Cloning AtPCS1 gene and Agrobacterium-mediated transformation of AtPCS1 into Medicago sativa.L

利用植物修复重金属污染土壤是一种廉价并且可行的修复技术。大部分已发现的超富集植物生物量小，限制了其在植物修复方面的应用。本实验借助基因工程手段将重金属富集、耐受相关的基因AtPCS1转入生物量大且适应性广的苜蓿中，以期获得能够富集重金属的苜蓿，为今后的工作提供基础材料。首先，比较了六种不同基因型苜蓿（阿尔岗金、三得利、陇东、甘农一号、大花及天兰）愈伤组织的形成及分化能力。以叶片为外植体，在含1.0mg/L2，4-D、0.1mg/LKT的B5培养基上诱导胚性愈伤组织，将愈伤组织置于无激素的B5培养基上诱导植株再生。六种不同基因型的苜蓿愈伤形成率均在90％以上，品种对苜蓿愈伤组织形成能力差异并不显...Plants have many natural properties that make them ideally suited to clean up polluted soil in the process called phytoremediation. Most native hyperaccumulators can not be used broadly for their slow growth and little biomass. We tried to use transgenic approach for transferring the metal-accumulate related gene (AtPCS1) to the plant with advantages of high biomass and easy grown — alfalfa, to ob...学位：理学硕士院系专业：生命科学学院生物化学与生物技术系_细胞生物学学号：20032607

A Convenient Method for Condon Adjustment by Using High-fidelity DNA Polymerase Amplification Property

在基因工程研究中,经常要涉及到对目的基因读码框进行调整的研究。本文报道了利用高保真DNA聚合酶扩增DNA后产生平末端的特性,及限制性内切酶对一重组的原核表达载体pET28a-HA进行读码框调整的研究,测序结果显示读码框按照预期设计正确调整,表明高保真DNA聚合酶可以用于DNA 3'凹陷端的补平,在DNA 3'凹陷端平滑化时多了一个有效的选择。The reading frame adjustment of target genes was often occurred in the study of gene engineering.The results of pET28-HA gene reading frame exactly adjustment showed high-fidelity DNA polymerase can be used generate blunt end from a DNA 5'-protruding end.The convenient method can be selected in the blunt of DNA 5'-protruding end

Study on transformation system of alfalfa and constructed AtPCS1 plant expression vector

通过对6种不同基因型苜蓿愈伤组织的形成能力、愈伤组织的分化能力的比较发现,不同苜蓿品种间的分化率存在显著差异(P<0.05),其中甘农1号分化率最高(42.9%);对甘农1号叶片、下胚轴及子叶等外植体的分化再生能力研究发现,叶片起源的愈伤组织分化时间短,且丛生芽形成数量高于下胚轴和子叶;再生芽可在B5培养基上成苗,并在蔗糖浓度为10 g/L的1/2 MS培养基上顺利生根.据此认为,苜蓿甘农1号叶片是适于做遗传转化的理想材料.利用RT-PCR方法扩增拟南芥螯合肽合成酶(AtPCS1)基因全序列构建了AtPCS1的植物表达载体pBI121-AtPCS1,为下一步AtPCS1基因转化苜蓿奠定了基础.Six cultivars of alfalfa with different genotypes were compared with each other in their callus induction capacity and plant regeneration ability.The significant differences were observed(P<0.05) in regeneration ability of these genotypes,and the cultivar Medicago sativa'Gannong No.1' has that the highest regeneration ability.Then the callus induction and plant regeneration ability of different explants as leaf,hypocotyls and cotyledon of Gannong No.1 were studied,and found that the calluses coming from leaf differentiated much earlier and had more regeneration buds than others.The in vitro regenerated plants were successfully rooted in 1/2 MS medium and 10 g/L sugar.The results above suggested that the leaves of the cultivar 'Gannong No.1' would be fit for the ideal explants in transgenic researches.Meantime,the full length of AtPCS1 gene was amplified by RT-PCR from Arabidopsis thaliana(ecotype Columbia).Furthermore,the plant expression vector pBI121-AtPCS1 was constructed,and that were as basic for transgenic alfalfa research in future.福建省自然科学基金(D041004);; 甘肃省自然科学基金(3ZS042-B25-011);; 兰州理工大学博士基金(SB08200602)

Comparison of four promoters for transient expression of RFP reporter gene in cultured Bombyx mori cells (Bm-e-HNU5)

