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Review: Plant Binary Vectors of Ti Plasmid in Agrobacterium tumefacienswith a Broad Host-Range Replicon of pRK2, pRi, pSa or pVS1

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American Journal of Plant Sciences 2013年第4期4卷 932-939页

作者： norimoto murai Department of Plant Pathology and Crop Physiology Louisiana State University and LSU AgCenter Baton Rouge USA

This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli. A general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues. A second trend is to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in E. coli. The broad host-range replicon of VS1 was shown to be a choice of replicon over those of pRK2, pRi and pSA because of the superior stability and of small well-defined replicon. Newly developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene, and the T-DNA region (152 bp).

关键词： Agrobacterium tumefaciens Binary Vectors pRK2 pRi pSA pVS1 T-DNA Ti Plasmid

Review: Plant Growth Hormone Cytokinins Control the Crop Seed Yield

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American Journal of Plant Sciences 2014年第14期5卷 2178-2187页

作者： norimoto murai Department of Plant Pathology and Crop Physiology Louisiana State University and LSU Agricultural Center Baton Rouge USA

This review chronicles the development of the cytokinin research during the last 30 years. Cytokinin and auxin are the two major plant growth hormones that control virtually all aspects of growth and development in higher plants. The pathways for cytokinin biosynthesis and metabolism have been characterized by the identification of isopentenyl pyrophosphate transferase, cytokinin oxidases, cytokinin hydroxylase, zeatin cis-/trans-isomerase, cytokinin phosphoribosyl hydrolases, cytokinin-specific riboside phosphorylase, and others enzymes. Loss-of function mutant phenotypes of cytokinin degradation/activating enzymes indicate the regulation of concentration and spatial distribution of bio-active cytokinin plays a pivotal role in the increase in panicle size, in the numbers of floral organs, and eventually in seed yield. One of the most fundamental questions in the cytokinin field is one concerning the prevalence of cis-zeatin in monocotyledonous crops (rice and maize) and in dicotyledonous legumes (pea, chickpea) and potato/sweet potato. A hypothesis is that cis-zeatin is synthesized by the cis-specific hydroxylation of the terminal methyl group of N6-isopentenyl side chain of N6-isopentenyl adenosine (i6Ado) or of their mono-, di-, or tri-phosphates catalyzed by the cis-specific hydroxylase. A second potential pathway is the isomerization of trans-zeatin to cis-zeatin by zeatin cis-/trans-isomerase. A second fundamental question to be addressed is the physiological role of cis-zeatin. Some have argued for a special function of cis-zeatin to account for the prevalence of the cis-zeatin in the plant kingdom from algae to higher plants.

关键词： Cytokinins Biosynthesis and Metabolism cis-and trans-Zeatin Cytokinin Oxidase

Low Co-Cultivation Temperature at 20°C Resulted in the Reproducible Maximum Increase in Both the Fresh Weight Yield and Stable Expression of GUS Activity after Agrobacterium tumefaciens-Mediated Transformation of Tobacco Leaf Disks

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American Journal of Plant Sciences 2012年第4期3卷 537-545页

作者： Guiying Su Sunjung Park Seokhyun Lee norimoto murai Department of Plant Pathology and Crop Physiology Louisiana State University and LSU AgCenter Baton Rouge USA

The importance of controlled temperature during the four-days co-cultivation period was evaluated under the most physiologically relevant conditions for Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cv. Xanthi (nn, Smith)) leaf disks. We compared the effect of temperatures ranging from 15°C, 18°C, 20°C, 22°C to 25°C on the stable expression of β-glucuronidase (GUS) activity of 14 days old hygromycin-selected leaf disks, and on the increase in the fresh weight yield of 28 days old kanamycin-selected calli. The highest average of GUS activity was obtained at 20°C among the five temperatures tested although the difference between the 18°C and 20°C treatment was not statistically significant. The GUS activity at 15°C was statistically lower than those at 18°C and 20°C. The GUS activity in 22°C treatment was an intermediate between the highest (18/20°C) and second highest averages (15°C), and was not statistically significantly different. The lowest average of GUS activity was observed at 25°C. The highest increase in the plate average of fresh weight yield was obtained at 20°C among the five temperature tested. The 20°C treatment was statistically significantly better than the 15°C and 18°C treatments. The 20°C co-cultivation treatment resulted in the higher FW yield than 22°C and 25°C even though the differences were not statistically significant. In conclusion, low co-cultivation temperature at 20°C resulted in the reproducible maximum increase in both the fresh weight yield and stable expression of GUS activity after transformation of tobacco leaf disks.

