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A novel tomato SUMO E_3 ligase,SlSIZ_1,confers drought tolerance in transgenic tobacco

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Journal of Integrative Plant Biology 2017年第2期59卷 102-117页

作者： Song Zhang Kunyang Zhuang Shiju Wang Jinlian Lv Na'na Ma Qingwei Meng State Key Laboratory of Crop Biology College of Life SciencesShandong Agricultural UniversityTai'an 271018China

SUMOylation is an impo^ant post-translational modification process that regulates different cellular functions in eukaryotes. SIZ/PIAS-type SAP and Mizl （SIZ0 proteins exhibit SUMO E3 ligase activity, which modulates SUMOylation. However, SIZI in tomato has been rarely investigated. In this study, a tomato SIZI gene （SISIZI） was isolated and its molecular characteristics and role in tolerance to drought stress are described. SISIZI was upregulated by cold, sodium chloride （NaCI）, polyethylene glycol （PEG）, hydrogen peroxide （H20~） and abscisic acid （ABA）, and the corresponding proteins were localized in the nucleus. The expression of SISIZI in Arabidopsis thaliana （Arabidopsis） sizl-2 mutants partially complemented the phenotypes of dwarf, cold sensitivity and ABA hypersensitivity. SISIZI also exhibited the activity of SUMO E3 ligase to promote the accumulation of SUMO conjugates. Under drought stress, the ectopic expression of SISIZl in transgenic tobacco lines enhanced seed germination and reduced the accumulation of reactive oxygen species..SISIZl overexpression conferred the plants with improved growth, high free proline content, minimal malondialdehyde accumulation and increased accumulation of SUMO conjugates. SISIZ1 is a functional homolog of Arabidopsis SIZ1 with SUMO E3 ligase activity. Therefore, overexpression of SISIZ1 enhanced the tolerance of transgenic tobacco to drought stress.

关键词： drought stress SUMO E3 ligase SlSIZ1 SUMO conjugates tomato transgenic tobacco

Expression of a Modified Cry1Ie Gene in E. coli and in transgenic tobacco Confers Resistance to Corn Borer

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Acta Biochimica et Biophysica Sinica 2004年第4期36卷 309-313页

作者： Yun-Jun LIU, Fu-Ping SONG1, Kang-Lai HE1, Yuan YUAN, Xiao-Xia ZHANG, Peng GAO, Jian-Hua WANG, and Guo-Ying WANG * State Key Laboratory for Agrobiotechnology, National Center for Maize Improvement, China Agricultural University, Beijing 100094, China 1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, CAAS, Beijing 100094, China State Key Laboratory for Agrobiotechnology National Center for Maize Improvement China Agricultural UniversityBeijing 100094 China State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionCAAS Beijing 100094 China

The wild-type Cry1Ie gene from Bacillus thuringiensis was modified for its efficient expression in transgenic plants. Modified Cry1Ie gene (designated as Cry1Iem) was cloned into prokaryotic expression vector pET28b and its expression in E. coli was confirmed by SDS-PAGE analysis. Bioassays using crude expression products in E. coli revealed that Cry1Iem protein had a similar toxicity to corn borer as wild-type Cry1Ie. Cry1Iem gene was then inserted downstream of the maize ubiquitin-1 promoter in plant expression vector p3301. transgenic tobacco plants carrying Cry1Iem showed insecticidal activity against corn borer.

关键词： Bacillus thuringiensis Cry1Iem transgenic tobacco insect-resistance

Obtainment of transgenic tobacco Harboring phbA , phbB and phbC Genes by Twice Transformation

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Acta Botanica Sinica 2001年第1期43卷 59-62页

作者：张景昱叶梁李丽宋艳茹中国科学院植物研究所北京100093

To avoid the long time required for conventional sexual crossing, transgenic tobacco (Nicotiana tabacum L.) plants harboring phbA gene (encoding 3_ketothiolase) were used as the target plant for the second transformation mediated by Agrobacterium tumefaciens (Smith et Townsend) Conn LBA4404 containing pZCB which was constructed by linking phbB (encoding acetoacetyl_CoA reductase), phbC (encoding PHB synthase) and ctp sequence to pBIB_HYG under the control of CaMV 35S promoter. The hygromycin resistant transformants were morphologically normal and stable integration of phbB and phbC was confirmed by PCR and PCR_Southern. Moreover, RT_PCR_DNA hybridization analysis showed that 6.67% of the transformed tobacco plants could express both phbB and phbC at transcriptional level.

