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Kaiwun Yeh - One of the best experts on this subject based on the ideXlab platform.
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Neomycin: An Effective Inhibitor of Jasmonate-Induced Reactions in Plants
Journal of Plant Growth Regulation, 2019Co-Authors: Jyothilakshmi Vadassery, Kaiwun Yeh, Meredith C Schuman, Daniel Ballhorn, Steven Fleming, Christian Mazars, Shree P. Pandey, Axel Schmidt, Ayufu Yilamujiang, Axel MithoferAbstract:Jasmonates are important phytohormones involved in both plant developmental processes as well as defense reactions. Many JA-mediated plant defense responses have been studied in model plants using mutants of the jasmonate signaling pathway. However, in plant species where JA-signaling mutants are not accessible, the availability of a tool targeting JA signaling is crucial to investigate jasmonate-dependent processes. Neomycin is a poly-cationic aminoglycoside antibiotic that blocks the release of Ca 2+ from internal stores. We examined the inhibitory activities of neomycin on different jasmonate-inducible responses in eight different plant species: Intracellular calcium measurements in Nicotiana tabacum cell culture, Sporamin gene induction in Ipomoea batatas, PDF2.2 gene expression in Triticum aestivum, Nepenthesin protease activity measurement in Nepenthes alata, extrafloral nectar production in Phaseolus lunatus, nectary formation in Populus trichocarpa, terpene accumulation in Picea abies, and secondary metabolite generation in Nicotiana attenuata. We are able to show that neomy-cin, an easily manageable and commercially available compound, inhibits JA-mediated responses across the plant kingdom.
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sweet potato nac transcription factor ibnac1 upregulates Sporamin gene expression by binding the swre motif against mechanical wounding and herbivore attack
Plant Journal, 2016Co-Authors: Shipeng Chen, Winnie I Lin, Xuanyang Chen, Yinhao Huang, Shiaochi Chang, Kaiwun YehAbstract:Sporamin is a tuberous storage protein with trypsin inhibitory activity in sweet potato (Ipomoea batatas Lam.), which accounts for 85% of the soluble protein in tubers. It is constitutively expressed in tuberous roots but is expressed in leaves only after wounding. Thus far, its wound-inducible signal transduction mechanisms remain unclear. In the present work, a 53-bp DNA region, Sporamin wound-response cis-element (SWRE), was identified in the Sporamin promoter and was determined to be responsible for the wounding response. Using yeast one-hybrid screening, a NAC domain protein, IbNAC1, that specifically bound to the 5'-TACAATATC-3' sequence in SWRE was isolated from a cDNA library from wounded leaves. IbNAC1 was constitutively expressed in root tissues and was induced earlier than Sporamin following the wounding of leaves. Transgenic sweet potato plants overexpressing IbNAC1 had greatly increased Sporamin expression, increased trypsin inhibitory activity, and elevated resistance against Spodoptera litura. We further demonstrated that IbNAC1 has multiple biological functions in the jasmonic acid (JA) response, including the inhibition of root formation, accumulation of anthocyanin, regulation of aging processes, reduction of abiotic tolerance, and overproduction of reactive oxygen species (ROS). Thus, IbNAC1 is a core transcription factor that reprograms the transcriptional response to wounding via the JA-mediated pathway in sweet potato.