关键词： Agrobacterium tumefaciens Co-Cultivation Temperature Fresh Weight Yield Stable GUS Gene Expression Tobacco Leaf Disks Transformation

Tetracycline-Based Binary Ti Vectors pLSU with Efficient Cloning by the Gateway Technology for Agrobacterium tumefaciens-Mediated Transformation of Higher Plants

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American Journal of Plant Sciences 2013年第7期4卷 1418-1426页

作者： Seokhyun Lee Guiying Su Eric Lasserre norimoto murai Department of Plant Pathology and Crop Physiology Louisiana State University and LSU AgCenter Baton Rouge USA

We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated transformation of higher plants. The Gateway Technology vectors are kanamycin-based, thus tetracycline-based destination and expression vectors are easily selected for the antibiotic resistance in the Escherichia coli media. We reduced the size of the tetracycline resistance gene TetC from pBR322 to 1468 bp containing 1191 bp of the coding region, 93 bp of 5’-upstream, and 184 bp 3’-downstream region. The final size of binary Ti vector skeleton pLSU11 is 5034 bp. pLSU12 and 13 have the kanamycin resistance NPTII gene as a plant-selectable marker. pLSU13?and 15 contain the hygromycin resistance HPH gene as a selection marker. pLSU13 and 15 also have the β-glucuronidase (GUS) reporter gene in addition to the plant selection marker. We also constructed a mobilizable version of tetracycline-based binary Ti vector pLSU16 in which the mob function of ColE1 replicon was maintained for mobilization of the binary vector from E. coli to A. tumefaciens by tri-parental mating. The final size of binary Ti vector skeleton pLSU16 is 5580 bp. New tetracycline- based binary Ti vectors pLSU12 were found as effective as kanamycin-based vector pLSU2 in promoting a 10-fold increase in fresh weight yield of kanamycin-resistant calli after A. tumefaciens-mediated transformation of tobacco leaf discs. Using the Gateway Technology we introduced the plant-expressible GUSgene to the T-DNA of binary Ti vector pLSU12. Expression of the β-glucuronidase enzyme activity was demonstrated by histochemical staining of the GUS activity in transformed tobacco leaf discs.

关键词： Agrobacterium tumefaciens Binary Ti Vectors Gateway Technology pLSU Tobacco Leaf Disk Transformation Tetracycline Resistance

Sweet Potato Leaf Curl Virus: Coat Protein Gene Expression in Escherichia coliand Product Identification by Mass Spectrometry

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American Journal of Plant Sciences 2015年第19期6卷 3013-3024页

作者： Dina Lida Gutierrez Reynoso Rodrigo A. Valverde norimoto murai Department of Plant Pathology & Crop Physiology College of Agriculture and LSU Agricultural Center Louisiana State University Baton Rouge USA

Sweet potato is one of the first natural GMOs, genetically modified 8000 years ago by Agrobacterium rhizogenes as reported recently by Kyndt et al. A section of 10 kbp long DNA (Transferred- DNA or T-DNA) of the Ri (Root-inducing) plasmid was transferred to the plant genome by A. rhizo-genes and has been maintained in all 291 hexaploid sweet potato cultivars of the world. The maintenance in the sweet potato genome and expression of two T-DNA genes for tryptophan-2-monooxygenease (iaaM) and for indole-3-acetamide hydrolase (iaaH) are likely to be physiologically significant since these enzymes convert tryptophan to indole-3-acetic acid, a major plant growth hormone auxin. Sweet potato (Ipomoea batatas (L.) Lam) is ranked the third most important root crop after potato and cassava, and the seventh in global food crop production with more than 126 million metric tons. Although sweet potato originated in Central or South America, China currently produces over 86% of world production with 109 million metric tons. In the United States, North Carolina is the leading producer with 38.5% of the 2007 sweet potato production, followed by California, Mississippi, and Louisiana with 23%, 19%, and 15.9%, respectively. Leaf curl virus diseases have been reported in sweet potato throughout the world. One of the causal agents is Sweet potato leaf curl virus (SPLCV) belonging to the genus Begomovirus (family Geminiviridae). Although SPLCV does not cause symptoms on Beauregard, one of the most predominant sweet potato cultivars in the US, it can reduce the yield up to 26%. Serological detection of SPLCV is not currently available due to the difficulties in obtaining purified virions that can be used as antigen for antiserum production. In attempts to obtain the coat protein (CP) of SPLCV for antibody production, primers were designed to amplify the CP gene. This gene was cloned into the expression vector pMAL-c2E as a fusion protein with maltose-binding protein, and transformed into E