关键词： poly (3_hydroxybutyrate) (PHB) twice transformation transgenic tobacco

transgenic tobacco plants harboring tomato proteinase inhibitor II gene and their insect resistance

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Progress in Natural Science:Materials International 2002年第3期 40-45页

作者： XIE Xianzhi, HUANG Meijuan and WU Naihu( Institute of Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China College of Life Sciences, Peking University, Beijing 100871, China) Institute of Developmental Biology Chinese Academy of Sciences Beijing 100080 China College of Life Sciences Peking University Beijing 100871 China

The plant expression vectors pBCT2 and pBT2 were constructed with the cDNA sequence (tin2) and genomic DNA sequence (tin2i) of tomato proteinase inhibitor II gene respectively. Then the two expression vectors were transferred into tobacco via the Agrobacterium tumefaciens strain LBA4404, and transgenic tobacco plants were generated. Molecular analysis and trypsin activity assay showed that both cDNA and genomic DNA were expressed properly in the transgenic plants. Insecticidal activities in these transgenic plants indicated that transgenic tobacco plants carrying tin2i sequence were more resistant to 2-instar larvae of Heliothis armigera Hubner than those carrying tin2 sequence. Therefore the intron of tin2i sequence might be a contributor to insecticidal activity of the transgenic tobacco.

关键词： tomato proteinase inhibitor II gene transgenic tobacco insect-resistance.

Expression of Human Papillomavirus Type 16 L1 Protein in transgenic tobacco Plants

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Acta Biochimica et Biophysica Sinica 2005年第3期37卷 153-158页

作者： Hong-Li LIU Wen-Sheng LI Ting LEI Jing ZHENG Zheng ZHANG Xiao-Fei YAN Zhe-Zhi WANG Yi-Li WANG L-Sheng SI Institute of Cancer Research,School of Life Science & Technology,Xi'an Jiaotong University.Xi'an 710061,China First Hospital of Xi'an Jiaotong University Xi'an 710061,China Shaanxi Normal University,Xi'an 710062China InstituteofCancerResearch SchoolofLifeScience&TechnologyXi'anJiaotongUniversityXi'an710061China

To develop a plant expression system for the production of the human papillomavirus type 16(HPV16)vaccine,we investigated whether the HPV16 L1 protein can be expressed in tobacco plants andwhether it can be used as the cheapest form of edible *** HPV16 L1 coding sequence was amplifiedby PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence,andsubcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plantexpression plasmid *** T-DNA regions of the pBI-L1 binary vector contained the constitutiveCauliflower mosaic virus(CaMV)35S promoter and the neomycin phosphotransferase npt Ⅱ gene,whichallowed the selection of transformed plants using *** tobacco plants were transformed by co-cultivating them,using the leaf disc method,with Agrobacterium tumefaciens LBA4404,which harbored theplant expression *** regenerated transgenic tobacco plants were selected using kanamycin,andconfirmed by *** results of the Southern blot assay also showed that the HPV16 L1 gene was inte-grated stably into the genome of the transformed tobacco *** Western blot analysis showed that thetransformed tobacco leaves could express the HPV16 L1 ***,it was demonstrated byELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein,wasable to form 55 nm virus-like particles compatible with HPV virus-like particle(VLP),and induced mouseerythrocyte hemagglutination in *** present results indicate that the HPV16 L1 protein can be ex-pressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16L1 protein,implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.

关键词： human papillomavirus(HPV) virus-like particle transgenic tobacco plant vaccine Agrobacterium tumefaciens Ll major capsid protein

Cloning of Glutamate Dehydrogenase cDNA from Chlorella sorokiniana and Analysis of transgenic tobacco Plants

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Acta Botanica Sinica 2002年第3期44卷 318-324页

作者：黄国存孟颂东王荣杨怀义田波中国科学院微生物研究所北京100080

A full_length cDNA has been cloned encoding nicotinamide adenine dinucleotide phosphate_specific glutamate dehydrogenase (NADP_GDH) from Chlorella sorokiniana with the RT_PCR method. The complete nucleotide sequence of NADP_GDH gene had 94% homology to the previously reported one . The NADP_GDH gene was constructed into a vector highly expressed in plants. The specific activity of NADP_GDH in transformants was detected, but not in the control plants. All transformed shoots on MS medium containing lower concentration of nitrogen and the transformed seedlings grown in lower concentration of nitrogen vermiculite had higher growth rate and more leaves than the control plants. Transformed leaf discs cultured on MS medium containing different nitrogen concentrations had more chlorophyll contents compared to the controls. These results suggested that exogenous NADP_GDH may enhance the absorption and utilization to ammonium in plants. The increased weight of transformed leaf discs cultured on medium supplemented with different concentrations of phosphinothricin (PPT) was more than that of control discs. 0.5 μg/mL PPT could be used as a selecting drug instead of kanamycin to develop the transformants. These results suggested that the NADP_GDH gene might be used as a new selecting gene in the future research of plant gene engineering.

