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Generation of selectable marker-free transgenic rice resistant to chewing insects using two co-transformation systems

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Progress in Natural Science:Materials International 2009年第11期19卷 1485-1492页

作者： Hengxiu Yu~a,Quanhong Yao~b,Ling Wang~(a,c),Zhipeng Zhao~(a,b),Zhiyun Gong~a, Shuzhu Tang~a,Qiaoquan Liu~(a,*),Minghong Gu~(a,*) a key laboratory of Plant Functional Genomics of the Ministry of Education,Jiangsu key laboratory of Crop genetics and Physiology, Yangzhou University,Yangzhou 225009,China b shanghai key laboratory of agricultural genetics and breeding,shanghai Academy of agricultural Science,shanghai 201106,China c China National Rice Research Institute,Hangzhou 310006,China key laboratory of Plant Functional Genomics of the Ministry of Education Jiangsu Key Laboratory of Crop Genetics and Physiology Yangzhou University Yangzhou 225009 China shanghai key laboratory of agricultural genetics and breeding Shanghai Academy of Agricultural Science Shanghai 201106 China China National Rice Research Institute Hangzhou 310006 China

To produce selectable marker-free(SMF) transgenic rice resistant to chewing insects,the Bacillus thuringiensis cryIA(c) gene(Bt) was introduced into two elite japonica rice varieties by using two Agrobacterium-mediated co-transformation *** system is with a single mini-twin T-DNA binary vector in one Agrobacterium strain,which consists of two separate T-DNA regions,one carrying the Bt while the other contains the selectable marker gene,hygromycin resistant gene(HPT).The other system uses two separate binary vectors in two separate Agrobacterium cultures,containing the Bt or HPT gene on individual plasmids.A lot of independent transgenic rice lines harboring both Bt and selectable marker genes were *** results showed that the co-transformation frequency of the Bt gene and HPT gene was much higher by using the mini-twin T-DNA vector system(29.87%) than that by the two separate binary vector systems (4.52%).However,the frequency of the SMF transgenic rice plants obtained from the offspring of co-transgenic plants(21.74%) was lower for the mini-twin T-DNA vector system than that for the latter(50-60%).The data of ELISA implied that the expressed Bt proteins were quantitated as 0.025-0.103%of total leaf soluble proteins in the transgenic ***,several elite transgenic rice lines, free of the selectable marker gene,were *** results from both in vitro and in vivo insect bioassays indicated that the SMF transgenic rice was shown to be highly resistant to the striped stem borer and rice leaf ***,in a natural field condition without any insecticide applied,all the transgenic rice plants were found to be not injured by the rice leaf folder,whereas the wild types were impaired seriously.

关键词： Oryza sativa L. Bacillus thuringiensis cryIA(c) gene Selectable marker-free(SMF) Co-transformation Insect resistance

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Genome-wide SNP in silico detection of Lentinula edodes via whole genome sequencing

Genome-wide SNP in silico detection of Lentinula edodes via ...

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中国菌物学会第五届会员代表大会暨2011年学术年会

作者： CHEN Xiang BAO Da-Peng (National Research Center for Edible Fungi Biotechnology and Engineering key laboratory of Applied Mycological Resources and Utilization,Ministry of Agriculture,China shanghai key laboratory of agricultural genetics and breeding Institute of Edible Fungi,shanghai Academy of agricultural Sciences,shanghai,China)

＜正＞Analysis of single nucleotide polymorphisms（SNPs） emerges as an important tool in evolutionary biology and *** we report the in silico discovery and analysis of SNPs across the whole genome of two protoplast monokaryons（TPMs） of basidiomycete fungi Lentinula edodes via the high-throughput sequencing *** our knowledge,this is a novel study in which different protoplast monokaryons are utilized for SNPs detection and *** sequence information including 210M and 250M nucleotide bases was generated from TPMs yielding approximately 80%of whole genome *** used surpassed 380K and 410K reads of TPMs for the de novo assembly of two contig sets with an overall estimated depth of 5.67-fold and 6.05-fold coverage,*** of the contigs between TPMs identified 108,235 putative SNPs owning a high probability of at least 10-fold *** SNPs reveal TPMs having similar intra-strain SNP frequencies,and the ratio of transition to transversion among these SNPs being similar to the ratio reported in other *** verify the confidence of detected SNPs,25,044 SNPs were mapped onto 43,912 positions that uniformly distributed over the sequenced chromosomes of Coprinopsis *** result shows high sequence conservation among closely related fungal *** addition,the gene structure of TPMs was constructed for SNPs classification according to their distribution in genome ***,this study indicates the utility of using next generation sequencing technologies to identify large numbers of reliable ***,the method combination of SNP detection and novel algorithm allows an efficient approach for the development of high-density genotyping platforms in fungal species.