以红色荧光蛋白基因(RFP)为报告基因,构建含4种不同启动子的重组表达质粒,用脂质体介导法转染家蚕Bombyxmori细胞(Bm-e-HNU5),观察家蚕细胞质肌动蛋白4基因启动子(A4)、α微管蛋白基因启动子(α-tub)、蚕丝心蛋白重链基因启动子(Fib)和家蚕核型多角体病毒早期即刻蛋白基因启动子(IE)4种启动子调控RFP报告基因在家蚕细胞内的瞬时表达情况。构建的重组表达质粒pDsRed-α-tub、pDsRed-A4、pDsRed-IE和pDsRed-Fib经双酶切和PCR鉴定正确无误。转染和转录实验结果表明,除了pDsRed-A4外,其他3种重组质粒在Bm-e-HNU5细胞中都得到高转染率,α-tub、IE和Fib可依次增强RFP报告基因在家蚕细胞内的瞬时表达活性。The red fluorescent protein reporter gene (RFP) was used to construct recombinant plasmids containing four different promoters, i. e., the cytoplasmic actin4 promoter (A4), α-tubulin promoter (α-tub)from silkworm, the Bombyx mori nuclear polyhedrosis virus immediate early protein promoter (IE) and the fibroin heavy chain gene promoter (Fib), respectively. These recombinant plasmids, i. e., pDsRed-A4,pDsRed-α-tub,pDsRed-Fib and pDsRed-IE, had been constructed successfully by restriction enzyme digestion and PCR analysis, and then were transfected into B. mori cell lines (Bm-e-HNU5) by lipid-mediated method to observe the ability of the four promoters to drive RFP reporter gene transient expression in cells. Transfection and transcription experiments indicated that except pDsRed-A4, the other three kinds of recombinant plasmids all transfected Bm-e-HNU5 obviously. The promoters of α-tub, IE and Fib enhanced the transient expression activity of RFP reporter gene in the Bm-e-HNU5, and their activity strengthened sequentially.国家自然科学基金项目(39870410

Prokaryotic expression of the recombinant AtPCS1 and preparation of polyclonal anti-serum against AtPCS1

利用RT-PCR扩增拟南芥螯合肽合成酶(AtPCS1)基因全序列,构建原核表达载体pET28a- AtPCS1并测序.将该载体转化大肠杆菌BL21,用0.75 mmol/LIPTG诱导融合蛋白AtPCS1-His的表达,纯化该融合蛋白.用纯化的融合蛋白免疫小鼠,ELISA法测定抗血清的效价可达1:2 000.抗血清与融合蛋白及拟南芥总蛋白的western-blot结果表明:制备的抗血清具有较高的活性和特异性.The full length of AtPCS1 gene was amplified by RT-PCR.After sequencing,AtPCS1 expression plasmid pET28a-AtPCS1 was constrasted.The E.coli BL21 containing the expression plasmid was induced by 0.75 mmol/L IPTG and the fusing protein was purified.Mouse was immunized with the purified protein. The results of ELISA show that the titer of the anti-serum is about 1:2 000 and the western-blot analysis of the anti-serum with purified protein AtPCS1-His or the total protein of Arabidopsis thaliana show that the polyclonal anti-AtPCS1-His serum has high activity and specialty.甘肃省自然科学基金(3ZS042-B25-011);; 兰州理工大学博士基金(SB08200602);; 甘肃省教育厅研究生导师基金(0703-11)资

