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Overexpression of a modified AM79 aroA gene in transgenic maize confers high tolerance to glyphosate

Journal of Integrative Agriculture 2015年第3期14卷 414-422页

作者： REN Zhen-jing CAO Gao-yi ZHANG Yu-wen LIU Yan LIU Yun-jun Institute of Crop Science Chinese Academy of Agricultural Sciences College of Agriculture and Biotechnology China Agricultural University

It has previously been shown that a bacterial 5-enolpyruvylshikimate-3-phosphate synthase（EPSPS） encoding gene AM79 aroA can be a candidate gene to develop glyphosate-tolerant transgenic crops（Cao et al. 2012）. In this study, AM79 aroA was redesigned using the plant biased codons and eliminating the motifs which would lead to the instability of mRNA, to create a synthetic gene that would be expressed highly in plant cells. The redesigned and artificially synthesized gene, named as mAM79, was cloned into plant expression vector pM3301 Ubi Sp AM79, where mAM79 is fused with signal peptide sequence of pea rib-1,5-bisphospate carboxylase（rbcS） small subunit and controlled by ubiquitin promoter. The plasmid was transformed into maize（Zea mays） immature embryos using Agrobacterium-mediated transformation method. Total 74 regenerated plants were obtained and PCR analysis showed that these transgenic plants had the integration of mAM79. Southern blot analysis was performed on the genomic DNA from four transgenic lines, and the result showed that one or two copies of mAM79 were integrated into maize genome. RT-PCR analysis result indicated that mAM79 was highly transcribed in transgenic maize plants. When sprayed with glyphosate, transgenic maize line AM85 and AM72 could tolerate 4-fold of commercial usage of glyphosate; however, all the non-transgenic maize plants were killed by glyphosate. The results in this study confirmed that mAM79 could be used to develop glyphosate-tolerant maize, and the obtained transgenic maize lines could be used for the breeding of glyphosate-tolerant maize.

关键词： AM79 aroA modify transgenic maize glyphosate tolerance

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The Influence of Bt-transgenic maize Pollen on the Bacterial Diversity in the Midgut of Chinese Honeybees, Apis cerana cerana

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Journal of Integrative Agriculture 2013年第3期12卷 474-482页

作者： JIANG Wei-yu GENG Li-li DAI Ping-li LANG Zhi-hong SHU Chang-long LIN Yi ZHOU Ting SONG Fu-ping ZHANG Jie State Key Laboratory for Biology of Plant Diseases and Insect Pests/Institute of Plant Protection Chinese Academy of Agricultural Sciences College of Chemical Engineering Huaqiao University Key Laboratory of Pollinating Insect Biology Ministry of Agriculture/Institute of Apicultural Research Chinese Academy of Agricultural Sciences Biotechnology Research Institute Chinese Academy of Agricultural Sciences

Using culture-independent technique polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） and conventional culture techniques, ecological risk of transgenic maize pollen on gut bacteria of the Chinese honeybee, Apis cerana cerana, was assessed. Honeybees were fed with Bt-transgenic maize pollen, non-transgenic near isoline pollen, linear crylAh gene （800 ng mL^-1） and supercoiled plasmid DNA （800 ng mL^-1） under laboratory conditions. The DGGE profile showed that the number of DGGE bands varied from 10.7 to 14.7 per sample, and the Shannon＇s index ranged from 0.85 to 1.00. The similarity calculated by PAST was mostly above 92%, indicating no obvious changes among treatments or within replicates. 14 bacterial strains affiliated with Alphaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were isolated and characterized on media under aerobic and anaerobic conditions. These results demonstrated that transgenic crylAh maize pollen did not induce significant changes of the honeybee gut bacterial community composition under laboratory conditions.

关键词： Bacillus thuringiensis intestinal bacteria horizontal gene transfer transgenic maize Apis cerana biosafety

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Developing transgenic maize(Zea mays L.) with insect resistance and glyphosate tolerance by fusion gene transformation

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Journal of Integrative Agriculture 2015年第2期14卷 305-313页

作者： SUN He LANG Zhi-hong LU Wei ZHANG Jie HE Kang-lai ZHU Li LIN Min HUANG Da-fang Biotechnology Research Institute Chinese Academy of Agricultural Sciences State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences

Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.

