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Bachchu, Mohammad Adnan Al,Jin, Seong-Beom,Park, Jeong-Won,Boo, Kyung-Hwan,Sun, Hyeon-Jin,Kim, Yong-Woo,Lee, Hyo-Yeon,Riu, Key-Zung,Kim, Jae-Hoon The Korean Society for Applied Biological Chemistr 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.1
Miraculin is a taste modifying plant protein, which displays a peculiar property of being able to modify sour taste into sweet taste. Thus, there has been an increasing interest in miraculin due to this amazing property. In the present study, miraculin gene was introduced into the Miyagawa Wase Satsuma mandarin (Citrus unshiu Marc.) callus by Agrobacterum-mediated transformation to produce a citrus transgenic plant and express the recombinant miraculin protein under the control of 35S promoter. Satsuma mandarin is one of the choicest citrus varieties grown widely and commercially in Korea, especially in Jeju Island, and Japan. Expression of this protein in the transgenic plant resulted in the accumulation of a significant amount of the miraculin protein in the leaves. To investigate whether the expressed protein was correctly modified, the dimerization and N-glycosylation of recombinant miraculin in the transgenic plants were analyzed. The recombinant protein also showed a sufficient biological activity. These results open up a new way of expression system in woody plants such as citrus and can provide a suitable alternative for producing recombinant miraculin.
Mohammad Adnan Al Bachchu,Seong-Beom Jin,Jeong-Won Park,부경환,선현진,Yong-Woo Kim,Hyo-Yeon Lee,Key-Zung Riu,Jae Hoon Kim 한국응용생명화학회 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.1
Miraculin is a taste modifying plant protein, which displays a peculiar property of being able to modify sour taste into sweet taste. Thus, there has been an increasing interest in miraculin due to this amazing property. In the present study, miraculin gene was introduced into the Miyagawa Wase Satsuma mandarin (Citrus unshiu Marc.) callus by Agrobacterum-mediated transformation to produce a citrus transgenic plant and express the recombinant miraculin protein under the control of 35S promoter. Satsuma mandarin is one of the choicest citrus varieties grown widely and commercially in Korea, especially in Jeju Island, and Japan. Expression of this protein in the transgenic plant resulted in the accumulation of a significant amount of the miraculin protein in the leaves. To investigate whether the expressed protein was correctly modified, the dimerization and N-glycosylation of recombinant miraculin in the transgenic plants were analyzed. The recombinant protein also showed a sufficient biological activity. These results open up a new way of expression system in woody plants such as citrus and can provide a suitable alternative for producing recombinant miraculin.
Md. Adnan Al Bachchu,진성범,박정원,선현진,윤수현,이효연,이동선,Quan Chun Hong,김용우,류기중,김재훈 한국원예학회 2011 Horticulture, Environment, and Biotechnology Vol.52 No.2
Agrobacterium-mediated transformation in Satsuma mandarin (Citrus unshiu Marc.) cv. Miyagawa wase was achieved with reasonable transformation efficiency of about 22%, which was the percentage of transgenic plantlets confirmed by genomic PCR (37 plantlets/168 hygromycin-resistant calli). Embryogenic calli of Miyagawa wase were infected with Agrobacterium tumefaciens strain EHA105 harboring binary vector pCAMBIA1300 that contained miraculin gene (a taste-modifying protein) and hygromycin as a selection marker. After 5 days of co-culture in the medium containing 100 μM acetosyringone, calli were transferred to the liquid half embryogenic cell culture medium (half concentration of Murashige and Tucker’s (MT) medium modified with the addition of 500 mg・L-1 malt extract, 50 g・L-1 sucrose and 1.55 g・L-1 glutamine) with 15 mg・L-1 hygromycin and 250 mg・L-1 cefotaxime, and were cultured for two weeks. Then, the calli were grown on the solid selection medium with 20 mg・L-1 hygromycin for four weeks and 25 mg・L-1 hygromycin for another four weeks. The resistant embryos were selected and transferred to the embryo maturation medium. After 3 weeks of culture, the heart shaped embryos were transferred to the MT medium containing 1.0 mg・L-1 GA3, 20.0 mL・L-1 coconut water, 0.02 mg・L-1 NAA and 0.0146 mg・L-1 coumarin for embryo germination. Finally the germinated embryos were cultured on MT medium containing 3.0% sucrose and 0.8% agar for growing to the normal plant. Stable integration of the transgene in the plant genome was confirmed by PCR and Southern blot analysis.
