李明刚
植物及微生物分子生物学、基因工程、分子遗传学等
个性化签名
- 姓名:李明刚
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学术头衔:
博士生导师,
- 职称:-
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学科领域:
物理海洋学
- 研究兴趣:植物及微生物分子生物学、基因工程、分子遗传学等
李明刚博士,天津市遗传学会理事长,南开大学生命科学学院教授,遗传学与细胞生物学系主任,分子生物学博士生导师,1992-1993年作为访问学者赴日本东京大学进行合作研究,1994-1997年赴日本北海道大学攻读博士学位,1997年3月于北海道大学生物化学与分子生物学专业博士研究生毕业,并获得博士学位归国。
课题组长在植物及微生物分子生物学、基因工程、分子遗传学等领域具有较深造诣,首次证明磷饥饿能诱导植物根系分泌植酸酶,对其诱导机制、基因克隆等进行了深入系统研究,其成果在Plant and soil, Soil Sci. Plant Nutr.,J. Crop Sci.,Biotechnol. Appl. Biochem.,Journal of Applied Microbiology, Biosystem studies等国际性学术刊物发表论文15篇(SCI收录),近年来共发表论文50多篇,获省级科技进步二等奖一项,国家发明专利三项。出版了《植物基因操作原理与技术》(全书53.3万字,2000年天津科学技术出版社出版)、《真核基因表达调控》(76万字,2002年科学出版社出版),《GeneVI中译本》电子版(150万字,生物信息网),《高级分子遗传学》(140万字,2004年科学出版社出版)等专著。
“十五”期间曾主持“863”“多功能基因工程菌的构建及应用”,国家转基因研究与产业化专项“磷高效利用型转基因大豆研究”,云南省院省校合作项目“植酸酶工程菌肥研究”和天津市农业重点攻关项目“植酸酶基因克隆与大豆遗传转化研究”等科研项目,均顺利完成并取得多项成果。
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成果数
16
【期刊论文】植酸酶基因表达与调控机制研究II. 番茄幼苗cDNA文库构健与植酸酶基因筛选
李明刚, 张敏, 刘迅, 龚雪琴, 郝亚桓, 姬生健
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-1年11月30日
采用异硫氰酸胍法从发芽3-4天的番茄幼苗中提取出总RNA,用Oligo(dT)纤维素亲和层析分离纯化mRNA。以mRNA为模板,Oligo(dT)为引物用逆转录法合成双链cDNA。将cDNA与EcoRI接头连接后克隆到表达载体λgt11的单一EcoRI位点,经体外包装后感染宿主菌Y1090,成功地构建了完整的番茄幼苗cDNA文库。用颜色筛选法筛选重组子,然后用植酸酶抗体做探针进行免疫筛选,得到两个阳性克隆。挑取阳性克隆噬菌斑扩增后纯化DNA,经琼脂糖凝胶电泳鉴定,显示确有外源DNA存在。该插入片断序列测定正在进行。
番茄, 植酸酶, cDNA文库, 克隆
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【期刊论文】缺磷条件下作物根系分泌特性的初步研究I.分泌性磷酸酶与有机酸的诱导
李明刚, 邹超亚, 但野利秋
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-1年11月30日
本文选择低磷耐性较强的羽扇豆和低磷耐性较弱的番茄两种作物,在不同磷水平下水培,对其根系分泌特性进行了比较研究。结果表明,在低磷条件下,羽扇豆酸性磷酸酶和有机酸分泌能力比番茄高2-3倍。番茄体内磷的生理临界浓度显著比羽扇豆为高,可能是其低磷耐性较低的重要原因。
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李明刚, 李桂兰, , 晏晶, 杨少辉, 薄涛, 姜洪州, 李明刚*
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-1年11月30日
黑芥子酶是一类催化水解芥子油苷的同工酶,Pyk10是在拟南芥根和下胚轴中特异表达的黑芥子酶基因,Pyk10基因的启动子具有根组织特异性。根据已发表的序列,用PCR方法从拟南芥基因组中扩增并克隆了Pyk10基因启动子片段。序列分表明:该片段全长1454bp,与已发表序列同源性达到98%。该启动子片段中含有多种在其他植物启动子中发现的通用启动子元件,并含有器官和组织特异性转录因子的结合位点有ACGT-、CANNTG-、GATA-以及I盒等顺式元件。Pyk10根特异性启动子的成功克隆,为培育抗根部病虫害和营养高效利用型转基因植物奠定了基础。
拟南芥, pyk10, 根特异性, 启动子
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【期刊论文】Construction of a recombinant Pichia Pastoris strain secreting insulin
李明刚, Shao-Hui Yang, Long-Fei Wang, Gui-Lan Li, , Hou-Jian Hua, Tao Bo, Gong Chen, Xin Li, Hong-Zhou Jiang and Ming-Gang Li*
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-1年11月30日
An intact insulin gene was synthesized by half-enzymic method and cloned into expression vector pPIN9K. After transferring the expression vector pPINS319 into Pichia pastoris GS115, two Pichia pastoris strains secreting high-level human insulin were obtained. The expressed human insulin proteins of recombinant 4 and 9 were 76 and 62 times higher than that of the negative control, respectively. Moreover 88%-90% of the insulin was secreted out of the cells. The actual insulin products of recombinant 4 and 9 calculated according to the sample volume reached 0.563g/l and 0.441g/l, respectively.