关键词： Lentinula edodes whole genome sequencing contig assembly SNPs

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ACC氧化酶和ACC合成酶反义RNA融合基因导入番茄和乙烯合成的抑制

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实验生物学报 2003年第6期36卷 428-434页

作者：熊爱生姚泉洪李贤范惠琴彭日荷上海市农业遗传育种重点实验室上海市农业科学院生物技术研究中心上海 201106

从番茄品种强力米寿的总DNA中克隆番茄果实特异启动子2A11,以番茄成熟果实的RNA为模板,进行RT-PCR扩增,克隆番茄全长的ACC氧化酶基因和ACC合成酶基因片段。完成两个基因的克隆和测序后,将888bp的番茄ACC氧化酶基因和943bp的ACC合成酶基... 详细信息

从番茄品种强力米寿的总DNA中克隆番茄果实特异启动子2A11,以番茄成熟果实的RNA为模板,进行RT-PCR扩增,克隆番茄全长的ACC氧化酶基因和ACC合成酶基因片段。完成两个基因的克隆和测序后,将888bp的番茄ACC氧化酶基因和943bp的ACC合成酶基因片段串联,构成全长1837bp的融合基因。将该融合基因以反义的方向插入植物双元载体pYPX145中番茄果实表达特异启动子下游,获得ACC氧化酶基因和ACC合成酶基因融合的植物双元载体pOSACC。该载体外源基因表达单元的两端含两个烟草SAR序列,利于转基因的稳定遗传。以番茄栽培品种合作903子叶和下胚轴为外植体,利用根癌农杆菌进行基因转化,通过200mg/L卡那霉素选择和GUS检测,获得了105株番茄GUS阳性植株,转基因番茄果实在当代表现明显耐贮特点。经过4代的耐贮和果实农艺性状的综合选择,获得了两个表现良好的株系DR-1和DR-2,两株系果实乙烯释放量显著下降,是未转基因材料的9.5%,番茄的贮存期在50天以上。

关键词： ACC氧化酶基因 ACC合成酶基因反义基因融合根癌农杆菌乙烯番茄

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Molecular diversity and differential expression of starch-synthesis genes in developing kernels of three maize inbreds

Molecular diversity and differential expression of starch-sy...

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上海市植物生理学会成立三十周年庆祝活动暨第三届上海市植物生理学青年学术研讨会

作者： Xiangzhen Ding , Beiguo Wang, Qinghua Gao, Qiong Zhang , Guiqin Yan, Ke Duan, Jianhua Huang shanghai key laboratory of agricultural genetics and breeding, Institute of Biological Technique, shanghai Academy of agricultural Sciences(SAAS), BeidiRoad2901, MinhangZone, 201106 shanghai ,Chin

The maize genome remains abundant in molecular diversity, and the rich genetic diversity of maize starch-synthesis genes is crucial for controlling various graintraits. To explore the unique

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DNA Fingerprinting of Six Pleurotus nebrodensis Strains Cultivated Commercially in China

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食用菌学报 2006年第1期 20-23页

作者： TAN Qi 1 ,ROMAINE CP 2 ,SCHLAGNHAUFER C 2 ,CHEN Ming-jie 1 , GUO Qian 1 ,GEML J 2 and GARZON C 2 1 Institute of Edible Fungi, shanghai Academy of agricultural Sciences, shanghai key laboratory of agricultural genetics and breeding, shanghai 201106, P.R. China 2 Department of Plant Pathology, The Pennsylvania StateUniversity, University Park, Pennsylvania 16802, USA

The distribution of a 621 bp sequence of the ITS region of nuclear rDNA, and a 519 bp fragment of the protein-coding translation elongation factor gene (EF-1α), among six commercial strains of Pleurotus nebrodensis collected from the main cultivation area of China confirmed that the six strains belonged to the same species. RAPD data indicated that five strains were very closely related, with character differences rated at or below 3.4% using the UPGMA method of PAUP 4.0. Character differences between P. nebrodensis strain No.6 and the five other strains were rated at 16.9%, and a specific primer for this strain has been identified.