关键词： nicotinamide adenine dinucleotide phosphate_specific glutamate dehydrogenase (NADP_GDH) transgenic tobacco ammonium absorption phosphinothricin resistance

Novel Halophyte EREBP/AP2-type DNA Binding Protein Improves Salt Tolerance in transgenic tobacco

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Acta Botanica Sinica 2003年第1期45卷 82-87页

作者：沈义国闫冬青张万科杜保兴张劲松刘强陈受宜中国科学院遗传和发育生物学研究所北京100101 西北农林科技大学生命科学学院陕西杨凌712100 清华大学生物科学和生物技术系北京100084

EREBP/AP2-type proteins are members of a large DNA binding protein (DBP) family found in plants. Some members like APETALA2 and AtDREB/CBF can regulate flower development and response to environmental stresses, respectively. To characterize transcription factors involved in plant responses to salt stress, we constructed cDNA library from salt-treated halophyte (Atriplex hortensis) and isolated a novel gene encoding EREBP/AP2-type protein from this library. This cDNA contained an ORF of 723 bp and a long 3'-Untranslated-Region (UTR) of 655 bp. The deduced amino acid sequence showed one conserved DNA binding domain of EREBP/AP2, thus the corresponding gene was named AhDREB1 with a calculated molecular mass of 26.1 kD. AhDREB1 under the control of CaMV 35S promoter was then transformed into tobacco and nine independent transgenic lines were obtained and subjected to long term salt stress. The results suggested that overexpression of AhDREB1 improved the salt tolerance in transgenic tobacco through functioning as a regulatory molecule in response to salt stress. Analysis of Arabidopsis genome in database resulted in dozens of EREBP/AP2-type homologous proteins, of which seven members showed high similarity to AhDREB1. Secondary structure analysis predicted similar arrangement of a-helix in their DNA binding domains.

关键词： Atriplex hortensis EREBP/AP2-type DNA binding protein transgenic tobacco

Cloning of cDNA Encoding Choline Monooxygenase from Suaeda liaotungensis and Salt Tolerance of transgenic tobacco

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Acta Botanica Sinica 2003年第2期45卷 242-247页

作者：李秋莉刘大伟高晓蓉苏乔安利佳大连理工大学生物工程系

Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is catalyzed by choline monooxygenase (CMO). In this study, the full-length CMO cDNA (1 820 bp) was cloned from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. It included a 123 bp 5' UTR, a 368 bp 3' UTR and a 1 329 bp open reading frame encoding a 442-amino-acid polypeptide with 77%, 72% and 74% sequence identity compared to CMOs from spinach, sugar beet and Atriplex hortensis, respectively. The CMO open reading frame (ORF) was cloned and the plant expression vector pBI121-CMO was constructed. It was transferred into tobacco ( Nicotiana tabacum L. ev. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO gene was integrated into tobacco genome. transgenic tobacco plants contained higher amount of betaine than that of control plants and were able to survive on MS medium containing 250 mmol/L NaCl. Relative electronic conductivity demonstrated less membrane damage in transgenic plants as in the wild type.

关键词： Suaeda liaotungensis choline monooxygenase gene cloning transgenic tobacco salt tolerance

Isolation of a Genomic DNA for Gastrodia Antifungal Protein and Analyses of Its Promoter in transgenic tobacco

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Acta Botanica Sinica 2003年第2期45卷 229-233页

作者：萨其拉王义琴李文彬张利明孙勇如中国科学院遗传与发育生物学研究所北京100101

A genomic DNA containing 5'-upstream region and complete open reading frame of a Gastrodia antifungal protein was isolated by screening of a genomic library from Gastrodia elata B1. To investigate the promoter activity, the 5'-flanking region - 1 157 lip upstream from the putative transcription start site was fused to the coding sequence of beta-glucuronidase (GUS) gene and transformed into Nicotiana tabacum. The strongest GUS activity was detected in the roots of transgenic tobacco, followed by stems. The leaves only showed a low GUS activity. Furthermore, the promoter established inducible expression pattern in transgenic tobacco upon fungus Trichoderma viride inoculation and jasmonic acid and salicylic acid treatments.

关键词： Gastrodia elata Bl. promoter transgenic tobacco organ-specific induced expression