expression, human insulin gene, Pichia pastoris GS115, secretion
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【期刊论文】Cloning and Expression of Quail Ovalbumin cDNA in Pichia. Pastoris
李明刚, Gong Chen, Xing-Chun Yu, Jiatong Chen, Xin Li and Minggang Li∗
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-1年11月30日
The cDNA encoding quail (coturnix) ovalbumin was cloned from quail oviduct by the method of RT-PCR, which was inserted into the P. pastoris genome downstream of the methanol inducible 5' alcohol oxidase (AOX) promoter to replace the AOX1 gene using the plasmid vector pPIC9, and a recombinant P. pastoris strain efficiently secreting quail ovalbumin was successfully constructed. ELISA analysis using a polyclonal antibody raised against quail ovalbumin showed that, induction by 0.75% methanol for 48h led to synthesis of secreted quail ovalbumin to give around 5.45 g l-1, respectively. The recombinant ovalbumin was purified into homogeneity later through ion exchange and gel filtration chromatography. SDS-PAGE analysis revealed that, compared to natural ovalbumin, the recombinant ovalbumin is glycosylated in the similar extent by P. pastoris.
AOX promoter, insertion, secretion, induction, SDS-PAGE
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【期刊论文】Construction of Multi-fuctional Bacillus Mucilaginosus Strain Secreting Phytase
李明刚, Xin Li, Shaohui Yang, Xinchun Yu, Zhaoxia Jin, Weidong Li, Li Li, Jun Li, and Minggang Li*
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-1年11月30日
Silicate bacteria or Bacillus mucilaginosus, which is a special Bacillus species producing a rich variety of exo-polysaccharides and have some capability of dissolving potassium, phosphorus and silicate from minerals, may have wide potential uses in agriculture and industry. In order to increase its capability on hydrolyzing phytate phosphorus so as to make a microbial fertilizer, a transgenic Bacillus strain highly secreting phytase was constructed by the particle bombardment method using a phytase secreting expression vector pSP43 with a mini-tn5 transposon and a phytase expression cassette. In this study, the vector pSP43 was triumphantly transferred into the Bacillus mucilaginosus, and three transgenic strains with a stable copy of phytase expression cassette integrated into the chromosomal of the Bacillus mucilaginosus by tn5 transposition were selected. The phytase activity of engineering strains obtained increased 36-46times in comparison with the start strain D4B1. This is the first report made a transgenic Bacillus strain by the particle bombardment method successfully.