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differential activation of Sporamin expression in response to abiotic mechanical wounding and biotic herbivore attack in the sweet potato
BMC Plant Biology, 2014Co-Authors: Senthilkumar Rajendran, Iwinnie Lin, Meiju May Chen, Chienyu Chen, Kaiwun YehAbstract:Plants respond differently to mechanical wounding and herbivore attack, using distinct pathways for defense. The versatile sweet potato Sporamin possesses multiple biological functions in response to stress. However, the regulation of Sporamin gene expression that is activated upon mechanical damage or herbivore attack has not been well studied. Biochemical analysis revealed that different patterns of Reactive oxygen species (ROS) and antioxidant mechanism exist between mechanical wounding (MW) and herbivore attack (HA) in the sweet potato leaf. Using LC-ESI-MS (Liquid chromatography electrospray ionization mass spectrometry analysis), only the endogenous JA (jasmonic acid) level was found to increase dramatically after MW in a time-dependent manner, whereas both endogenous JA and SA (salicylic acid) increase in parallel after HA. Through yeast one-hybrid screening, two transcription factors IbNAC1 (no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC)) and IbWRKY1 were isolated, which interact with the Sporamin promoter fragment of SWRE (Sporamin wounding-responsive element) regulatory sequences. Exogenous application of MeJA (methyl jasmonate), SA and DIECA (diethyldithiocarbamic acid, JAs biosynthesis inhibitor) on sweet potato leaves was employed, and the results revealed that IbNAC1 mediated the expression of Sporamin through a JA-dependent signaling pathway upon MW, whereas both IbNAC1 and IbWRKY1 coordinately regulated Sporamin expression through JA- and SA-dependent pathways upon HA. Transcriptome analysis identified MYC2/4 and JAZ2/TIFY10A (jasmonate ZIM/tify-domain), the repressor and activator of JA and SA signaling among others, as the genes that play an intermediate role in the JA and SA pathways, and these results were further validated by qRT-PCR (quantitative real-time polymerase chain reaction). This work has improved our understanding of the differential regulatory mechanism of Sporamin expression. Our study illustrates that sweet potato Sporamin expression is differentially induced upon abiotic MW and biotic HA that involves IbNAC1 and IbWRKY1 and is dependent on the JA and SA signaling pathways. Thus, we established a model to address the plant-wounding response upon physical and biotic damage.
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multiple biological functions of Sporamin related to stress tolerance in sweet potato ipomoea batatas lam
Biotechnology Advances, 2012Co-Authors: Rajendran Senthilkumar, Kaiwun YehAbstract:The initial investigation of the nature of the proteins in the tuber of sweet potato (Ipomoea batatas Lam.) revealed a globulin-designated "ipomoein," which was reported by Jones and Gersdorff, (1931). Later, "ipomoein" was renamed "Sporamin" and was found to be a major storage protein that accounted for over 80% of the total protein in the tuberous root. To date, Sporamin has been studied by a series of biochemical and molecular approaches. The first purification of Sporamin into two major fractions, A and B, was successfully completed in 1985. Several characteristics of the protein, such as the diversification of the nucleotide sequences in the gene family, the protein structure, the biological functions of storage, defense, inhibitory activity and ROS scavenging, were identified. In the past decade, Sporamin was classified as a Kunitz-type trypsin inhibitor, and its insect-resistance capability has been examined in transgenic tobacco and cauliflower plants, indicating the multiple functions of this protein has evolved to facilitate the growth and development of sweet potato. Sporamin is constitutively expressed in the tuberous root and is not normally expressed in the stem or leaves. However, this protein is expressed systemically in response to wounding and other abiotic stresses. These dual expression patterns at the transcriptional level revealed that the complex regulatory mechanism of Sporamin was modulated by environmental stresses. The versatile functions of Sporamin make this storage protein a good research model to study molecular evolution, regulatory mechanisms and physiological functions in plants. This review summarizes and discusses recent approaches and future perspectives in agricultural biotechnology.
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new gene construction strategy in t dna vector to enhance expression level of sweet potato Sporamin and insect resistance in transgenic brassica oleracea
Plant Science, 2006Co-Authors: Huaiju Chen, Shujen Wang, Chiencheng Chen, Kaiwun YehAbstract:Sporamin, an abundant storage protein in tuberous roots of sweet potato, possesses strong inhibitory activity against trypsin and pest-resistance. To promote consistent high-level expression of Sporamin and insect resistance in transgenic Brassica plants, a wound-responsive Sporamin promoter (Pspoa) alone or combined with matrix-attached-region-like DNA segment (spoMAR) were constructed for driving Sporamin cDNA. The results showed the transgenic plants containing Pspoa-drived Sporamin and spoMAR displayed the highest level and low inter-transformant variability of Sporamin expression, and the ability of insect resistance of transformants positively correlated with Sporamin activity. Furthermore, expressions of Pspoa-drived Sporamin especially combined with the spoaMAR retains high and steady levels in the T1 and T2 generations, in marked contrast to the variable expression patterns observed in CaMV35S promoter-driven transformants. This study evidently indicates that the Pspoa and spoaMAR would be very efficient for high transgene expression in plants and obtaining inherently stable transformants in consecutive progenies.