关键词： Affinity Chromatography Purification Coat Protein Escherichia coli Mass Spectrometry Maltose Binding Protein Sweet Potato Leaf Curl Virus

Modified Bean Seed Protein Phaseolin Did Not Accumulate Stably in Transgenic Tobacco Seeds after Methionine Enhancement Mutations

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American Journal of Plant Sciences 2015年第5期6卷 640-650页

作者： Eric Lasserre T. S. Ko John M. Dyer norimoto murai Department of Plant Pathology and Crop Physiology Louisiana State University and LSU Agricultural Center Baton Rouge LA USA Laboratoire Genome et Developement des Plantes Universite de Perpignan Perpignan France Plant Physiology and Genetic Research Unit US Arid-Land Agricultural Research Center USDA-ARS Maricopa AZ USA

The major seed storage protein phaseolin of common bean (Phaseolus vulgaris L.) is deficient in methionine, an essential amino acid for human and animal health. To improve the nutritional quality of common bean, we designed methionine enhancement of phaseolin based on the three-dimensional structure of protein, de novo design principles and genetic information. Amino acid substitution and loop insertion were targeted to the interior and exterior, respectively, of the protein’s β-barrels. First, we introduced the methionine enhancement mutations into phaseolin cDNA, expressed cDNA in Escherichia coli and purified monomeric non-glycosylated proteins. Biophysical analysis of E. coli-expressed proteins demonstrated a similar structural stability of wild-type and mutant phaseolin monomers. Here, we attempted to test the structural stability of the methionine-enhanced phaseolin by introducing phaseolin cDNA to tobacco via Agrobacterium tumefaciens-mediated transformation of leaf disks, regenerating transgenic tobacco plants, and examining the accumulation of phaseolin protein in mature transgenic tobacco seeds. We used seven constructs containing different extents of methionine enhancement, ranging from the original 3 to maximum 33 methionines per 397 amino acid residues. ELISA analyses indicated that the methionine-enhanced phaseolins did not accumulate as stably in mature transgenic tobacco seeds as the wild-type phaseolin. It seems likely that the methionine-enhanced phaseolin proteins were under the stringent scrutiny of the protein quality control mechanism in the endoplasmic reticulum (ER), Golgi complex and/or vacuolar protein bodies. The protein degradation is probably to occur in the vacuolar protein bodies due to the instability of the trimer assembly caused by the methionine enhancement mutations targeting either amino-acids substitutions or/and loop insertions to the interior β-sheets and tum/loop regions, respectively, of N- and C-barrel structures.

关键词： Common Bean Glycosylation Methionine Enhancement Phaseolin Polypeptide Folding Trimer Assembly

Design and strategy for manufacturing kidney organoids

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Bio-Design and Manufacturing 2020年第1期3卷 7-14页

作者： Masaki Nishikawa Yasuyuki Sakai norimoto Yanagawa Department of Chemical System Engineering School of EngineeringUniversity of TokyoRm.5A01Engineering Bldg.3Hongo 7‑3‑1BunkyoTokyo 113‑8656Japan Medical and Research Services Greater Los Angeles Veterans Affairs Healthcare System at SepulvedaNorth HillsCAUSA David Geffen School of Medicine University of California at Los AngelesLos AngelesCAUSA

Despite a continuing increase in the number of patients suffering from chronic kidney disease,currently available treatments for these patients,such as dialysis and kidney transplantation,are *** kidney is also a critical target organ vulnerable to the toxicity of various new drugs,and the lack of a reliable in vitro culture model imposes a severe limitation on drug *** the development of induced pluripotent stem cells(iPSCs)revolutionized strategies in biomedical fields,the complexity of the kidney imposed additional challenge to the application of this technology in kidney ***,the recent advancement in our understanding on the developmental origin of kidney progenitor cells and the mechanisms of their reciprocal induction and self-organization has boosted research in kidney *** since then has demonstrated that kidney organoids derived from iPSCs can serve as a useful model for drug discovery and toxicity screening,as well as for disease modeling,especially in combination with gene editing ***,attempts at kidney organoid implantation in animals have suggested their potential as an alternative source of kidney *** this review,we summarize recent progress on the generation of kidney organoids,as well as the obstacles that remain.