Production of Recombinant Miraculin Protein Using Transgenic Citrus Cell Suspension Culture System
Jin, Seong Beom,Sun, Hyeon Jin,Bachchu, Md Adnan Al,Chung, Sung Jin,Lee, Jongwoo,Han, Song-I,Yun, Jeong Hun,Boo, Kyung Whan,Lee, Dongsun,Riu, Key Zung,Kim, Jae-Hoon The Korean Society for Applied Biological Chemistr 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.3
Miraculin gene containing the N-terminal signal peptide was introduced into navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) callus cells by Agrobacterum-mediated transformation. Transgenic somatic embryos were screened on the shoot induction medium containing 25 mg hygromycin $L^{-1}$. Citrus callus cells were reproduced from the green color somatic embryos on the callus reproduction medium. The obtained transgenic cells were cultured in Murashige and Tucker's liquid medium containing 50 g sucrose $L^{-1}$ in a shaking incubator. Similar to the native miraculin, the secreted recombinant miraculin protein formed a disulfide-linked dimer and retained taste-modifying activity. The stability of recombinant protein expression was confirmed over nine generations of callus. This production system can be an excellent alternative for producing various recombinant proteins as well as miraculin.
Production of Recombinant Miraculin Protein Using Transgenic Citrus Cell Suspension Culture System
Seong-Beom Jin,선현진,Adnan Al Bachchu,정성진,이정우,한송이,윤정훈,부경환,이동선,류기중,김재훈 한국응용생명화학회 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.3
Miraculin gene containing the N-terminal signal peptide was introduced into navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) callus cells by Agrobacterum-mediated transformation. Transgenic somatic embryos were screened on the shoot induction medium containing 25 mg hygromycin L−1. Citrus callus cells were reproduced from the green color somatic embryos on the callus reproduction medium. The obtained transgenic cells were cultured in Murashige and Tucker’s liquid medium containing 50 g sucrose L−1 in a shaking incubator. Similar to the native miraculin, the secreted recombinant miraculin protein formed a disulfide-linked dimer and retained taste-modifying activity. The stability of recombinant protein expression was confirmed over nine generations of callus. This production system can be an excellent alternative for producing various recombinant proteins as well as miraculin.
Expression Profiling of Cultivar-related Genes in Satsuma Mandarins, Miyagawa Wase and Ueno Wase
Jeong-Won Park,부경환,Seong-Beom Jin,Md. Adnan Al Bachchu,Yong-Woo Kim,Dong-Sun Lee,Hyo-Yeon Lee,Key-Zung Riu,Su-Hyun Yun,Jae-Hoon Kim 한국응용생명화학회 2010 Applied Biological Chemistry (Appl Biol Chem) Vol.53 No.6
Satsuma mandarin (Citrus unshiu Marc.) is characterized by its tender peel and seedless nature. The representative cultivar of Satsuma mandarin is Miyagawa wase. Another cultivar, Ueno wase was developed from the bud mutation of a Miyagawa wase tree. Compared to Miyagawa wase fruit, Ueno wase fruit is known to have less albedo, higher sugar content, and to mature earlier. To find genes related to this bud mutation, a citrus 300 k DNA microarray was developed for gene expression analyses of the fruits and leaves of each cultivar. When up-regulated genes were classified in the Cluster of Orthologous Groups of proteins database, the number of genes included differed significantly between the two cultivars. Ten genes, including cytochrome P450, α-mannosidase, S-adenosylmethionine decarboxylase, carboxyesterase, anthranilate synthase, glycosyl hydrolase family-17,ethylene response factor subfamily, and alcohol dehydrogenase,showing significant differences in gene expression by DNA microarray were selected, and their expression patterns were examined by RT-PCR. The information gained from genes that are up (or down)-regulated upon bud mutation may explain the observed phenotypes, while also increasing the understanding of genes related to citrus bud mutation.