Bacillus mucilaginosus, microbial fertilizer, phytase, Tn5 transposition.,
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【期刊论文】A high efficient method for filling-in the cohesive ends of double-stranded DNA
李明刚, Shao-Hui Yang, Xin Li, Weidong Li, Jianhua Hou, and Ming-Gang Li*
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-1年11月30日
This paper reports a high efficient method for making cohesive end of double-stranded DNA into blunt end. Klenow fragment and Pfu DNA polymerase were used to fill-in the cohesive ends, and their illing-in efficiency and the corresponding cloning efficiency were compared. The results indicated the filling-in efficiency of Pfu DNA polymerase is 1.89 times that of Klenow fragment, and the difference was significant (p﹤0.01).
Klenow fragment, Pfu DNA polymerase, Filling-in
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【期刊论文】The construction of a transgenic yeast strain secreting phytase
李明刚, ZHU Jianhong, SUN Jian, CHEN Jia, LI Guilan, WU Zuowei, WANG Longfei, CHEN Gong, Marc G. Fortin, LI Minggang
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-1年11月30日
1.4-kb phytase gene amplified by RT-PCR from A. ficuum total RNA was successfully cloned into the expression vector pYES2, subsequently the recombinant expression vector pYPA1 was obtained. By the transformation with pYPA1, a transgenic S. cerevisiae that could express extracellular functional phytase was constructed. The successful construction of this engineering yeast strain made the roundwork for research on feed additive and microbial fertilizer.
phytase, S., cerevisiae, secretion
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【期刊论文】Cloning of the phytase gene phyA from Aspergillus ficuum 3.4322 and its expression in yeast
李明刚, Li Gui-Lan, Zhu Jian-Hong, Sun Jian, Wu Zuo-Wei, Chen Jia, Yan Jing, Wang Long-Fei, Chen Gong, Jiang Hong-Zhou, Li Ming-Gang*
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-1年11月30日
A phyA gene was cloned from Aspergillus ficuum 3.4322 by reverse transcription polymerase chain reaction. The amplified fragment was cloned into the pMD18 T-vector and sequenced. Nucleotide equence analysis of the phyA gene showed that it comprised 1347bp without the signal peptide sequence and encodes a polypeptide of 448 amino acids. The phyA sequence has been deposited in GenBank (accession number: AF537344). Expression vectors pYPA1 and pYPA2 were constructed by cloning the phyA gene with and without the signal peptide sequence into the yeast expression vector pYES2. The recombinant plasmids were transformed into Saccharomyces cerevisiae INVSc1 by the method of LiAc. Phytase activity was found in pYPA2 (about 11.55IU/ml) endocellular fluid and in pYPA1 supernatant (about 11.60IU/mL) by galactose inducing. The results demonstrated that the phyA gene had been expressed in Saccharomyces cerevisiae and the signal sequence of Aspergillu ficuum3.4322 could facilitate the phytase secretion from S. cerevisiae efficiently.
Aspergillus ficuum 3., 4322, Saccharomyces cerevisiae INVSc1, phyA, phytase, expression, secretion
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【期刊论文】A Study on the Transgenic Soybean Plants Containing Lower Phytate
李明刚, Minggang Li, Hongzhou Jiang, Jianhong Zhu, Guilang Li, Jian Sun, Gongchen, Longfei Wang
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-1年11月30日
Phyt1 gene was successfully transferred into soybean (Glycine max L. Merr. c.v. Jilin35, China) by Agrobacterium mediated method in this study. The results of molecular detections (Southern, Nouthern and Western) demonstrated that the phyt1 transgene was expressing in the transgenic soybean plant To and T1 generations, thereby caused a higher phytase activity and a lower phytate content in the ransgenic plants. This study is the first report about the transgenic soybean plants containing lower phytate.
Glycine max L., Merr., ,, transgene, low phytate
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