Kenzo Nakamura - One of the best experts on this subject based on the ideXlab platform.
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Different Sensitivity to Wortmannin of Two Vacuolar Sorting Signals Indicates the Presence of Distinct Sorting Machineries in Tobacco Cells
2013Co-Authors: Ken Matsuoka, Natasha V Raikhel, Diane C. Bassham, Kenzo NakamuraAbstract:Abstract. Vacuolar matrix proteins in plant cells are sorted from the secretory pathway to the vacuoles at the Golgi apparatus. Previously, we reported that the NHz-terminal propeptide (NTPP) of the Sporamin precursor and the COOH-terminal propeptide (CTPP) of the barley lectin precursor contain information for vacuolar sorting. To analyze whether these propeptides are interchangeable, we expressed constructs consisting of wild-type or mutated NTPP with the mature part of barley lectin and Sporamin with CTPP and mutated NTPP in tobacco BY-2 cells. The vacuolar localization of these constructs indicated that the signals were interchangeable. We next analyzed the effect of wortmannin, a specific inhibitor of mammalian phosphatidylinositol (PI) 3-kinase on vacuolar delivery by NTP
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Two cis-acting regulatory elements are involved in the sucrose-inducible expression of the Sporamin gene promoter from sweet potato in transgenic tobacco
Molecular Genetics and Genomics, 2005Co-Authors: Atsushi Morikami, Rie Matsunaga, Yoshimi Tanaka, Satomi Suzuki, Shoji Mano, Kenzo NakamuraAbstract:In this study, we generated transgenic tobacco plants that express the β-glucuronidase (GUS) gene under the control of the 305-bp 5′-upstream region of a gene coding for Sporamin A of sweet potato. Expression of GUS in excised tobacco leaves was induced by sucrose, mimicking the sugar-inducible expression of the endogenous Sporamin genes in sweet potato. Deletion of the sequences extending from position −305 (relative to the transcription start site) to −283 and from −146 to −87 resulted in an approximately 40-fold enhancement in GUS reporter expression. Furthermore, the sequence from −282 to −165 conferred sucrose-inducibility on the −89 core promoter of the Cauliflower Mosaic Virus 35S RNA gene. Analysis of internal deletions, linker scanning and the introduction of base substitutions in the sequence between positions −282 and −165 indicated that sucrose-responsiveness conferred by this region was dependent on the presence of two cis -acting elements, termed CMSREs (carbohydrate metabolite signal responsive elements) 1 and 2, which are separated by a spacer. A sequence similar or identical to the core of CMSRE-1 (TGGACGG) is also present in the promoters of several other sugar-inducible genes, and sequences encopassing the TGGACGG-related motifs from two of these could functionally replace the CMSRE-1 in the truncated Sporamin A promoter. These results suggest that the TGGACGG element plays an important role in the sucrose-inducible expression of a group of plant genes.
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large alkyl side chains of isoleucine and leucine in the npirl region constitute the core of the vacuolar sorting determinant of Sporamin precursor
Plant Molecular Biology, 1999Co-Authors: K Matsuoka, Kenzo NakamuraAbstract:The N-terminal propeptide of the Sporamin precursor contains vacuolar targeting information within the Asn-26/Pro-27/Ile-28/Arg-29/Leu-30 (NPIRL) sequence. An Agrobacterium-mediated transient expression assay with tobacco BY-2 cells was employed to investigate the role of each amino acid of the NPIRL region in vacuolar targeting. Replacement of Asn-26, Pro-27, Ile-28 and Leu-30 with several amino acids caused secretion of the mutant proSporamin. Leu was the only amino acid that could be substituted for Ile-28 without affecting transport. Exchange of Leu-30 for amino acids with small side-chains abolished vacuolar delivery. These results indicate that the consensus composition of the NPIRL sequence is [preferably Asn]-[not acidic]-[Ile or Leu]-[any amino acid]-[large and hydrophobic] and suggest that the large alkyl side-chains of Ile-28 and Leu-30 constitute the core of the vacuolar sorting determinant.
