• 한국잡초학회지
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• 제18권3호
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• pp.237-245
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• 1998

Bacillus subtilis protoporphyrinogen oxidase 유전자를 Agrobacterium을 매개체로 이용하여 전이 시킨 후 cauliflower mosaic virus 35S promoter하에서 발현케 한 형질전환 담배를 유기하였다. 이러한 형질전환 담배의 diphenyl ether계 제초제 oxfluorfen에 대한 생리적 반응과 여러 환경 조건에서의 생장 반응을 재배종 담배와 비교하였다. Oxyfluorfen의 처리에 의해 나타나는 세포내 구성물질의 누출과 지질과산화작용은 재배종 담배에서보다 형질전환 담배에서 더 작게 이루어졌다. 형질전환 담배의 생장을 여러 온도, 광도 및 수분 조건에서 조사한 결과, 저광도와 포화수분 조건에서의 생장이 재배종 담배에 비해 다소 저하되는 현상이 나타났을 뿐 기타 조건에서의 생장은 재배종 담배의 생장과 거의 동일하였다. 따라서 B. subtilis protoporphyrinogen oxidase 유전자를 전이시켜 cauliflower mosaic virus 35S promoter하에서 발현케 한 형질전환 담배는 oxyfluorfen에 대해 비교적 높은 저항성을 나타내지만 형질전환에 따른 생장 변화는 크게 일어나지 않음을 알 수 있었다. 한편 이러한 형질전환 담배의 oxyfluorfen에 대한 저항성 기작에 대해서도 논의하였다.

• The Plant Pathology Journal
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• 제17권1호
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• pp.22-28
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• 2001

Barley yellow dwarf virus PAV (BYDV-PAV) has a 5.7-kb positive-sense single-stranded RNA genome that contains six open reading frames (ORFs). BYDV-PAV produces three subgenomic RNAs (sgRNAs). The largest of which encodes the coat, 17-kDa, and readthrough proteins from two initiation codons. To investigate the role of intergenic and 3'-proximal noncoding regions (NCRs) in coat protein (CP) expression and BYDV-PAV replication, a full-length infectious cDNA of the RNA genome of an Illinois isolate of BYDV-PAV was constructed downstream of the Cauliflower mosaic virus-35S promoter. Linear DNA molecules of these cDNAs were infectious, expressed the 22-kDa CP, and produced both genomic RNA sgRNAs in ratios similar to those observed in protoplasts inoculated with viral RNA. The portion of 5'NCR of sgRNA1 between ORFs 2 and 3 was not required for, but enhanced translation of CP from ORF3. Mutants containing deletions in the NCR downstream of ORF5 failed to replicate in oat protoplasts. These results indicate that an intact 3$^1$NCR is required for BYDV-PAV replication.

• Journal of Plant Biology
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• 제32권1호
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• pp.23-32
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• 1989

We have developed a plasmid vector, pKCH1, for the purpose of higher plant transformation. It contains the promoter region of cauliflower mosaic virus 35S transcript (P35s) and the terminator region of nopaline synthase gene (Tnos) with unique cloning sites, Bam HI and Xba I, between them. After inserting a foreing gene at the cloning sites, P35s-foreign gene-Tnos cassette can be recovered by using a restriction enzyme Hind III.

• Journal of Plant Biology
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• 제37권1호
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• pp.17-25
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• 1994

Two potato (Solanum tuberosum L.) cultivars were transformed by Agrobacterium tumefaciens harboring cauliflower mosaic virus (CaMV) 35S promoter and $\beta$-glucuronidase (GUS) gene. Expression patterns of the CaMV 35S promoter according to tissue types and developmental stages, and genetic stability of GUS gene were investigated in the clonal progenies of transgenic potatoes. Kanamycin-resistant shoot emerged from tuber disc after 4 weeks of culture, and root was induced 6 weeks after culture on the selection medium. Shooting frequency of cvs. Superior and Dejima were 43% and 27%, respectively. Mature transformants and their clonal progenies showed no phenotypical abnormality. GUS activity was expressed primarily at parenchymatous cells of phloem tissue around the vascular cambium in the stem and root, and higher activity was found at the apical meristem of shoot, root and adventious shoot bud. GUS activity was higher at tubers of young explants than at stored tubers. These facts indicate that expression level of the CaMV 35S promoter differed according to tissue types and developmental stages of the organs. The GUS gene was stably inherited to each clonal progeny and normally expressed.

