의 저항개체를 발견하였으며 이 개체들은 현재 계속 연구가 진행 중에 있다.

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분자생물학적 TMV 및 PVY 저항성 연초 육종 (Molecular Breeding of Tobacco Plants Resistant to TMV and PVY)

  • E.K. Pank;Kim, Y.H.;Kim, S.S.;Park, S.W.;Lee, C.H.;K.H.Paik
    • 한국연초학회:학술대회논문집
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    • 한국연초학회 1997년도 담배과학 국제학술대회
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    • pp.134-152
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    • 1997
  • 담배 모자이크 바이러스(TMV)와 감자 바이러스 Y(PVY) 등 식물바이러스병은 잎담배 생산에 심한 경제적 손실을 초래하고 있다. 바이러스병 방제를 위한 여러 가지 경종적 방법은 충분한-방제 효과를 얻지 못하는 경우가 많아 바이러스의 방제에는 우수한 저항성 품종의 사용이 가장 바람직한 것으로 알려져 있다. 그러나 기존의 육종 방법이 저항성 품종 개발에 항상 바람직한 것은 아닌데 그 이유는 저항성 유전자원이 없는 경우가 많고 또한 육종을 통해 유전자원의 열등한 특성이 도입될 수 있기 때문이다. 따라서 본 연구실에서는 TMV와 PVY의 여러 가지 형태의 외피단백질(CP) (비전사부분 포함 또는 제거, 비 전사부분 등) 및 복제유전자(Nlb) (3' 및 5' 결손 유무, 돌연변이)를 상용 담배 품종인 NC 82와 Burley 21에 형질전환시켜 바이러스 저항성 개발을 시도하였다. 각각의 유전자 cDNA를 1-2개의 35S promotor를 가진 식물발현벡타에 클로닝한 후 Agrobacteriupn (upnefaciens LBA 4404를 이용하여 식물의 잎조직에 도입시킨 후 kanamycin 함유 MS 배지에서 식물체를 재닥화하였다. 재분화 식물체는 TMV와 PVY에 대한 저항성 검정을 하였다. 그 결과 TMV에 저항성인 TMV CP 형질전환 식물체와 8 가지 Nlb 형질전환 식물체 계통 중에 6 계통의 저항성 형질전환 식물체를 획득하였다. 여기에서는 TMV와 PVY의 접종시험을 통하여 각각의 바이러스에 대한 형질전환 담배의 계통별 저항성 정도를 조사하고, 저항성 형질전환 식물체에서의 도입 유전자 확인하며, 세대별 저항성의 유전 및 안정성을 살펴보고자 한다. 또한 유망 저항성 형질전환 계통의 식물체 내에서의 바이러스 증식 및 이동과 관련된 저항성 기작, 여러 가지 PVY 계통에 대한 저항성 유무, 수량, 생육 특성 및 주요 화학 성분 함량 등을 발표하고자 한다.-glucose로 구성된 다당류 이었다. 아미노산은 Asp 및 Glu의 산성 아미노산과 Ala, Leu 등의 함량이 높게 나타났으며, 비알칼리 추출물에서 Ser과 Thr의 함량이 높게 나타났다. 다당류 T-AS는 평균 분자량이 2,000 kD와 12kD에서 주 peak를 나타냈으며, 수용성 분획의 평균 분자량은 12kD이고 비수용성 분획은 36~2,000 kD의 평균 분자량 분포를 갖는 것으로 나타났다. IR과 NMR 분석 결과 890 cm-1에서 흡수 peak를 나타내어 $\beta$-(1,3)0glucan과 $\beta$-(1,6)-glucan의 구조를 갖는 다당류로 확인 되었다. T-AS 분획은 C:H:O:N의 함량비가 38.9:5.7:49.6:1.84%이며, 이 물질의 융점은 163 $^{\circ}C$로 연한 갈색을 나타낸다. 분리된 GLG의 항암활성 기전 규명을 위해, in vivo 항암실험, 항보체 활성능, 항체 생성능, serum protein 분비능, 대식세포의 탐식능과 활성능 및 세포간 물질 분비 등의 상관관계를 조사하였다. 다당류 GLG 분획물들 가운데 항보체의 활성이 높았던 분획은 sarcome 180에 대한 항암 활성이 높게 나타났다. 다당류 T-AS의 보체 활성화 기작은 classical과 alternative complement pathway의 양 경로를 통해 활성화 되었다. T-AS 분획은 mouse내의 특정 혈청단백을 증가시켰으며, 항체 생성능의 증가가 관찰되어 effect T 세포의 활성화가 나타나고 있음을 알 수 있었다. T-AS는 생체내 투여시에 대식세포의 탐식능이 증진되었으며, 대식세포 기능 저해제에 의한 대식세포의 기능 저해 현상이 회복되었다. 이와 같은 결과들로부터, T-AS의 항암 활성은 활성화된 보체 성분 및 당 수용체들이 존재하는 대식세포의 개입을 시사한다.가능성과

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RNA silencing-mediated resistance is related to biotic / abiotic stresses and cellular RdRp expression in transgenic tobacco plants

  • Wu, Xiao-Liang;Hou, Wen-Cui;Wang, Mei-Mei;Zhu, Xiao-Ping;Li, Fang;Zhang, Jie-Dao;Li, Xin-Zheng;Guo, Xing-Qi
    • BMB Reports
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    • 제41권5호
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    • pp.376-381
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    • 2008
  • The discovery of RNA silencing inhibition by virus encoded suppressors or low temperature leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRp) has been previously characterized to be essential for transgene-mediated RNA silencing. Here we showed that low temperature led to the inhibition of RNA silencing, the loss of viral resistance and the reduced expression of host RdRp homolog (NtRdRP1) in transgenic T4 progeny with untranslatable potato virus Y coat protein (PVY-CP) gene. Moreover, RNA silencing and the associated resistance were differently inhibited by potato virus X (PVX) and tobacco mosaic virus (TMV) infections. The increased expression of NtRdRP1 in both PVX and TMV infected plants indicated its general role in response to viral pathogens. Collectively, we propose that biotic and abiotic stress factors affect RNA silencing-mediated resistance in transgenic tobacco plants and that their effects target different steps of RNA silencing.