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constitutive expression of feedback-insensitive cystathionine γ-synthase increases methionine levels in soybean leaves and seeds

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Journal of Integrative Agriculture 2018年第1期17卷 54-62页

作者： YU Yang HOU Wen-sheng Yael Hacham SUN Shi WU Cun-xiang Ifat Matityahu SONG Shi-kui Rachel Amir HAN Tian-fu Key Laboratory of Soybean Biology (Beijing) Ministry of Agriculture/Institute of Crop Sciences Chinese Academy of Agricultural Sciences Beijing 100081 P.R.China Laboratory of Plant Science Migal Galilee Technology Center Kiryat Shmona 12100 Israel TeI-Hai College Upper Galilee 11016 Israel

Soybean （Glycine max （L.） Merr.） is a major crop that provides plant-origin protein and oil for humans and livestock. Al- though the soybean vegetative tissues and seeds provide a major source of high-quality protein, they suffer from low con- centration of an essential sulfur-containing amino acid, methionine, which significantly limits their nutritional quality. The level of methionine is mainly controlled by the first unique enzyme of methionine synthesis, cystathione y-synthase （CGS）. Aiming to elevate methionine level in vegetative tissues and seeds, we constitutively over-expressed a feedback-insensitive Arabidopsis CGS （AtD-CGS） in soybean cultivars, Zigongdongdou （ZD） and Jilinxiaoli 1 （JX）. The levels of soluble methionine increased remarkably in leaves of transgenic soybeans compared to wild-type plants （6.6- and 7.3-fold in two transgenic ZD lines, and 3.7-fold in one transgenic JX line）. Furthermore, the total methionine contents were significantly increased in seeds of the transgenic ZD lines （1.5- to 4.8-fold increase） and the transgenic JX lines （1.3- to 2.3-fold increase） than in the wild type. The protein contents of the transgenic soybean seeds were significantly elevated compared to the wild type, suggesting that the scarcity of methionine in soybeans may limit protein accumulation in soybean seeds. The increased protein content did not alter the profile of major storage proteins in the seeds. Generally, this study provides a promising strategy to increase the levels of methionine and protein in soybean through the breeding programs.

关键词： soybean (Glycine max (L.) Merr.) constitutive expression cystathionine y-synthase (CGS) methionine contentstorage protein

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constitutive expression of human coagulating factor IX in HeLa cells by homologous recombination of the promoter

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Science China(Life Sciences) 2001年第1期44卷 18-24页

作者：杨晓青王飞王颖卢大儒邱信芳薛京伦 1. State Key Laboratory of Genetics Engineering Institute of Genetics School of Life Sciences Fudan University 200433 Shanghai China

constitutive expression of hFIX protein in nonhepatocytes wasstudied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenous hFIX promoter was replaced with an hCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression of hFIX in the modified HeLa cells, 11.2 ng/106 cell/24 h, strongly suggested that hFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.

关键词： human clotting factor IX constitutive expression homologous recombination promoter.

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constitutive expression of codon optimized Trichoderma reesei TrCel5A in Pichia pastoris using GAP promoter

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Systems Microbiology and Biomanufacturing 2022年第3期2卷 498-506页

作者： Yun Hu Renhui Bai Shaohua Dou Zhimeng Wu Ali Abdulkhani Mohammad Ali Asadollahi Abd El-Fatah Abomohra Fubao Sun Key Laboratory of Carbohydrate and Biotechnology and Key Laboratory of Industrial BiotechnologyMinistry of EducationSchool of BiotechnologyJiangnan UniversityWuxi 214122China School of Life Science and Biotechnology Dalian UniversityDalian 116622China Department of Wood and Paper Sciences and Technology Faculty of Natural ResourcesUniversity of Tehran31585-4314 KarajIran Department of Biotechnology Faculty of Biological Science and TechnologyUniversity of Isfahan81746-73441 IsfahanIran New Energy and Environmental Laboratory School of Architecture and Civil EngineeringChengdu UniversityChengdu 610106China

To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinant TrCel5A was screened out after gene optimization,synthesis,and *** biochemical and enzymatic properties of the new recombinant were *** a result,optimization of shake-flask fermentation of the recombinant was obtained at 28℃,2%inoculum volume,an initial pH of 6.0,as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L,*** the above-optimized conditions,the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of *** further enhance enzyme production,pilot-scale cultivation was evaluated using 5-L *** high-cell-density fermentation,the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/*** addition,the kinetic factors,including K_(m) and V_(max) values for TrCel5A,were detected to be 5.1 mg/mL and 265.9μmol/(***),***,TrCel5A was effectively expressed in *** under the GAP promoter,and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.