关键词： Pleurotus nebrodensis ITS Protein-coding translation elongation factor gene (EF-1α) RAPD Specific primer

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Molecular Diversity and Differential Expression of Starch-Synthesis Genes in Developing Kernels of three Maize Inbreds

Molecular Diversity and Differential Expression of Starch-Sy...

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中国植物生理学会第十次会员代表大会暨全国学术年会

作者： Xiang-Zhen Ding1,2*, Bei-Guo Wang1*, Qing-Hua Gao3, Qiong Zhang1, Gui-Qin Yan2, Ke Duan1*, Jian-Hua Huang1* 1shanghai key laboratory of agricultural genetics and breeding, Institute of Biological Technique, shanghai Academy of agricultural Sciences (SAAS), shanghai, 201106, China 2College of Life Sciences, Shanxi Normal University, Linfen, 041104, China 3shanghai key laboratory of Protected Horticultural Technology, Forest & Fruit Research Institute of SAAS, shanghai, 201106, China.

＜正＞Maize （Zea mays L.） is one of the most important agricultural crops in the grass family, and also is a source of tremendous phenotypic and molecular diversity[1,2]. Starch composition （the ratio of amylose/am... 详细信息

关键词： Zea mays starch-synthesis gene expression pattern nucleic polymorphism mis-splicing event mutation

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通过DNA改组技术获得高活性β-葡萄糖苷酸酶

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中国生物化学与分子生物学报 2003年第2期19卷 162-167页

作者：熊爱生姚泉洪彭日荷陈建民李贤范惠琴上海市农业遗传育种重点实验室上海市农业科学院生物技术研究中心上海201106 扬州大学生物科学与技术学院扬州225009

β 葡萄糖苷酸酶是在植物转基因中广泛应用的报告基因 .以质粒pBI12 1中的GUS基因为基础 ,利用DNA改组方法 ,经DNaseⅠ降解 ,PrimerlessPCR ,PrimerPCR对GUS基因进行了突变和改组 ,然后将改组的GUS基因连接到原核表达载体pG2 5 1中 ,构... 详细信息

β 葡萄糖苷酸酶是在植物转基因中广泛应用的报告基因 .以质粒pBI12 1中的GUS基因为基础 ,利用DNA改组方法 ,经DNaseⅠ降解 ,PrimerlessPCR ,PrimerPCR对GUS基因进行了突变和改组 ,然后将改组的GUS基因连接到原核表达载体pG2 5 1中 ,构建了库容为 10 8的突变体库 .经过活性的筛选 ,得到活性提高的克隆 ,再以此为基础 ,经过新的改组、筛选得到活性大幅度提高的克隆GUS2 4 .基因测序显示 ,GUS2 4与GUS基因之间的同源性为 99 7% ,共有 6个核苷酸位点发生了改变 ,分别是 :379位的A突变为G ,396位的T突变为C ,711位的G突变为A ,95 8位T突变为C ,990位的T突变为C ,1649位的A突变为G .核苷酸序列推导的氨基酸序列显示 ,3个氨基酸发生了突变 ,12 7位的Ser突变为Gly ,32 0位的Trp突变为Arg ,5 5 0位的Asn突变为Ser.X gluc染色检测和荧光测活结果显示GUS2 4基因表达的 β 葡萄糖苷酸酶基较GUS基因表达产物活性提高

关键词： β-葡萄糖苷酸酶植物转基因报告基因 DNA改组技术酶活性体外分子进化

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拟南芥热激因子cDNA的克隆及转化烟草研究

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上海农业学报 2003年第1期19卷 6-10页

作者：熊爱生彭日荷李贤范惠琴姚泉洪上海市农业遗传育种重点实验室

拟南芥热激因子是一重要的耐热基因。从拟南芥幼苗中提取总RNA ,根据报道设计引物 ,利用RT PCR的方法扩增出大约 1.4kb的拟南芥热激因子基因Athsfc。将其克隆入pBluescriptSK载体的XbaI和SacI位点 ,测序结果显示核苷酸序列与国外H櫣ｂｅ... 详细信息