关键词： LP4/2A gene stacking transgenic maize insect resistance glyphosate tolerance

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The Inheritance and Expression of cry1A Gene in transgenic maize

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Acta Botanica Sinica 2003年第3期45卷 253-256页

作者：刘允军王国英中国农业大学农业生物技术国家重点实验室北京100094

We investigated the inheritance and expression of cry1A gene in transgenic maize ( Zea mays L.) by Southern blotting analysis and enzyme_linked immunosorbent assays (ELISA). The results showed that cry1A had been transmitted to progeny of transgenic maize as a single gene. Contents of cry1A insecticidal protein were significantly different among transgenic maize lines and various tissues of the same transgenic lines. High expression of cry1A protein occurred in green tissues, such as leaf and husk leaf, and low expression occurred in pith, tassel, ear pith, pollen and silk. The results also showed that the contents of cry1A insecticidal protein in leaves of transgenic maize increased with the advance of development and there was no significant difference in cry1A expression level among various generations of transgenic maize.

关键词： cry1A gene transgenic maize inheritance expression insecticidal protein

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Screening Method for transgenic maize with Kanamycin Resistance

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Agricultural Science & Technology 2011年第7期12卷 954-957页

作者： LU Meng-yu DONG Fu-shuang ZHANG Jun-min WANG Hai-bo Hebei Center of Plant Genetic Transformation Institute of Genetic and PhysiologyHebei Academy of Agricultural and Forestry SciencesShijiazhuang 050051

[Objective]The paper was to establish simple and effective method to screen marker gene in maize with kanamycin resistance.[Method]Using inbred line "Chang 7-2" and hybrid "Zhengdan 958" of maize as test materials,their seeds were soaked with different concentrations and volumes of kanamycin for3 and 4 d,respectively,the rate of albino seedlings and average seedling height after sowing for10 d were investigated.[Result]The rate of albino seedlings not only was related to kanamycin concentration,but also had relationship with solution volume during soaking *** difference between inbred line and hybrid was no *** 100 ml of kanamycin solution with concentration of 200 mg/L was used to soak seeds for 3 d,the rate of albino seedlings basically could reach 100%.When 100 ml of kanamycin solution with concentration of 100 mg/L was used to soak 20 seeds for 3 d to carry out resistance screening,the accuracy was up to 84.8% after verifying the screening test of T1 transgenic maize plants.[Conclusion]The method was feasible,which could be used as a simple method for screening transgenic gene maize with kanamycin resistance.

关键词： transgenic maize Kanamycin Screening method

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Overexpression of Phosphatidylinositol Synthase Gene(ZmPIS) improves the drought tolerance of transgenic maize

Overexpression of Phosphatidylinositol Synthase Gene(ZmPIS) ...

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“细胞活动生命活力”——中国细胞生物学学会全体会员代表大会暨第十二次学术大会

作者： Xiuxia Liu,Shumei Zhai,Aifang Yang,Juren Zhang School of Life Science,Shandong University,27 Shanda South Road,Jinan,250100,China

The phosphatidylinositol signaling pathway(PI pathway) plays critical roles in plant responses to abiotic *** PI metabolic pathway begins with Ptdlns synthesis,which is performed by Ptdlns synthase using CDP-diacylglycerol(CDP-DG) and free cytoplasmic inositol as *** this paper,An Ptdlns synthase gene,ZmPIS cloned from maize cDNA in sense or antisense orientation with a maize ubiquitin promoter was transferred into *** and Southern blotting showed the transgene was integrated into maize ***-level expression of the transgene was confirmed by real-time *** the tolerance to drought stress of transgenic maize plants was also investigated at pre-flowering stages of maize *** results demonstrated that the sense transgenic plants had better osmotic adjustment ability, greater photosynthesis rate,less cell membrane damage,higher grain yield than the non-transgenic plants (WT);whereas those expressing the antisense transgene exhibited inferior traits compared with the WT. Moreover,the data of the global gene expression between WT and sense transgenic lines detected by the Digital gene expression profile also supported that the overexpression of ZmPIS enhanced the drought tolerance of maize.

关键词： Drought stress ZmPIS transgenic maize

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Assembly and shifts of the rhizosphere bacteria of transgenic maize line carrying mcry1Ab and mcry2Ab genes at different developmental stages

Assembly and shifts of the rhizosphere bacteria of transgeni...