• The Plant Pathology Journal
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• 제20권2호
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• pp.158-163
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• 2004

Chlorella is a eukaryotic microalgae which shares metabolic pathways with higher plants. These charac-teristics make chlorella a potential candidate for eukaryotic overexpression systems. Recently, a foreign flounder growth hormone gene was stably introduced and expressed in transformed Chlorella ellipsoidea by using a modified plant transformation vector that contains cauliflower mosaic virus (CaMV) 35S pro-moter and the phleomycin resistant Sh ble gene as a selection marker. In this study, this same vector was modified by incorporating a promoter and a 3' UTR region of the 33kDa peptide gene from a chlorella virus that was isolated in our laboratory. The 33kDa gene promoter was used to replace the 35S promoter and the 3' UTR was introduced to separate the target gene and downstream Sh ble gene. Three different chlorella transformation vectors containing human erythropoietin (EPO) gene were constructed. The mp335EPO vector consists of a promoter from the 33kDa peptide gene, whereas the mp3353EPO vector contains the same promoter from the 33kDa peptide gene and its 3' UTR. The mp35S33pEPO vector contains the 35S promoter and the 3' UTR from the 33 kDa peptide gene. There was no significant difference in the expression levels of EPO protein in chlorella cells transformed with either of three of the transformation vectors. These data indicate that the promoters from the chlorella virus are comparable to the most common CaMV 35S promoter. Furthermore, these data suggest that other promoters from this virus can be used in future construction of chlorella transformation system for higher expression of target proteins.

• Journal of Plant Biology
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• 제37권3호
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• pp.371-380
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• 1994

To determine the patterns and the levels of expression of the cauliflower mosaic virus (CaMV 35S) promoter and the rice actin 1 (Act1) promoter in rice, transgenic rice plants containing CaMV 35S-$\beta$-glucuronidase (GUS) and Act1-GUS constructs were generated and examined by fluorometric and histochemical analyses. The fluorometric analysis of stably transformed calluses showed that the activity of the rice Act1 promoter was stronger than that of the CaMV 35S promoter in rice cells. In a histochemcial study of the transgenic rices, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in parenchymal cells of vascular tissues of leaves and roots and mesophyll cells of leaves. These results are similar to those of potato, a dicot plant. In contrast, rice plant transformed with Act1-GUS fusion construct revealed strong GUS activity in parenchymal cells of vascular tissue, mesophyll cells, epidermal cells, bulliform cells, guard subsidiary cells of leaves and most cells of the root, suggesting that the rice Act1 promoter is more constitutive than the CaMV 35S promoter. It was also confirmed that in both types of transgenic rice little or no staining was localized in metaxylen tracheary elements of vascular tissue from leaves or roots. These results indicate that the rice Act1 promoter can be utilized more successfully for expression of a variety of foreign gene in rice than the CaMV 35S promoter.

• Applied Biological Chemistry
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• 제38권3호
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• pp.224-231
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• 1995

형질전환 식물체에서 외래 단백질의 발현을 높이기 위하여, 외래 단백질 유전자를 TMV RNA 또는 Gy2 5'-untranslated leader 부위와 재조합시킨 plasmid들을 제조하였다. pGA643에서 유래된 이들 plasmid에는 cauliflower mosaic virus의 35S promoter와 Gy2 terminator를 포함하고 있고, 외래 유전자로는 옥수수의 10kDa zein (10kZ) 유전자를 사용하였다. Gy2 또는 TMV RNA의 leader 부위는 promoter 부위와 단백질 coding sequence 사이에 삽입하였다. Agrobacterium을 매개로 하는 leaf disc 형질전환법을 이용하여 재조합 단백질들을 담배에 도입하였다. Leader를 포함하지 않은 도입 유전자의 전사 효율이 leader를 포함하는 도입 유전자들 보다 높았지만, leader를 포함한 mRNA들이 보다 효율적으로 전사되었다. 이는 5'-untranslated sequence의 길이와 AUG codon 주위의 context에 의한 영향보다는 이들 leader sequence의 특이적 염기서열에 의한 영향이 큼을 의미한다. 이러한 결과는 형질전환된 담배에서 외래 유전자의 전이효율을 조절하는 데 있어서 Gy2와 TMV의 leader sequence들이 enhancer로서 역할을 하고 있음을 의미한다.

