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The Crop Journal 2014年第Z1期2卷 164-169页

作者： baoqing dun Xujing Wang Wei Lu Ming Chen Wei Zhang Shuzhen Ping Zhixing Wang Baoming Zhang Min Lin Institute of Crop Sciences The National Key Facility for Crop Gene Resources and Genetic ImprovementChinese Academy of Agricultural Sciences Biotechnology Research Institute Chinese Academy of Agricultural Sciences

The widely used herbicide glyphosate targets 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS).Glyphosate acetyltransferase(GAT)effectively detoxifies glyphosate by *** the aim of identifying a new strategy for development of glyphosate-tolerant crops,the plant expression vector pG2-GAT harboring gat and G2-aroA(encoding EPSPS)has been transformed into tobacco(Nicotiana tabacum)to develop novel plants with higher tolerance to *** from Southern and Western blotting analyses indicated that the target genes were integrated into tobacco chromosomes and expressed effectively at the protein *** tolerance was compared among transgenic tobacco plants containing gat,G2-aroA,or both *** containing both gat and G2-aroA genes were the most *** study has shown that a combination of different strategies may result in higher tolerance in transgenic crops,providing a new approach for development of glyphosate-tolerant crops.

关键词： Transgenic tobacco Glyphosate tolerance gat gene G2-aroA gene

Reconstruction of enzymatic activity from split genes encoding glyphosate-tolerant EPSPS protein of Psedomonas fluorescens G2 strain by intein mediated protein complementation

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Chinese Science Bulletin 2006年第13期51卷 1652-1654页

作者： dun baoqing LU Wei ZHANG Wei PING Shuzhen WANG Xujing CHEN Ming XU Yuquan JIN Dan WANG Jin ZHAO Zhonglin LIANG Aimin HOU Songna XU Ming-Qun LIN Min Biotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing 100081 China New England Biolabs Inc. Beverly Massachusetts 01915 USA

A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS pro- teins were identified by the ability to rescue growth of aroA-deleted mutant ER2799 on M9 minimal media. 12 unique sites, which can tolerate a 5-aa insertion, were identified. In all of the 12 sites, only F295/T296 site was found to split the G2-EPSPS properly by co-transformation of plasmids into E. coli ER2799. The G2-EPSPS gene was then divided into N-termi- nal and C-terminal from F295/T296 site which were fused to the N-terminal and C-terminal of *** intein, respectively, creating two plasmids pMEPS- N295IN and pKEPSc296Ic. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296Ic, rescu- ed growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Re- consituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.

关键词：断裂基因蛋白质再生转基因 EPSPS

A complete assembly of the sorghum BTx623 reference genome

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Plant Communications 2024年第6期5卷 13-16页

作者： Yuan Deng Peng Zhou Fei Li Jing Wang Kun Xie Hongkai Liang Chunchao Wang Bin Liu Zhenxing Zhu Wenbin Zhou baoqing dun Xiaochun Lu Xianmin Diao Qiang He State Key Laboratory of Crop Gene Resources and Breeding Institute of Crop SciencesChinese Academy of Agricultural SciencesBeijing 100081China Crop Molecular Improvement Lab Liaoning Academy of Agricultural SciencesShenyangLiaoning 110161China Zhongyuan Research Center Chinese Academy of Agricultural SciencesXinxiangHenan 453599China

Dear Editor,Sorghum(Sorghum bicolor),recognized as the world’s fifth most significant cereal crop,plays an essential role as a model for genomic comparison and functional genomics of C4 *** was domesticated in Ethiop... 详细信息

关键词： Sorghum environments

Microbial Transformation of Flavonoids by Isaria fumosorosea ACCC 37814

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Microbial Transformation of Flavonoids by Isaria fumosorosea...

Fusion Expression of Glucoamylase and Xylanase in Pichia pastoris

中国生物工程学会第十三届学术年会暨2019年全国生物技术大会

作者： Fangmin Dou Zhi Wang Guiying Li baoqing dun The National Key Facility for Crop Gene Resources and Genetic Improvement Institute of Crop SciencesChinese Academy of Agricultural Sciences

Glycosylation is an efficient strategy to modulate the solubility, stability, bioavailability and bioactivity of drug-like natural products. Biological methods, such as whole-cell biocatalyst, promise a simple but highly effective approach to glycosylate biologically active small molecules with remarkable regio-and stereo-selectivity. Herein, we use the entomopathogenic filamentous fungus Isaria fumosorosea ACCC 37814 to biotransform a panel of phenolic natural products, including flavonoids and anthraquinone, into their glycosides. Six new flavonoid(4-O-methyl)glucopyranosides are obtained and structurally characterized using high resolution mass and nuclear magnetic resonance spectroscopic techniques. These compounds further expand the structural diversity of flavonoid glycosides and may be used in biological study.