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江苏省遗传学会2019年学术研讨会

作者： Aliya Fazal Zhong-Ling Wen Yun-Ting Lu Min-Kai Yang Gui-Hua Lu Jin-Liang Qi Yong-Hua Yang State Key Laboratory of Pharmaceutical Biotechnology Institute of Plant Molecular Biology School of Life Sciences Nanjing University

Assessing effects of transgenic crops on rhizobacterial communities have catalyzed numerous studies in the recent past decades. To address further to this concern, we designed a research to examine the variations in the bacterial diversity profiles of transgenic insect-resistant maize 2 A-5（2 A5） carrying mcry1 Ab and mcry2 Abgenes and its control Zheng58（Z58）, at different developmental stages under natural field conditions utilizing next generation sequencing（NGS） technology. High-throughput sequencing of 16 S rRNA gene（16 S rDNA） V3-V4 hypervariable region via Illumina MiSeq revealed no significant shifts in the overall α and β-diversity of rhizosphere bacteria profiles. However, analysis of the relative abundances of microbial communities revealed some developmental stage-dependent discrepencies in the maize bacterial rhizobiome. Members of the phylum Proteobacteria were greatly enriched in the rhizosphere soil of 2 A5 and Z58 compared to bulk soil and surrounding soil with a relatively higher abundance at jointing stagethan the rest of two stages, indicating that the maize roots do exert effects on bacterial phyla. Some bacterial orders varied quantitatively between the two maize cultivars at different developmental stages e.g. Rhizobiales had a lower abundance at jointing stage compared with latter stages while the relative abundance of Sphingomonadales was maximum at the flowering stage compared to former and latter stages. The microbial community of Enterobacterialesbelonging to the class Gammaproteobacteria was significantly lower（P＜0.05） in the rhizospheric soil of 2 A5 compared to Z58. Moving towards the genus level, the rhizosphere soils of both 2 A5 and Z58 were highly enriched with the microbes belonging to Rahnella, Kaistobacter, Pseudomonas and Arthrobacterwhile de-riched with themicrobes belonging to Bacillus and Flavobacteriumat the jointing stage. In addition, the relative abundances of nitrogen fixing（NF） microbes i.e. Arthrobacter, Burkholderia

关键词： enrichment derichment mcry1Ab and mcry2Ab genes bacterial rhizobiome transgenic maize nitrogen fixing microbes

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Effect of three insect-resistant maizes expressing Cry1le,Cry1Ab/Cry2Aj and Cry1Ab on the growth and development of armyworm Mythimna separata(Walker)

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Journal of Integrative Agriculture 2020年第7期19卷 1842-1849页

作者： SU Hong-hua JIANG Tao SUN Yu GU Hui-jie WU Jiao-jiao YANG Yi-zhong School of Horticulture and Plant Protection Yangzhou UniversityYangzhou 225009P.R.China

Three transgenic maize events(IE09S034,Shuangkang 12--5 and C0030.3.5)produced Cry1le,Cry1Ab/Cry2Aj and G10-EPSPS,Cry1Ab and EPSPS,respectively,all of which target the Asian corn *** oriental armyworm Mythimna separata(Walker)is the secondary *** this study,the effects of the three Bt maizes on the development and survival of armyworm were *** results showed that IE09S034 had insecticidal activity against 1st instar larvae,and the survival rate of armyworm fed with Bt maize for 10 days was 462%,significantly lower than that of the *** larvae at 3rd--6th instar were more tolerant of the Bt toxin than the early instar ***,Shuangkang 12-5 had good insecticidal activity against 1st-5th instar *** mortality was nearly 100%when the larvae were fed with Shuangkang 12-5 before 3rd instar,and the toxin had quick acting *** event significantly ihibited the development of armyworm;that is,the larval duration of the 3rd and 4th instar larvae fed with Shuangkang 12-5 was prolonged by 4.5 and 3.0 days,*** pupal weight and egg number were also significantly lower than those of the *** C0030.3.5,it could control 1st--5th instar larvae *** mortality rates were all over 50%if 1st-3rd larvae were fed with this *** pupal weight of 4th-6th instar larvae fed with Bt maize were only 53.9,56.8 and 54.6%,respectively,compared to that of the *** number of eggs laid was significantly less than the *** results indicate that all three transgenic maize events exhibit the potential to provide effective control of early instar larvae of *** can be commercialized in future to control lepidoptera pests such as Asian corn borer and armyworm.