关键词： Trichoderma reesei endoglucanase(Cel5A) Pichia pastoris GAP promoter constitutive expression Fermentation optimization CMC enzyme activity

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Construction and characterization of a hrpG mutant rendering constitutive expression of hrp genes in Xanthomonas campestris ***

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Progress in Natural Science:Materials International 2006年第5期16卷 480-485页

作者： JIANG Bole, XU Rongqi, LI Xianzhen, WEI Hongyu, BAI Faan, HU Xi, HE Yongqiang and TANG Jiliang ( The Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization,College of Life Science and Technology of Guangxi University, Nanning 530004, China) [a]The Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization [b]College of Life Science and Technology of Guangxi University Nanning China

The expression of hrp genes in Xanthomonas is regulated by hrpG, an OmpR-type transcriptional activator, and can be induced by artificial mimic medium XVM2, but repressed in rich medium NYG. In this study, a site-specific mutation of hrpG in Xanthomonas campestris pv. campestris (Xcc) was generated by PCR-directed point mutagenesis and was characterized by its phenotype and transcriptional expression analyses. The results indicated that the E45K mutated hrpG rendered all the hrp genes expressing constitutively in the suppression medium NYG. Plant test showed that mutated hrpG enhanced the timing and intensity of hypersensitive reaction (HR) on the nonhost plant pepper, but did not affect the growth of Xcc in both culture medium and plants. The E45K mutation did not cause significant changes of production of exopolysaccharide, extracellular cellulase and amylase, but it inhibited the secretion of extracellular protease. This mutant 8004 is more suitable than the wild type for identifying hrp-dependent effectors in vitro and studying the transcriptional regulation genome-wide.

关键词： hrpG Xanthomonas campestris pv. campestris effector constitutive expression site-specific mutagenesis.

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构成性表达(constitutive expression)

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中华预防医学杂志 2003年第5期37卷 81-81页

作者：方福德中国医学科学院基础医学研究所 100005北京

也叫组成性表达。组成性表达是指不需要其他调节因子、RNA聚合酶与启动子相互作用而发生的基因表达。即基础表达。对于一个细胞来说，在任何时候都需要其表达一些基因通常是构成性的表达。这种表达基本上保持在恒定的水平，对于正常代... 详细信息

也叫组成性表达。组成性表达是指不需要其他调节因子、RNA聚合酶与启动子相互作用而发生的基因表达。即基础表达。对于一个细胞来说，在任何时候都需要其表达一些基因通常是构成性的表达。这种表达基本上保持在恒定的水平，对于正常代谢过程或转录过程必需的酶或蛋白因子的基因，都是构成性表达的。如管家基因。

关键词：组成性表达构成性 constitutive expression

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Deletion of 93 bp Far-upstream Fragment of Rice Cytosolic Fructose- 1, 6-Bisphosphatase Promoter Completely Alter Its expression Pattern

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Acta Botanica Sinica 2002年第11期44卷 1339-1345页

作者：司丽珍王力曹守云储成才中国科学院遗传与发育生物学研究所北京100101

The 1 195 bp 5′ flanking region of rice ( Oryza sativa L.) cytosolic fructose_1, 6_bisphosphatase (cyFBPase) can direct tissue, cell specific expression in transgenic rice. In order to identify sequence elements responsible for the regulation of mesophyll_specific expression, the 5′ flanking regions of -1 195 bp, -1 102 bp, -768 bp, and -644 bp upstream of the translation initiation ATG codon were fused to the reporter gene encoding β_glucuronidase (GUS) and transferred to rice via particle bombardment. Analysis of the 5′ promoter deletions identified that a 93 bp fragment between -1 195 bp and -1 102 bp is essential for directing mesophyll specific expression. High constitutive expression of GUS reporter gene was found in the -768 deletion lines and another two deletion series. These results indicate the great potential utility of the promoter in rice biotechnology.