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the n terminal propeptide of the precursor to Sporamin acts as a vacuole targeting signal even at the c terminus of the mature part in tobacco cells
Plant Physiology, 1997Co-Authors: Yasuhiro Koide, K Matsuoka, Hideyuki Hirano, Kenzo NakamuraAbstract:An asparagine-proline-isoleucine-arginine-leucine (NPIRL) and its related sequences in the N-terminal propeptides (NTPP) of several plant vacuolar proteins, including that of Sporamin from sweet potato (SPO) function as vacuole-targeting determinants in a manner that is distinct from the vacuole-targeting determinant in the CTPPs of other plant vacuolar proteins. When the mutant precursor to Sporamin, SPO-NTPP (in which NTPP was moved to the C terminus of the mature part), was expressed in tobacco (Nicotiana tabacum) cells, the pro-form was efficiently targeted to the vacuole and the NTPP was cleaved off. Unlike the results obtained with the wild-type precursor, substitution of the NPIRL sequence in the C-terminally located NTPP to asparagine-proline-glycine-arginine-leucine in the SPO-isoleucine-28-to-glycine mutant resulted in missorting of less than 20% of the pro-form to the medium. Unlike the vacuolar transport of SPO-NTPP, the vacuolar transport of SPO-isoleucine-28-to-glycine was strongly inhibited by 33 [mu]M wortmannin, which is similar to the C-terminal propeptide-mediated vacuolar transport. These results suggest that the vacuole-targeting function of the NPIRL sequence is not strictly dependent on its location at the N terminus of a protein and that the C-terminally located mutant NTPP acquired some physicochemical properties of the C-terminal vacuole-targeting sequence.
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o glycosylation of a precursor to a sweet potato vacuolar protein Sporamin expressed in tobacco cells
Plant Journal, 1995Co-Authors: K Matsuoka, Natsu Watanabe, Kenzo NakamuraAbstract:Sporamin, a vacuolar protein of the sweet potato, is synthesized as a precursor that contains signal peptide and an N-terminal propeptide that functions as a vacuolar targeting determinant. Sporamin, when expressed in tobacco cells, migrated as smeared bands on an SDS-polyacrylamide gel. The smearing was due to O-glycosylation of the precursor to Sporamin. The smeared bands were stained by a glycan-specific stain but no N-glycosylation site was found in the amino acid sequence of the precursor to Sporamin. The glycan attached to Sporamin contained galactose and arabinose as major sugar components. Mutations that altered the Pro36 or Ser39 residue of the precursor to Sporamin prevented glycosylation of the protein, and analysis by semiquantitative Edman degradation suggested that a glycan moiety was attached to Pro36 and, possibly, to Ser39. Pulse-labeling and cell-fractionation experiments revealed that the O-glycosylation of the precursor to Sporamin occurred in the Golgi apparatus. Thus, this modification serves as a good marker of the transport from the endoplasmic reticulum (ER) to the Golgi apparatus of the precursor to Sporamin. Treatment of transformed tobacco cells with brefeldin A (BFA) caused the intracellular accumulation of proSporamin that did not migrate as smeared bands. Thus, it appeared that BFA inhibited the transport of the precursor to Sporamin to the Golgi apparatus. This result provides the first biochemical evidence that BFA inhibits transport from the ER to the Golgi apparatus in plant cells.
Yih-ming Chen - One of the best experts on this subject based on the ideXlab platform.