拟南芥热激因子是一重要的耐热基因。从拟南芥幼苗中提取总RNA ,根据报道设计引物 ,利用RT PCR的方法扩增出大约 1.4kb的拟南芥热激因子基因Athsfc。将其克隆入pBluescriptSK载体的XbaI和SacI位点 ,测序结果显示核苷酸序列与国外H櫣ｂｅｌ(1994)报道的Athsf1基因间 99.8%同源 ,只有 3个核苷酸不同。序列分析显示 ,编码相同的氨基酸。将该基因构建入含内含子Km筛选标记的植物双元表达载体pYP12 0 3E中 ,构成了拟南芥热激因子基因 Athsfc植物表达载体pYP12 0 3E(Athsfc)。利用根癌农杆菌 (Agrobacteriumtumefaciens)对烟草进行Athsfc基因的转化 ,获得了GUS染色显示阳性的烟草转基因植株。

关键词：拟南芥热激因子 cDNA克隆基因转化转基因烟草耐热性热激蛋白

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儿茶酚双加氧酶基因大肠杆菌表达体系研究

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上海农业学报 2003年第2期19卷 1-4页

作者：熊爱生彭日荷耿其芳李贤范惠琴姚泉洪上海市农业遗传育种重点实验室

从恶臭假单孢菌 (Pseudomonassp .S 47)的DNA中扩增得到 92 4bp编码儿茶酚双加氧酶的基因片段 ,将其构建入 pBluescriptSK载体 BamHI和 SacI位点间。测序结果显示与Kim ( 2 0 0 0 )报道一致。将该基因插入带庆大霉素抗性基因启动子和t1t... 详细信息

从恶臭假单孢菌 (Pseudomonassp .S 47)的DNA中扩增得到 92 4bp编码儿茶酚双加氧酶的基因片段 ,将其构建入 pBluescriptSK载体 BamHI和 SacI位点间。测序结果显示与Kim ( 2 0 0 0 )报道一致。将该基因插入带庆大霉素抗性基因启动子和t1t2终止子载体pG2 5 1、分别带ompA和EAP信号肽、lpp和Gmr 启动子载体 pYF3 95和 pYF5 2 5 4中 ,构建组成型表达载体pG2 5 1,分泌型表达载体 pYFX1,pYFX2。酶活测定结果显示pG2 5 1为 665mu/mL ,pYFX1,pYFX2分别为 1815mu/mL ,10 15mu/mL。SDS PAGE电泳显示表达蛋白产物的分子量约为 3 5kD。分泌型表达载体pYFX1表达量大于 pYFX2 ,组成型表达载体

关键词：儿茶酚双加氧酶基因大肠杆菌表达体系基因克隆基因表达酶活分析环境污染

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Analysis of Bacterial Communities Present in Agaricus bisporus Phase II Compost Using Amplified Ribosomal DNA Restriction Analysis (ARDRA)(Ⅰ)——Construction of a Bacterial 16S rDNA Library

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食用菌学报 2008年第4期 16-19页

作者： HE Lihong~(1,2) YU Rongli~1 CHEN Mingjie~(1*) PAN Yingjie~3(~1key laboratory of Applied Mycological Resources and Utilization,Ministry of Agriculture,the People's Republic of China,shanghai Research Center for Edible Fungi Biotechnology and Engineering,shanghai key laboratory of agricultural genetics and breeding,Institute of Edible Fungi,shanghai Academy of agricultural Sciences,shanghai 201106,China ~2Department of Microbiology,College of Life Sciences,Nanjing agricultural University,Nanjing,Jiangsu 210095,China ~3shanghai Ocean University,shanghai 200090,China)

An efficient and cost effective method for obtaining good quality DNA from composts containing high levels of organic matter has been *** protocol consisted of sample washing with sodium phosphate and EDTA,cell wall disruption using combined chemical and enzyme treatment prior to extraction,removal of humic acid and other inhibitors in high salt buffer containing polyvinyl polypyrrolidone (PVPP),and precipitation of DNA with polyethylene glycol (PEG-8000).The method was used to extract 16S ribosomal DNA from four samples taken at different stages in the preparation of PhaseⅡcompost used for Agaricus bisporus *** DNA was sufficiently free of contaminants to allow PCR amplification of products derived from bacteria,actinomycetes and *** products derived from PCR amplification of the extracted 16S rDNA using primers F27 and R1498 were used to construct 16S rDNA *** amplification of ten randomly selected positive clones using primer M13 revealed that the 16S rDNA insertion rate was higher than 90%.

关键词： Agaricus bisporus compost phaseⅡ bacterial 16S rDNA library

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