关键词： transgenic maize armyworm development survival

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Transcriptional Regulation of Expression of the maize Aldehyde Dehydrogenase 7 Gene (ZmALDH7B6) in Response to Abiotic Stresses

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Journal of Integrative Agriculture 2014年第9期13卷 1900-1908页

作者： AN Xia DUAN Feng-ying GUO Song CHEN Fan-jun YUAN Li-xing GU Ri-liang Department of Plant Nutrition Center for ResourcesEnvironment and Food SecurityChina Agricultural University

Aldehyde dehydrogenases（ALDHs） represent a large protein family, which includes several members that catalyze the oxidation of an aldehyde to its corresponding carboxylic acid in plants. Genes encoding members of the ALDH7 subfamily have been suggested to play important roles in various stress adaptations in plants. In this study, quantitative RT-PCR analysis revealed that a maize ALDH7 subfamily member（ZmALDH7B6） was constitutively expressed in various organs, including roots, leaves, immature ears, tassels, and developing seeds. The abundance of ZmALDH7B6 mRNA transcripts in maize roots was increased by ammonium, NaCl, and mannitol treatments. To further analyze tissue-specific and stress-induced expression patterns, the 1.5-kb 5′-flanking ZmALDH7B6 promoter region was fused to the β-glucuronidase（GUS） reporter gene and introduced into maize plants. In roots of independent transgenic lines, there was significant induction of GUS activity in response to ammonium supply, confirming ammonium-dependent expression of ZmALDH7B6 at the transcript level. Histochemical staining showed that GUS activity driven by the ZmALDH7B6 promoter was mainly localized in the vascular tissues of maize roots. These results suggested that ZmALDH7B6 is induced by multiple environmental stresses in maize roots, and may play a role in detoxifying aldehydes, particularly in vascular tissue.

关键词： abiotic stress aldehyde dehydrogenase gene expression promoter transgenic maize

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In maize,co-expression of GAT and GR79-EPSPS provides high glyphosate resistance,along with low glyphosate residues

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aBIOTECH 2023年第4期4卷 277-290页

作者： Shengyan Li Pengcheng Li Xiangyin Li Ning Wen Yinxiao Wang Wei Lu Min Lin Zhihong Lang Biotechnology Research Institute Chinese Academy of Agricultural SciencesBeijingChina National Nanfan Research Institute(Sanya) Chinese Academy of Agricultural SciencesSanyaHainanChina

Herbicide tolerance has been the dominant trait introduced during the global commercialization of genetically modified(GM)***-tolerant crops,especially glyphosate-resistant crops,offer great advantages for weed management;however,despite these benefits,glyphosate-resistant maize(Zea mays L.)has not yet been commercially deployed in *** develop a new bio-breeding resource for glyphosate-resistant maize,we introduced a codon-optimized glyphosate N-acetyltransferase gene,gat,and the enolpyruvyl-shikimate-3-phosphate synthase gene,gr79-epsps,into the maize variety *** selected a genetically stable high glyphosate resistance(GR)transgenic event,designated GG2,from the transgenic maize population through screening with high doses of glyphosate.A molecular analysis demonstrated that single copy of gat and gr79-epsps were integrated into the maize genome,and these two genes were stably transcribed and *** trials showed that the transgenic event GG2 could tolerate 9000 g acid equivalent(a.e.)glyphosate per ha with no effect on phenotype or yield.A gas chromatography-mass spectrometry(GC–MS)analysis revealed that,shortly after glyphosate application,the glyphosate(PMG)and aminomethylphosphonic acid(AMPA)residues in GG2 leaves decreased by more than 90%compared to their levels in HGK60 transgenic plants,which only harbored the epsps ***,PMG and its metabolic residues(AMPA and N-acetyl-PMG)were not detected in the silage or seeds of GG2,even when far more than the recommended agricultural dose of glyphosate was *** co-expression of gat and gr79-epsps,therefore,confers GG2 with high GR and a low risk of herbicide residue accumulation,making this germplasm a valuable GR event in herbicide-tolerant maize breeding.

关键词： transgenic maize Glyphosate resistance Gr79-epsps gene Gat gene Low residues

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