• 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.

• 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.

• 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.

• KSBB Journal
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• 제7권3호
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• pp.155-160
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• 1992

식량으로 쓰이는 식물 단백질은 공통적으로 Isoleucine, Lysine, Methionine, Threonine, Tryptoplan등 5가지 필수아미노산이 결핍되어있다. 본 연구에서는 이러한 필수아미노산을 다량 함유한 단백질을 발현시킬 수 있는 합성유전자를 fekaqo에서 높은 수준으로 발현시키고자 강한 식물 promoter로 알려진 CaMV 35S, CaMV duplicate 35S promoters를 사용하였다. 형질 전환 및 재분화된 식물을 분석한 결과 본 합성 유전자가 식물 nuclear genome 안으로 도입을 안정하여 잘되었고 mRNA수준까지는 유의적인 증가를 보였으나 단백질 수준에서는 유의적 수준의 증가를 관찰할 수 없었다.

• 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 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.

• 식물조직배양학회지
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• 제25권5호
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• pp.323-327
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• 1998

페튜니아에서의 안토시아닌 생합성 경로는 하나의 중요한 유전적인 모델시스템으로 연구되어 왔다. 본 연구에서는 이 경로에 대한 유전자를 연구하기 위하여 CaMV 35S promoter와 가지로부터 분리된 flavonoid 3',5'-hydroxylase cDNA를 pBI121 플라스미드에서 fusion gene 시스템을 만들었다. 형질전환된 페튜니아를 얻기 위한 최적조건이 페튜니아 절간을 IAA 0.2 ㎎/mL, BA 3 ㎎/mL를 혼용한 MS배지에서 얻었다. 효과적인 형질전환을 위하여 페튜니아 절간을 IAA 0.2㎎/L BA 3㎎/L를 혼용한 BM배지에서 Agrobacterium tumefaciens를 접종하기 전에 절편체를 preculture하였다 형질전환체는 50㎎/L kanamycin과 cefotaxim 300㎎/L을 포함하는 배지에서 선발하였다. PCR과 Southern hybridization분석에 의하여 형질전환체를 검증하였다.

• Food Science and Biotechnology
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• 제17권5호
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• pp.1092-1096
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• 2008

Commercial food products from major cities of Coahuila, Mexico were screened to identify residues of transgenic deoxyribonucleic acid (DNA) and/or proteins. After performed, an inventory on all products that contained a soybean-based ingredient in a commercial grocery store in the city of Saltillo, Coahuila, Mexico, 245 food products were identified and grouped in 15 classes according to the soybean ingredient as well as the manufacturing process used for their elaboration. Similar sampling was made for the different food classes in the cities of Monclova, Piedras Negras, and Torreon. A total of 88 samples were analyzed and DNA was extracted by the hexadecyltrimethyl-ammonium bromide (CTAB) technique with slight modification to obtain better DNA quality (1). In addition, segments of the transgenic genes one that codifies for 5-enolpyruvylshikimate-3-phosphate synthase (epsps), cry 1A, and the cauliflower mosaic virus (CaMV) promoter were amplified using polymerase chain reaction (PCR). The transgenic proteins 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) and insecticidal crystal protein (Cry 1Ab/Ac) were identified using double antibody sandwich-enzymatic linked immunoassay analysis (DAS-ELISA). Presence of transgenic genes and/or proteins was identified in 35.3% of the commercial products samples.

• 한국육종학회지
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• 제41권4호
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• pp.385-390
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• 2009

A resveratrol synthase (RS) gene was isolated from peanut (Arachis hypogaea, L. cv. Jinpoong) plants. This gene was placed under the control of the cauliflower mosaic virus 35S promoter (CaMV35S) and introduced into two Korean varieties of potato (Solanum tuberosum L. cvs. Jasim and Jowon) plants by Agrobacterium-mediated gene transfer. Putative transformants were screened by PCR with primers designed from CaMV 35S promoter, NOS terminator and RS gene. Most of selected transgenic potato plants showed the amplification of expected fragments by PCR of genomic DNA with gene-specific primers, while they were absent in untransformed control plants. Expression of the resveratrol synthase gene was also examined by northern blot analysis. The transformants showed a band which was lacking in the control plant, confirming that the introduced gene is transcribed into mRNA in the transformants. The strength of the band, which reflected the level of mRNA expression, differed among the individual transformants. Among the transformants obtained, the highest trans-resveratrol content in the transgenic young leaves of purple-fleshed "Jashim" was $2.11{\mu}gg^{-1}$ fresh weight and that in the microtubers in vitro of purple fleshed "Jashim" was $8.31{\mu}gg^{-1}$ fresh weight. This amount of resveratrol may have a positive biological effect on human health.

• 식물조직배양학회지
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• 제27권3호
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• pp.169-173
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• 2000

Amaranthus hypochondriacus의 저장 단백질을 encoding 하는 AmA1 유전자를 RT-PCR 방법을 이용하여 분리하고 특성화하였다. AmAl 유전자를 담배에 형질전환 시키기 위해 CaMV 35S promoter와 3'NOS를 가지고 있는 식물 발현 vector에 subcloning 하고 이 재조합 vector를 이용하여 Agrobacterium-mediated형질전환 방법을 이용하여 담배에 도입시켰다 신초는 0.1 mg/L NAA, 1.0 mg/L BA, 100 mg/L kanamycin 그리고 250 mg/L cefotaxime이 첨가된 MS 선발 배지에서 선발했고, 선발된 신초는 식물 생장 조절제를 첨가 하지 않고 200 mg/L kanamycin과 250 mg/L cefotaxime이 첨가된 MS배지에서 뿌리를 유도하였다. 선발된 담배의 게놈내의 AmA1 유전자의 존재는 PCR 방법과 hybridization을 이용하여 확인되었고, AmA1 유전자의 발현은 RT-PCR방법과 Southern blot hybridization을 사용하여 확인되었다.