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Wound-response regulation of the sweet potato Sporamin gene promoter region
Plant Molecular Biology, 2002Co-Authors: Shujen Wang, Yi-ching Lan, Shih-fung Chen, Yih-ming Chen, Kaiwun YehAbstract:Sporamin, a tuberous storage protein of sweet potato, was systemically expressed in leaves and stems by wound stimulation. In an effort to demonstrate the regulatory mechanism of wound response on the Sporamin gene, a 1.25 kb Sporamin promoter was isolated for studying the wound-induced signal transduction. Two wound response-like elements, a G box-like element and a GCC core-like sequence were found in this promoter. A construct containing the Sporamin promoter fused to a β- glucuronidase ( GUS ) gene was transferred into tobacco plants by Agrobacterium -mediated transformation. The wound-induced high level of GUS activity was observed in stems and leaves of transgenic tobacco, but not in roots. This expression pattern was similar to that of the Sporamin gene in sweet potatoes. Exogenous application of methyl jasmonate (MeJA) activated the Sporamin promoter in leaves and stems of sweet potato and transgenic tobacco plants. A competitive inhibitor of ethylene (2,5-norbornadiene; NBD) down-regulated the effect of MeJA on Sporamin gene expression. In contrast, salicylic acid (SA), an inhibitor of the octadecanoid pathway, strongly suppressed the Sporamin promoter function that was stimulated by wound and MeJA treatments. In conclusion, wound-response expression of the Sporamin gene in aerial parts of plants is regulated by the octadecanoid signal pathway.
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site directed mutagenesis evidence for a negatively charged trypsin inhibitory loop in sweet potato Sporamin
FEBS Letters, 2001Co-Authors: Peili Yao, Yih-ming Chen, Mingjing Hwang, Kaiwun YehAbstract:Abstract Sporamin, a sweet potato tuberous storage protein, has trypsin inhibitory activity. Sequence comparison with other plant trypsin inhibitors (TIs) of the Kunitz family reveals that, instead of the conserved Arg or Lys found in other Kunitz TIs, Sporamin contains a negatively charged residue (Asp70 or Glu72) at the P1 reactive site. Using site-directed mutagenesis, six mutants were generated containing substitutions at the reactive site and at one of the disulfide bonds, and the recombinant proteins were assayed for TI activity. Mutants Asp70Val and Glu72Arg were found to have only 2–3% of the wild-type activity. These results provide the first evidence for a negatively charged trypsin inhibitory loop and a new mechanism of trypsin inhibition in the Kunitz family.
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isolation and characterization of new Sporamin gene members from sweet potato ipomoea batatas lam
TAIWANIA, 1997Co-Authors: Jenchih Chen, Yih-ming Chen, Kaiwun YehAbstract:Two full-length and two partial cDNAs encoding Sporamin A have been isolated from sweet potato tuberous roots. Sequence comparisons show that they are very similar with 94-98% homology at nucleotide level, and 80-88% at protein level. All four cDNAs possess multiple alternate polyadenylation signals in the 3' untranslated region (3'-UTR). Genomic Southern blot analysis indicates the presence of a Sporamin multigene family in sweet potato. High levels of Sporamin mRNAs were detected in developing tuberous roots, but they disappeared at the sprout-germinating stage. Differential expression of these genes was obvious as their mRNAs were present specifically in developing roots, rarely in stems and not in leaves.
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functional activity of Sporamin from sweet potato ipomoea batatas lam a tuber storage protein with trypsin inhibitory activity
Plant Molecular Biology, 1997Co-Authors: Kaiwun Yeh, Yih-ming Chen, Jenchih Chen, Meiin Lin, Chuyung LinAbstract:Sporamin accounts for about 60% to 80% of total soluble protein in sweet potato tubers, and the predicted protein sequence of Sporamin shares significant amino acid sequence identity with some Kunitz-type trypsin inhibitors. We constructed three recombinant plasmids with cDNAs that encode preproSporamin, proSporamin, and Sporamin, and these three were expressed in Escherichia coli cells as fusion proteins. All three forms of Sporamin expressed in E. coli were shown to have strong inhibitory activity to trypsin in vitro, suggesting that post-translational modifications are not essential for trypsin inhibitory activity. Northern blot analysis showed that Sporamin transcripts could be systemically induced in leaf tissue of sweet potato by wounding. Therefore, Sporamin may have a defense role as a protease inhibitor, in addition to its role as a storage protein.
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c 1997 Kluwer Academic Publishers. Printed in Belgium.
