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Agrobacterium tumefaciens-mediated Hairy Root Production from Seedlings of Chinese Cabbage
( Yun Xiang Zang ),( Doo Hwan Kim ),( Seung Beom Hong ) 한국식물학회 2008 Journal of Plant Biology Vol.51 No.4
Cruciferous hairy roots are often used for improving drought adaptability, peroxidase production, and in vitro subculturing of Plasmodiophora brassicae. For metabolic engineering, Agrobacterium tumefaciens-mediated systems have previously been developed for hairy root production in other plant species. Here, we used the rolABC gene binary construct in A. tumefaciens strain GV3101 to establish cultures of Chinese cabbage hairy roots. On both solid and liquid media, the rolABC hairy root lines exhibited a wild-type hairy root syndrome in terms of their growth and morphology. This demonstrates that those three genes are sufficient to induce high-quality hairy roots in Chinese cabbage. Such a system could be useful for the stable production of secondary metabolites in that species.
Yun Xiang Zang,Seung Beom Hong,Dhandapani Murugesan,Jong Gu Lim,Doo Hwan Kim 한국원예학회 2006 Horticulture, Environment, and Biotechnology Vol.47 No.4
Two common bermudagrass (Cynodon dactylon L. Pers.) cultivars, ‘Sundevil Ⅱ’ and ‘Mirage’, were used to improve tissue culture responses in embryogenesis and regeneration. Embryogenic calli were induced from the seeds of the two cultivars. Callus started forming at the base of scutellum of germinating seeds about 7 days after culturing in callus induction medium. Callus induced on MS medium with 2 ㎎ㆍL?¹ -12,4-dichlorophenoxyacetic acid only was not regenerable. However, when 0.1 to 0.5 ㎎ㆍL?¹ 6-benzylaminopurine was added to the above medium, compact and yellowish callus formed and was regenerable. The induced embryogenic callus produced shoots about 5 days after transferring to regeneration medium with regeneration rates of more than 70% for the two cultivars. All of the regenerated plants rooted within 3 days and were morphologically normal after transferring to rooting medium.
Yun-Xiang Zang,Doo-Hwan Kim,박범석,홍승범 한국생물공학회 2009 Biotechnology and Bioprocess Engineering Vol.14 No.4
Indole glucosinolates (IG), a group of secondary metabolites found almost extensively in the order Brassicales, play an important role in the interaction between plant and insect or microorganism. In order to explore the possibility of IG metabolic engineering in Chinese cabbage hairy roots, three Arabidopsis cDNAs CYP79B2, CYP79B3, and CYP83B1 combined with rolABC were introduced into Chinese cabbage by Agrobacterium-mediated transformation. Overexpression of CYP79B2, CYP79B3, or CYP83B1 alone did not affect the accumulation levels of IG in transgenic hairy roots. However, when CYP83B1 was overexpressed together with CYP79B2 and/or CYP79B3, some of the transgenic hairy roots accumulated higher levels of glucobrassicin (GBC) or 4-methoxy glucobrassicin (4-OMeGBC) than control hairy root line carrying rolABC vector. With regard to the IG accumulation, overexpression of all three cDNAs showed no better than overexpression of both cDNAs. Both 4-OMeGBC and neoglucobrassicin (neo-GBC) were found to be the main components of IG that comprise about 90% of total IG in all types of Chinese cabbage hairy root lines. In transgenic hairy root lines rB3B1-8 and rB2B1B3-5, 4-OMeGBC increased to 2 and 1.5-fold, while neo-GBC decreased to 0.5 and 0.6-fold, respectively. This suggests that an increased production of 4-OMeGBC causes a reduction of neo-GBC level since the two types derive from a common precursor GBC. However, in terms of the total IG level, the transgenic hairy roots did not show significant differences from controls.