• The Plant Pathology Journal
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• 제30권2호
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• pp.159-167
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• 2014

Molecular and biological characteristics of an isolate of Cucumber mosaic virus (CMV) from Glycine soja (wild soybean), named as CMV-209, was examined in this study. Comparison of nucleotide sequences and phylogenetic analyses of CMV-209 with the other CMV strains revealed that CMV-209 belonged to CMV subgroup I. However, CMV-209 showed some genetic distance from the CMV strains assigned to subgroup IA or subgroup IB. Infectious full-genome cDNA clones of CMV-209 were generated under the control of the Cauliflower mosaic virus 35S promoter. Infectivity of the CMV-209 clones was evaluated in Nicotiana benthamiana and various legume species. Our assays revealed that CMV-209 could systemically infect Glycine soja (wild soybean) and Pisum sativum (pea) as well as N. benthamiana, but not the other legume species.

• Journal of Plant Biology
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• 제35권3호
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• pp.237-245
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• 1992

감자 (Solanum tuberosum L. cv. Superior)에서 cauliflower mosaic virus (CaMV) 35S promoter의 발현 양상 및 외래 유전자의 발현에 미치는 intron fragment의 효과를 조사하기 위하여 옥수수의 alcohol dehydrogenase 1-S (Adh1-S) intron 1의 249 base pairs 와 ${\beta}-glucuronidase$ (GUS) 유전자를 결합한, CaMV 35S/deleted Adh1 intron-GUS 구조의 유전자 전달벡터를 제조하고 이를 Agrobacterium tumefaciens를 매개로 형질전환을 유도하였다. 유전자 전달벡터인 pLS201는 17.7 kilobase pairs로서 형질전환의 초기 선별에 용이한 kanamycin 저항성 유전자와 GUS 유전자를 갖는 구조로 제조되었다. 형질전환된 개체의 조직화학적 분석 결과 CaMV 35S promoter에 의한 GUS 유전자는 모든 기관에서 발현되었고, 줄기 및 뿌리에서는 세포분열이 활발한 유관속 형성층을 중심으로 강한 발현을 나타내었다. GUS 유전자의 발현에 미치는 intron fragment의 효과를 조사하기 위하여 CaMV 35S/GUS 구조의 plasmid (pBI121)를 형질전환된 개체를 대조구하여 GUS 활성을 조사한 결과 pLS201의 잎, 줄기, 뿌리에서 각각 30, 34, 42배 높은 활성을 보여, 옥수수 탈수소 효소 유전자의 절단된 인트론이 GUS 유전자의 발현을 증가시킴을 알 수 있었다.

• Journal of Plant Biotechnology
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• 제6권1호
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• pp.9-13
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• 2004

Transient uidA expression was used to optimize parameters required for biolistic transformation of suspension cells of Easter lily, Lilium longiflourm. Maximum uidA expression occurred following bombardment with gold particles as compared to tungsten. A 3hr pre-treatment of suspension cells with 0.125M osmoticum resulted in a 1.5X increase in uidA expression. A helium pressure of 1550 psi combined with a particle travelling distance of 6cm resulted in maximum uidA expression as compared to either 1100, 1200, or 1800 psi. Transient transformation resulted in up to 493 uidA expressing cells/Petri plate. For stable transformation suspension cells of Lilium longiflorum, were co-bombarded with plasmid DNA containing cucumber mosaic virus (CMV) replicase under the rice actin (Act1) promoter and either the bar or PAT genes under the cauliflower mosaic virus (CaMV 355) promoter. Ten regenerated plants contained the transgene as analyzed by PCR, and two of the ten plants were confirmed to contain the transgene by Southern hybridization. The two transgenic plants were independent transformants, one containing the bar gene and the other both the CMV replicase and bar genes. Plants were sprayed at the rosette stage and found to be resistant to 1000 mg/L of phosphinothricin (Trade name-Ignite) indicating expression of the bar gene throughout the leaves when bar was under control of the CaMV 35S promoter.