Social Capital,Political Connections and Corporate Investment Decision

本文引入"社会资本"这一社会学概念,首次从微观视角实证研究了我国各省社会资本水平差异对上市公司的对外投资决策、股权投资类型选择以及多元化投资决策的影响,并进而深入探讨了社会资本与公司政治关系在影响公司投资决策方面的相互替代作用。结果发现:在社会资本水平较高的省份,上市公司更倾向于对外投资,也更愿意与其他企业组成共同控制的合营企业,并且其多元化投资的意愿更强。而且社会资本与政治关系在公司投资决策中所起的作用是可相互替代的,即当公司没有政治关系可资利用时,社会资本对公司投资决策的影响程度更强,反之反是。本文的研究不仅丰富了社会资本在财务学领域的研究内容,而且开拓了社会资本与政治关系交叉研究的新领域。 This paper introduces the sociological concept of / social capital0 , and for the first time ever, empirically studies the influence of the different levels of social capital of Chinese provinces on outward investment decisions, selection of the desired type of equity investment, and investment diversification decisions, from a micro perspective. Furthermore, we conduct an in- depth analysis of the interchangeability of the roles of social capital and corporate political connections in corporate investment decisions. We find that in provinces with higher levels of social capital, public- listed companies are more inclined towards outward investment and more willing to form joint ventures with other enterprises and show a stronger desire to diversify their investments. We also find that the roles of social capital and political connections are interchangeable in corporate investment decisions; in other words, when no political connections are available, social capital tends to have a stronger influence on the company. s investment decision, and vice versa. Our study has not only enriched the research of social capital in the context of financial science, but also opened up a new field in the interdisciplinary research of social capital and political connections国家自然科学基金项目(70902039);教育部人文社会科学研究项目(01JA910002);福建省社科基金项目(2009C009)的资

Study on constructed AtPCS1 plant expression vector and transformation of Medicago sativa

扩增拟南芥(ArAbIdOPSIS THAlIAnA)螯合肽合成酶(ATPCS1)全长基因;构建ATPCS1植物表达载体PbⅠ121-ATPCS1,进一步转化农杆菌EHA105;利用转化的农杆菌EHA105侵染甘农一号苜蓿(MEdICAgO SATIVA)叶片,经过80--100 d的筛选与培养,获得57株再生转基因植株。随机抽取其中9株进行PCr检测,其中6株为阳性。初步结果表明,ATPCS1基因已整合到苜蓿基因组中。The full length of AtPCS1 gene was amplified from Arabidopsis thaliana(ecotype Columbia),and AtPCS1 plant expression vector pBI121-AtPCS1 was constructed.Furthermore,the expression vector was transferred into Agrobacterium EHA105,and AtPCS1 was transferred into alfalfa(Medicago sativa) Gannong No.1 by leaf infection method.Fifty-seven transgenic alfalfa plants have been obtained after a period of 80-100 d after transformation.Six in nine random chosen plants were positive transgenic plants identified by AtPCS1-specific PCR.Results from this study showed that AtPCS1 has been transferred into genome of alfalfa.甘肃省教育厅研究生导师基金(0703-11);兰州理工大学博士基金(SB08200602

Agrobacterium-mediated transformation of AtPCS1 into Medicago sativa.L

利用RT-PCR扩增拟南芥螫合肽合成酶(AtPCS1)基因全序列.进一步构建AtPCS1的植物表达载体pB I 121-AtPCS1,转化农杆菌EHA105;然后用转化的农杆菌EHA105以叶盘法侵染苜蓿甘农一号叶片,在50mg/L Kan的筛选压下,经过约80～100d的筛选,获得57棵再生苗.随机取其中9棵再生苗进行PCR检测,其中6棵为阳性.初步鉴定表明AtPCS1基因已整合到苜蓿基因组中.The full length of AtPCS1 gene was amplified by RT-PCR from Arabidopsis thaliana(ecotype Columbia).Furthermore,AtPCS1 plant expression vector pBⅠ121-AtPCS1 was constructed and transfered into Agrobacterium EHA105 and AtPCS1 gene was transferred into alfalfa Gannong No.1 by leaf infection method.Transgenic alfalfa plants have been regenerated under the 50 mg/L Kan concentration pressure after 80～100 days.Six putative transgenic plants were screened by AtPCS1-specific PCR in nine randomly chosen samples.The primary results showed that the AtPCS1 has been transferred into alfalfa.甘肃省自然科学基金项目(3ZS042-B25-011);; 兰州理工大学博士基金项目(SB08200602);; 幅建省自然科学基金项目(D0410004)资助

低温制备N-Fe共掺杂TiO_2-SiO_2可见光催化复合薄膜

采用溶胶-凝胶结合低温(<100℃)热水后处理法,在塑料衬底上制得N-Fe共掺杂锐钛矿TiO_2-SiO_2复合薄膜。采用多种技术手段对薄膜样品进行了表征,并考察了薄膜样品在可见光下对罗丹明B的降解能力。研究结果表明,有机衬底上形成了锐钛矿TiO_2纳米晶弥散分布的复合薄膜,薄膜具有较高的可见光催化效率,150min后对罗丹明B的降解效率达到77.4%,其中矿化率达61%