关键词： Isaria fumosorosea flavonoids microbial transformation methylglycosylation

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Agricultural Biotechnology 2012年第5期1卷 48-51页

作者： Zhi WANG baoqing dun Jingang GU Xuan ZHAO Gu TIAN Ming LU Guiying LI National Key Facility for Crop Gene Resources and Genetic Improvement of Institute of Crop Sciences Chinese Academy of Agricultural Sciences Agricultural Culture Collection of China Institute of Agricultural Resources and Agricultural DivisionsChinese Academy of Agricultural Sciences College of Life Sciences Northeast Agricultural University

Employing RT-PCR amplification method, the mature pepfide coding sequence of glucoamylase （amyA） gene was amplified from total RNA of Talaro- myces emersonii and the xylanase （xyrut） gene from genomic DNA ofPaenibacillus sp. H10-3. The result showed that the mature peptide sequence of amyA is 1857 bp long and encodes 618 amino acids; and the mature peptide sequence of xynA is 636 bp long and encodes 211 amino acids. Then these two genes were spliced by overlap extension PCR （ SOE-PCR）, yielding fusion gene amyA-l-xynA. The fusion gene amyA-l-xynA was then cloned into a Pichia pastoris expression vector pPIC9 to produce the recombinant expression plasmid pPIC9-amyA-l-xynA. The recombinant plasmid pPICg-amyA-l-xynA was linearized and transformed into Pichia pastoris GSll5 via electric shock, yielding engineering strain ALX2. The maximum yields of glucoamylase and xylanase in ALX2 fermentation supernatant were determined as 10.7 and 51.8 U/ml, respectively.

关键词： Glucoamylase Xylanase Fusion expression Pichia pastoris

利用宏基因组文库筛选草甘膦不敏感的5-烯醇式丙酮莽草酰-3-磷酸合酶（EPSPS）基因

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高技术通讯 2006年第12期16卷 1284-1288页

作者：陆伟梁爱敏孟秀萍顿宝庆金丹穆文超林敏中国农业科学院生物技术研究所北京100081

为探索利用宏基因组文库筛选草甘膦不敏感的5-烯醇式丙酮莽革酰-3-磷酸合酶（EPSPS）基因，直接从土壤中提取细菌DNA，构建草甘膦污染土壤细菌宏基因组文库，并利用EPSP合酶缺失突变株ER2799筛选到4个能互补EPSP合酶功能的克隆，其中两... 详细信息

为探索利用宏基因组文库筛选草甘膦不敏感的5-烯醇式丙酮莽革酰-3-磷酸合酶（EPSPS）基因，直接从土壤中提取细菌DNA，构建草甘膦污染土壤细菌宏基因组文库，并利用EPSP合酶缺失突变株ER2799筛选到4个能互补EPSP合酶功能的克隆，其中两个克隆的转化子能够在含5mmol／L草甘膦的MOPS培养基上生长。测序结果表明它们均含有完整的EPSP合酶编码基因，GT-1aroA核苷酸长度为1335bp，编码445个氨基酸，GT-4aroA核苷酸长度为1350bp，编码450个氨基酸。利用PCR方法将两个基因克隆到原核表达载体pET28a上后，可以使宿主细胞BL21（DE3）在含100mmol／L草甘膦的MOPS培养基中良好生长。上述结果表明，利用宏基因组文库筛选方法可以得到高抗草甘膦的EPSPS基因。

关键词：宏基因组 5-烯醇式丙酮莽草酰 -3-磷酸合酶(EPSPS) 草甘膦

一种改良的热酚法高效快速提取酿酒酵母总RNA

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生物技术通报 2012年第12期28卷 163-166页

作者：李维维顿宝庆王智曲娟娟东北农业大学生命科学学院哈尔滨150030 中国农业科学院作物科学研究所中国农业科学院生物质能源研究中心北京100081

以酿酒酵母单倍体CE ***1与双倍体***2为研究对象,利用改良热酚法,快速高效地提取酿酒酵母总RNA。结果显示应用该方法提取的酿酒酵母***1和***2的总RNA经分光光度计测定浓度为7.63μg/μL和3.77μg/μL,OD_(260/280)分别为2.10和2.04,OD... 详细信息

以酿酒酵母单倍体CE ***1与双倍体***2为研究对象,利用改良热酚法,快速高效地提取酿酒酵母总RNA。结果显示应用该方法提取的酿酒酵母***1和***2的总RNA经分光光度计测定浓度为7.63μg/μL和3.77μg/μL,OD_(260/280)分别为2.10和2.04,OD_(260/230)分别为2.32和2.24,浓度与纯度均能达到后续分子生物学试验的要求。经PCR与荧光定量PCR检测,该方法提取的RNA无DNA污染,可作为PCR反应的模板,与Trizol方法提取RNA相比,该方法不仅浓度高并且可将提取时间由常规的2.5 h缩短为1.5 h,具有操作简单、高质、高效等优点,经多次试验证明,此方法同样适用于酿酒酵母总RNA的大量提取试验,有较高的实用价值。