关键词： Kidney organoid Kidney development iPSC Progenitor cell Self-organization Regenerative medicine

Rare and Precious Chinese Materia Medica: Pseudobulbus Cremastrae seu Pleiones

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Chinese Medicine and Culture 2021年第4期4卷 211-220页

作者： Hisayoshi norimoto Chiaki Murayama Feng Zhao Hong-Yan Wei Association of Promoting Sustainable Use of Medicinal Resources Kusazu CityShiga 525-0058Japan PuraPharm International Ltd. Hong Kong Science ParkHong Kong 999077China PuraPharm Japan Corporation Toyama CityToyama 930-0866Japan PuraPharm Research Institute Pharmaceutical Co.Ltd.Nanning 5300007GuangxiChina Gold Sparke(Guizhou)Chinese Medicine Co. Ltd.Danzhai 557500GuizhouChina

Shan Ci Gu(山慈菇Pseudobulbus Cremastrae seu Pleiones),a rare and precious traditional Chinese medicine,has attracted attention for the treatment of various cancers and bacterial *** to the Pharmacopoeia of the People's Republic of China,Pseudobulbus Cremastrae seu Pleionesis sourced from the pseudobulbs of three plants in the Orchidaceae family:Cremastra appendiculata(***)Makino,Pleione bulbocodioides(Franch.)Rolfe,and Pleione yunnanensis *** from Pseudobulbus Cremastrae seu Pleiones are used for the treatment of tumors,burns,and *** aims of this review are to provide information on the historical and herbological origins of Pseudobulbus Cremastrae seu Pleiones,to summarize research conducted on its phytochemical and biological activities over the last twenty years,and to detail planting efforts.

关键词： Biological activity Cremastra appendiculata phytochemistry planting Pleione bulbocodioides Pleione yunnanensis Shan Ci Gu Pseudobulbus Cremastrae seu Pleiones

Identification of HPLC Characteristic Chromatograms of Phenylethanoid Glycosides and Iridoid Glycosides in Herba Cistanche of Different Origins

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Medicinal Plant 2020年第5期11卷 41-43,48页

作者： Xiongmei HUANG Yan HUANG Xinwei NONG Minling WEI Hongyan WEI Hisayoshi norimoto PuraPharm(Nanning)Pharmaceutical Co. Ltd.Nanning 530007China

[Objectives]This study aimed to establish a quality evaluation method for Herba Cistanche based on the HPLC characteristic chromatograms of the phenylethanoid glycosides and iridoid glycosides,and compare the HPLC characteristic chromatograms of different origins of Herba Cistanche.[Methods]The chromatographic conditions used were as follows:column,Agilent ZORBAX Eclipse Plus C18 column(4.6 mm×250 mm,5μm);gradient elution,acetonitrile-0.1%phosphoric acid aqueous solution;flow rate,1.0 mL/min;detection wavelength,240 nm;column temperature,30℃.[Results]Cistanche tubulosa(Schenk)Wight showed 9 characteristic peaks,Cistanche deserticola *** showed 10 characteristic peaks,Cistanche salsa(***.)*** showed 7 characteristic peaks,and Cistanche sinensis *** showed 4 characteristic *** 6 was echinacoside,peak 7 was verbascoside,peak 8 was tubuloside A,peak 9 was isoacteoside and peak 10 was cistanoside *** characteristic chromatograms of the four different origins of Herba Cistanche were significantly different.[Conclusions]This characteristic chromatogram method has good reproducibility and can be used to distinguish 4 different origins of Herba Cistanche,*** ***,***(Schenk)Wight,*** *** and ***(***.)***.

关键词： Cistanche deserticola Y.C.Ma Cistanche tubulosa(Schenk)Wight Cistanche sinensis G.Beck Cistanche salsa(C.A.Mey.)G.Beck Characteristic chromatogram HPLC