Yun-Xiang Zang,Myung-Ho Lim,Beom-Seok Park,홍승범,Doo Hwan Kim 한국분자세포생물학회 2008 Molecules and cells Vol.25 No.2
Indole glucosinolates (IG) play important roles in plant defense, plant-insect interactions, and stress responses in plants. In an attempt to metabolically engineer the IG pathway flux in Chinese cabbage, three important Arabidopsis cDNAs, CYP79B2, CYP79B3, and CYP83B1, were introduced into Chinese cabbage by Agrobacterium-mediated transformation. Overexpression of CYP79B3 or CYP83B1 did not affect IG accumulation levels, and overexpression of CYP79B2 or CYP79B3 prevented the transformed callus from being regenerated, displaying the phenotype of indole-3-acetic acid (IAA) overproduction. However, when CYP83B1 was overexpressed together with CYP79B2 and/or CYP79B3, the transformed calli were regenerated into whole plants that accumulated higher levels of glucobrassicin, 4-hydroxy glucobrassicin, and 4-methoxy glucobrassicin than wild-type controls. This result suggests that the flux in Chinese cabbage is predominantly channeled into IAA biosynthesis so that coordinate expression of the two consecutive enzymes is needed to divert the flux into IG biosynthesis. With regard to IG accumulation, overexpression of all three cDNAs was no better than overexpression of the two cDNAs. The content of neoglucobrassicin remained unchanged in all transgenic plants. Although glucobrassicin was most directly affected by overexpression of the transgenes, elevated levels of the parent IG, glucobrassicin, were not always accompanied by increases in 4-hydroxy and 4- methoxy glucobrassicin. However, one transgenic line producing about 8-fold increased glucobrassicin also accumulated at least 2.5 fold more 4-hydroxy and 4- methoxy glucobrassicin. This implies that a large glucobrassicin pool exceeding some threshold level drives the flux into the side chain modification pathway. Aliphatic glucosinolate content was not affected in any of the transgenic plants.
Identification and characterization of the phytocystatin family from Brassica rapa
홍준기,황정은,박태호,Yun-Xiang Zang,이상춘,권수진,문정환,김현욱,김진아,진미나,김정선,이수인,임명호,허윤강,임채오,박범석 한국식물생명공학회 2008 식물생명공학회지 Vol.35 No.4
Phytocystatins, which are inhibitors of plant cysteine peptidases, are involved in the regulation of protein turnover and in the defense against insect pests and pathogens. Extensive searches in the Brassica rapa genome allowed the prediction of at least eight different phytocystatin genes on seven chromosomes in the B. rapa genome. Structure comparisons based on alignments of the all BrCYS (B. rapa phytocystatin) proteins using the CLUSTALW program revealed conservation of the three consensus motifs known to interact with the active site of cysteine peptidases. According to the phylogenetic analysis based on the deduced amino acid sequences, the eight BrCYS proteins were divided into several clusters related to the orthologous phytocystatin. The predicted three-dimensional structure models of the eight BrCYS proteins demonstrate that all of these proteins are similar to the reported crystal structure of oryzacystatin-I (OC-I). Digital northern and RT-PCR analyses indicated that the eight BrCYS genes exhibit different expression patterns in B. rapa tissues and respond differently to abiotic stimuli. The differences in gene structure and expression between the eight BrCYS genes suggest that these proteins may play diverse physiological roles in B. rapa and may interact with cysteine peptidases through different mechanisms.
Identification and characterization of the phytocystatin family from Brassica rapa
Hong, Joon-Ki,Hwang, Jung-Eun,Park, Tae-Ho,Zang, Yun-Xiang,Lee, Sang-Choon,Kwon, Soo-Jin,Mun, Jeong-Hwan,Kim, Hyun-Uk,Kim, Jin-A,Jin, Mi-Na,Kim, Jung-Sun,Lee, Soo-In,Lim, Myung-Ho The Korean Society of Plant Biotechnology 2008 식물생명공학회지 Vol.35 No.4
Phytocystatins, which are inhibitors of plant cysteine peptidases, are involved in the regulation of protein turnover and in the defense against insect pests and pathogens. Extensive searches in the Brassica rapa genome allowed the prediction of at least eight different phytocystatin genes on seven chromosomes in the B. rapa genome. Structure comparisons based on alignments of the all BrCYS ($\underline{B}$. $\underline{r}apa$ $phyto{\underline{cys}}tatin$) proteins using the CLUSTALW program revealed conservation of the three consensus motifs known to interact with the active site of cysteine peptidases. According to the phylogenetic analysis based on the deduced amino acid sequences, the eight BrCYS proteins were divided into several clusters related to the orthologous phytocystatin. The predicted three-dimensional structure models of the eight BrCYS proteins demonstrate that all of these proteins are similar to the reported crystal structure of oryzacystatin-I (OC-I). Digital northern and RT-PCR analyses indicated that the eight BrCYS genes exhibit different expression patterns in B. rapa tissues and respond differently to abiotic stimuli. The differences in gene structure and expression between the eight BrCYS genes suggest that these proteins may play diverse physiological roles in B. rapa and may interact with cysteine peptidases through different mechanisms.