关键词： RNA分离酿酒酵母热酚法 cDNA 荧光定量PCR

新型高抗草甘膦N-乙酰转移酶基因的分离及其在大肠杆菌中的表达

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高技术通讯 2006年第9期16卷 943-947页

作者：顿宝庆陆伟平淑珍张维陈明徐玉泉金丹赵仲麟林敏中国农业科学院生物技术研究所北京100081

利用草甘膦极端污染土壤总DNA,构建了元基因组文库,筛选到一个高抗草甘膦的N-乙酰转移酶（N-acetyltransferase,GAT）基因,并利用原核表达系统对该基因进行了功能鉴定,发现其在大肠杆菌BL21中能耐受高达300 mM的草甘膦.研究结果为进行N... 详细信息

利用草甘膦极端污染土壤总DNA,构建了元基因组文库,筛选到一个高抗草甘膦的N-乙酰转移酶（N-acetyltransferase,GAT）基因,并利用原核表达系统对该基因进行了功能鉴定,发现其在大肠杆菌BL21中能耐受高达300 mM的草甘膦.研究结果为进行N-乙酰化途径高抗草甘膦机理研究和新型高抗草甘膦转基因作物的开发提供了理论基础.

关键词：元基因组功能筛选 N-乙酰转移酶基因大肠杆菌表达

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甜高粱茎汁及茎渣同步糖化发酵工艺优化

农业工程学报 2016年第3期32卷 253-258页

作者：陈朝儒王智马强张丽娜奚亚军顿宝庆李桂英路明杜风光西北农林科技大学农学院杨凌712100 中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程北京100081 车用生物燃料技术国家重点实验室南阳473000

为了提高甜高粱秸秆乙醇生产中茎汁和茎渣的利用,以甜高粱茎汁及其渣为发酵原料,对茎汁茎渣混合原料同步糖化乙醇发酵的工艺条件进行优化研究。采用Plackett-Burman(PB)筛选设计试验筛选出影响甜高粱茎秆渣汁同步糖化乙醇发酵的显著因... 详细信息

为了提高甜高粱秸秆乙醇生产中茎汁和茎渣的利用,以甜高粱茎汁及其渣为发酵原料,对茎汁茎渣混合原料同步糖化乙醇发酵的工艺条件进行优化研究。采用Plackett-Burman(PB)筛选设计试验筛选出影响甜高粱茎秆渣汁同步糖化乙醇发酵的显著因素。采用响应面法建立了同步糖化发酵乙醇生产的乙醇产量数学模型。根据该模型进行了工艺参数的优化,以乙醇产量为指标,试验所得甜高粱茎秆渣汁同步糖化化乙醇发酵的优化工艺条件为:发酵温度36.58℃,混合纤维素酶添加量=23.5(FBU/m L)/35.25(CBU/m L),甜高粱渣汁质量体积比为8.2%,理论预测乙醇产量为89.2%,在此条件下进行验证试验,乙醇产量为88.98%,平均质量浓度,验证了数学模型的有效性,为提高甜高粱茎汁及茎渣混合原料同步糖化发酵产乙醇和提高发酵效率提供参考。

关键词：发酵乙醇优化甜高粱茎汁甜高粱茎渣同步糖化发酵响应面分析法

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甜高粱填充甘蔗榨期生产燃料乙醇的可行性

生物工程学报 2010年第7期26卷 974-981页

作者：李桂英葛耀相梁文育覃守贵王秀玲顿宝庆中国农业科学院作物科学研究所农作物基因资源与基因改良国家重大科学工程生物质能源研究中心北京100081 广西柳州市农业科学研究所柳州545003

为了探讨在甘蔗产区种植甜高粱填充甘蔗压榨期,利用现有压榨设备生产燃料乙醇的可行性,2008年选用15个早、中、晚熟甜高粱品种在广西柳州进行了分期播种试验,从3月到9月,共播种7次,研究不同播期对茎秆产量、籽粒产量、茎秆糖锤度、叶片... 详细信息

为了探讨在甘蔗产区种植甜高粱填充甘蔗压榨期,利用现有压榨设备生产燃料乙醇的可行性,2008年选用15个早、中、晚熟甜高粱品种在广西柳州进行了分期播种试验,从3月到9月,共播种7次,研究不同播期对茎秆产量、籽粒产量、茎秆糖锤度、叶片产量等的影响。研究表明,3～8月播种,所有参试品种均能正常生长,9月底播种,所有品种均不能正常成熟。茎秆鲜产最高的品种是Sart和PT3-S,平均单季茎秆产量分别为79.28 t/hm2和78.58 t/hm2,双季茎秆鲜产分别为157.95 t/hm2和155.25 t/hm2。从6月底开始,早熟品种开始成熟,之后,不同品种陆续成熟,一直到12月底,均有品种可以收获。年度双季乙醇产量最高产量可达9.14 t/hm2。此外,还估算了木质纤维素产量,讨论了甘蔗区发展甜高粱填充甘蔗空榨期生产燃料乙醇的可行性以及甜高粱综合利用的潜力。

关键词：甜高粱甘蔗压榨期燃料乙醇