Expression Profiling of Cultivar-related Genes in Satsuma Mandarins, Miyagawa Wase and Ueno Wase
Park, Jeong-Won,Boo, Kyung-Hwan,Jin, Seong-Beom,Al Bachchu, Md. Adnan,Yun, Su-Hyun,Kim, Yong-Woo,Lee, Dong-Sun,Lee, Hyo-Yeon,Riu, Key-Zung,Kim, Jae-Hoon The Korean Society for Applied Biological Chemistr 2010 Applied Biological Chemistry (Appl Biol Chem) Vol.53 No.6
Satsuma mandarin (Citrus unshiu Marc.) is characterized by its tender peel and seedless nature. The representative cultivar of Satsuma mandarin is Miyagawa wase. Another cultivar, Ueno wase was developed from the bud mutation of a Miyagawa wase tree. Compared to Miyagawa wase fruit, Ueno wase fruit is known to have less albedo, higher sugar content, and to mature earlier. To find genes related to this bud mutation, a citrus 300 k DNA micro array was developed for gene expression analyses of the fruits and leaves of each cultivar. When up-regulated genes were classified in the Cluster of Orthologous Groups of proteins database, the number of genes included differed significantly between the two cultivars. Ten genes, including cytochrome P450, ${\alpha}$-mannosidase, S-adenosylmethionine decarboxylase, carboxyesterase, anthranilate synthase, glycosyl hydrolase family-17, ethylene response factor subfamily, and alcohol dehydrogenase, showing significant differences in gene expression by DNA micro array were selected, and their expression patterns were examined by RT-PCR. The information gained from genes that are up (or down)-regulated upon bud mutation may explain the observed phenotypes, while also increasing the understanding of genes related to citrus bud mutation.
Expression Profiling of Cultivar-related Genes in Satsuma Mandarins, Miyagawa Wase and Ueno Wase
Jae Hoon Kim,Jeong Won Park,Kyung Hwan Boo,Seong Beom Jin,Md. Adnan Al Bachchu,Su Hyun Yun,Yong Woo Kim,Dong Sun Lee,Hyo Yeon Lee,Key Zung Riu 한국응용생명화학회 2010 Applied Biological Chemistry (Appl Biol Chem) Vol.53 No.6
Comparative Analysis among Four Citrus Species by DNA Microarray
Jeong Won Park,Seong Beom Jin,Kyung Hwan Boo,Sung Jin Chung,Su Hyun Yun,Adnan Al Bachchu,Jeong Hun Yun,Song I Han,Key Zung Riu,Jae Hoon Kim 한국육종학회 2012 한국육종학회지 Vol.44 No.3
Citrus is one of the major fruits produced in Korea. There are about 20 species mainly grown in Jeju Island, Korea. Four representative species, which are quite different in the shape of leaf and the taste of fruit, were selected and were used to profile the transcriptomes. These species are ‘Miyagawa Wase’ (C. unshiu Marcov.) satsuma mandarin, ‘Kiyomi’ (C. unshiu Marcov. x C. sinensis) mandarin hybrid, ‘Dangyuja’ (C. grandis) and ‘Natsudaidai’ (C. natsudaidai). Classification of the up-regulated and down-regulated genes using the Cluster of Orthologous Groups of proteins (COG) database reveals that the number of genes included in each group differed significantly among the four species. Several genes that showed significant differences in expression on the microarray were selected and their expression patterns were examined by reverse transcription- ploymerase chain reaction. Metabolic genes such as tyrosine decarboxylase and β-glucosidase ligase were found to be highly expressed in Miyagawa Wase, relative to other species. On the other hand, the expression level of mannose phosphate isomerase was lower in Miyagawa Wase. An efflux pump gene was found to be up-regulated in Kiyomi, whereas cinnamyl-alcohol dehydrogenase was down-regulated. β-carotene 15,15’-dioxygenase, which is involved in the vitamin metabolism, was up-regulated in Natsudaidai. Interspecific differentiations of gene expression are analyzed in terms of the metabolic pathways and their possible roles in citrus species.
( Hyo Yeon Lee ),( Jae Hoon Kim ),( Jeong Won Park ),( Key Zung Riu ),( Yong Woo Kim ),( Seong Beom Jin ),( Kyung Hwan Boo ),( Hyeon Jin Sun ),( Mohammad Adnan Al Bachchu ) 한국응용생명화학회 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.1
Miraculin is a taste modifying plant protein, which displays a peculiar property of being able to modify sour taste into sweet taste. Thus, there has been an increasing interest in miraculin due to this amazing property. In the present study, miraculin gene was introduced into the Miyagawa Wase Satsuma mandarin (Citrus unshiu Marc.) callus by Agrobacterum-mediated transformation to produce a citrus transgenic plant and express the recombinant miraculin protein under the control of 35S promoter. Satsuma mandarin is one of the choicest citrus varieties grown widely and commercially in Korea, especially in Jeju Island, and Japan. Expression of this protein in the transgenic plant resulted in the accumulation of a significant amount of the miraculin protein in the leaves. To investigate whether the expressed protein was correctly modified, the dimerization and N-glycosylation of recombinant miraculin in the transgenic plants were analyzed. The recombinant protein also showed a sufficient biological activity. These results open up a new way of expression system in woody plants such as citrus and can provide a suitable alternative for producing recombinant miraculin.