关键词： rice ( Oryza sativa ) cis _regulatory elements cytosolic fructose_1,6_bisphosphatase gene mesophyll_specific expression constitutive expression

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Characterization of two constitutive promoters RPS28 and EIF1 for studying soybean growth,development,and symbiotic nodule development

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aBIOTECH 2022年第2期3卷 99-109页

作者： Shengcai Chen Yaqi Peng Qi Lv Jing Liu Zhihua Wu Haijiao Wang Xuelu Wang College of Life Science and Technology Huazhong Agricultural UniversityWuhan 430070China State Key Laboratory of Crop Stress Adaptation and Improvement School of Life SciencesKey Laboratory of Plant Stress BiologyHenan UniversityKaifeng 475001China Sanya Institute of Henan University Sanya 572025China Hubei Provincial Key Laboratory for Protection and Application of Special Plant Germplasm in Wuling Area of China Key Laboratory of State Ethnic Affairs Commission for Biological TechnologyCollege of Life SciencesSouth-Central University for NationalitiesWuhan 430074China

Native promoters that can drive high and stable transgene expression are important tools for modifying plant *** several such promoters have been reported in soybean(Glycine max),few of them function at multiple growth and development stages and during nodule ***,we report that the promoters of 40S RIBOSOMAL PROTEIN SMALL SUBUNIT S28(RPS28)and EUKARYOTIC TRANSLATION INITIATION FACTOR 1(EIF1)are ideal for high expression of *** bioinformatic analysis,we determined that RPS28 and EIF1 were highly expressed during soybean growth and development,nodule development,and various biotic and abiotic *** of both RPS28 and EIF1 promoters,with or without their first intron,with the reporter geneβ-GLUCURONIDASE(uidA)in transgenic soybean,resulted in high GUS activity in seedlings,seeds,and *** GUS assays showed that the RPS28 promoter and the EIF1 promoter yielded high expression,comparable to the soybean Ubiquitin(GmUbi)***28 and EIF1 promoters were also highly expressed in Arabidopsis thaliana and Nicotiana *** results indicate the potential of RPS28 and EIF1 promoters to facilitate future genetic engineering and breeding to improve the quality and yield of soybean,as well as in a wide variety of other plant species.

关键词： Promoter Soybean(Glycine max) Ribosomal protein S28 Translation initiation factor EIF1 constitutive expression

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A finTRIM member 100 (FTR100) is unique to Otomorpha fish for constitutive regulation of IFN response

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Water Biology and Security 2023年第2期2卷 80-90页

作者： Wen-Hao Guo Cheng Dan Xiu-Ying Gong Yi-Lin Li Hao-Yu Sun Zi-Ling Qu Li-Li An Xiang Zhao Jie Mei Jian-Fang Gui Yi-Bing Zhang State Key Laboratory of Freshwater Ecology and Biotechnology Institute of HydrobiologyChinese Academy of SciencesWuhan430072China University of Chinese Academy of Sciences Beijing10049China College of Fisheries Huazhong Agricultural UniversityWuhan430070China The Innovation Academy of Seed Design Chinese Academy of SciencesWuhan430072China

Vertebrate interferon(IFN)expression is fine-tuned in order to avoid excessive tissue injury under normal conditions and during virus ***(fish novel TRIM,FTR)proteins are reported to regulate the fish IFN ***,we identify a novel finTRIM gene from yellow catfish(Pelteobagrus fulvidraco),which is sequentially named PfFTR100 according to the nomenclature rule in ***-wide analyses reveal that FTR100 is unique to Otomorpha fish,with a single copy in spite of additional genome duplication in some fish *** that few of the 99 finTRIM genes identified in zebrafish are conserved in main fish branches and most,such as FTR100,are unique to distinct branches due to lineage-specific expansion of finTRIM genes,we develop a nomenclature for newly cloned finTRIM genes from different fish ***100 mRNA is not induced by virus infection,with a relatively high expression level comparable to that of cellular IFN and some IFN-stimulated genes(ISGs)in virally-infected ***,ectopically-expressed PfFTR100 protein is attenuated in virally-infected cells through the proteasomal-dependent *** of PfFTR100 promotes SVCV replication by downregulating the constitutive and inducible IFN response via a mechanism by which PfFTR100 targets IRF3 and IRF7 to attenuate their mRNA levels rather than their protein *** results indicate that yellow catfish FTR100 is essential for homeostatic regulation of fish tonic IFN response.

关键词： constitutive expression FTR100 Homeostatic regulation Lineage-specific expansion Tonic IFN response

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