1996Co-Authors: Short Communication, Kaiwun Yeh, Yih-ming Chen, Jenchih Chen, Meiin Lin, Chuyung LinAbstract:Sporamin accounts for about 60 % to 80 % of total soluble protein in sweet potato tubers, and the predicted protein sequence of Sporamin shares significant amino acid sequence identity with some Kunitz-type trypsin inhibitors. We constructed three recombinant plasmids with cDNAs that encode preproSporamin, proSporamin, and Sporamin, and these three were expressed in Escherichia coli cells as fusion proteins. All three forms of Sporamin expressed in E. coli were shown to have strong inhibitory activity to trypsin in vitro, suggesting that post-translational modifications are not essential for trypsin inhibitory activity. Northern blot analysis showed that Sporamin transcripts could be systemically induced in leaf tissue of sweet potato by wounding. Therefore, Sporamin may have a defense role as a protease inhibitor, in addition to its role as a storage protein. Seed and tuberous roots of most plants contain a large amount of storage protein (ranging from 30 % to 80% of total protein), such as glycinins of soybean [14], zein of maize [33], patatin of potato tuber [26], dioscorins of yam (Dioscorea cayenensis) [7], and Sporamin of sweet potato (Ipomoea batatas Lam.) [21]. The majo
Natasha V Raikhel - One of the best experts on this subject based on the ideXlab platform.
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Different Sensitivity to Wortmannin of Two Vacuolar Sorting Signals Indicates the Presence of Distinct Sorting Machineries in Tobacco Cells
2013Co-Authors: Ken Matsuoka, Natasha V Raikhel, Diane C. Bassham, Kenzo NakamuraAbstract:Abstract. Vacuolar matrix proteins in plant cells are sorted from the secretory pathway to the vacuoles at the Golgi apparatus. Previously, we reported that the NHz-terminal propeptide (NTPP) of the Sporamin precursor and the COOH-terminal propeptide (CTPP) of the barley lectin precursor contain information for vacuolar sorting. To analyze whether these propeptides are interchangeable, we expressed constructs consisting of wild-type or mutated NTPP with the mature part of barley lectin and Sporamin with CTPP and mutated NTPP in tobacco BY-2 cells. The vacuolar localization of these constructs indicated that the signals were interchangeable. We next analyzed the effect of wortmannin, a specific inhibitor of mammalian phosphatidylinositol (PI) 3-kinase on vacuolar delivery by NTP
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The Plant Vacuolar Sorting Receptor AtELP Is Involved in Transport of NH 2-terminal Propeptide-containing Vacuolar Proteins in
2013Co-Authors: Arabidopsis Thaliana, Ken Matsuoka, Sharif U. Ahmed, Enrique Rojo, Valentina Kovaleva, Sridhar Venkataraman, James E. Dombrowski, Natasha V RaikhelAbstract:Abstract. Many soluble plant vacuolar proteins are sorted away from secreted proteins into small vesicles at the trans-Golgi network by transmembrane cargo receptors. Cleavable vacuolar sorting signals include the NH 2-terminal propeptide (NTPP) present in sweet potato Sporamin (Spo) and the COOH-terminal propeptide (CTPP) present in barley lectin (BL). These two proteins have been found to be transported by different mechanisms to the vacuole. We examined the ability of the vacuolar cargo receptor AtELP to interact with the sorting signals of heterologous and endogenous plant vacuolar proteins in mediating vacuolar transport in Arabidopsis thaliana. AtELP extracted from microsomes was found to interact with the NTPPs of barley aleurain and Spo, but not with the CTPPs of BL o
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The Plant Vacuolar Sorting Receptor AtELP Is Involved in Transport of NH 2-terminal Propeptide-containing Vacuolar Proteins in
2000Co-Authors: Arabidopsis Thaliana, Ken Matsuoka, Sharif U. Ahmed, Enrique Rojo, Valentina Kovaleva, Sridhar Venkataraman, James E. Dombrowski, Natasha V RaikhelAbstract:Abstract. Many soluble plant vacuolar proteins are sorted away from secreted proteins into small vesicles at the trans-Golgi network by transmembrane cargo receptors. Cleavable vacuolar sorting signals include the NH 2-terminal propeptide (NTPP) present in sweet potato Sporamin (Spo) and the COOH-terminal propeptide (CTPP) present in barley lectin (BL). These two proteins have been found to be transported by different mechanisms to the vacuole. We examined the ability of the vacuolar cargo receptor AtELP to interact with the sorting signals of heterologous and endogenous plant vacuolar proteins in mediating vacuolar transport in Arabidopsis thaliana. AtELP extracted from microsomes was found to interact with the NTPPs of barle
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colocalization of barley lectin and Sporamin in vacuoles of transgenic tobacco plants
Plant Physiology, 1993Co-Authors: Martin R Schroeder, Kenzo Nakamura, K Matsuoka, Olga N Borkhsenious, Natasha V RaikhelAbstract:Various targeting motifs have been identified for plant proteins delivered to the vacuole. For barley (Hordeum vulgare) lectin, a typical Gramineae lectin and defense-related protein, the vacuolar information is contained in a carboxyl-terminal propeptide. In contrast, the vacuolar targeting information of Sporamin, a storage protein from the tuberous roots of the sweet potato (Ipomoea batatas), is encoded in an amino-terminal propeptide. Both proteins were expressed simultaneously in transgenic tobacco plants to enable analysis of their posttranslational processing and subcellular localization by pulse-chase labeling and electron-microscopic immunocytochemical methods. The pulse-chase experiments demonstrated that processing and delivery to the vacuole are not impaired by the simultaneous expression of barley lectin and Sporamin Both proteins were targeted quantitatively to the vacuole, indicating that the carboxyl-terminal and amino-terminal propeptides are equally recognized by the vacuolar protein-sorting machinery. Double-labeling experiments showed that barley lectin and Sporamin accumulate in the same vacuole of transgenic tobacco (Nicotiana tabacum) leaf and root cells.
Li Huang - One of the best experts on this subject based on the ideXlab platform.
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stable expression of exogenous imported Sporamin in transgenic chinese cabbage enhances resistance against insects
Plant Growth Regulation, 2017Co-Authors: Jie Cui, Lin Qiu, Jiashu Cao, Li HuangAbstract:Cultivating insect pest-resistant varieties is one of the most effective ways to prevent or mitigate pest infestation in Chinese cabbage (Brassica campestris ssp. chinensis). Via the agrobacterium tumefaciens-mediated transformation method, this study introduced the protease inhibitor encoding gene Sporamin into two widely cultured cultivars ‘Youdonger’ and ‘Shanghaiqing’, of the common variety of Chinese cabbages (B. campestriss ssp. chinensis var. communis), getting transgenic plants with high Sporamin expression. In vitro insect bioassays indicated that, compared with the wild type plants, the transgenic plants exhibited improved resistance to diamondback moth (Plutella xylostella L.) The analysis of inheritance pattern of exogenous Sporamin in the progenies of single copy insertion transgenic lines demonstrated that Sporamin could be inherited and expressed stably in transgenic progenies. Field survey of the insect resistance under the normal culture condition confirmed the enhanced resistance in transgenic progenies to diamondback moth. Our results strongly suggest that Sporamin is an efficient candidate gene for insect-resistant genetic engineering in Chinese cabbage.
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high level expression of Sporamin in transgenic chinese cabbage enhances resistance against diamondback moth
Plant Molecular Biology Reporter, 2013Co-Authors: Lin Qiu, Jiashu Cao, Heng Dong, Li HuangAbstract:This study introduced the protease inhibitor-encoding gene Sporamin from sweet potato together with the wound-responsive promoter pMSPOA into the Chinese cabbage (Brassica campestris ssp. chinensis var. parachinensis) through Agrobacterium tumefaciens-mediated transformation. Transgenic lines were confirmed via PCR detection of both the Sporamin and hygromycin gene fragments in the cloning vector. The expression level of Sporamin in the different lines was determined using RT-PCR analysis. Leaves from four individual transgenic lines including T0-2, T0-3, T0-4 and T0-5, possessing higher levels of Sporamin than control plants, were then used to conduct insect feeding studies. Second instar larvae of diamondback moth (Plutella xylostella L.) were used in feeding assays. All four transgenic lines demonstrated high levels of resistance against diamondback based on average body weight of total incubated larvae following feeding assays and amount of leaf damage.