质粒体

Methods: Genes of MCP and DAF were connected by 10aa coding sequence of enriching Gly and Ser. Then insert into pcDAN3.1 vector, constituting pcDNA-MD3 expression plasmid of hMCP-DAF fusion gene. The genes were transformed by liposome into the pig endothelial cell; human serum handles masculine cloning, identify the function that restrain breaking of person alexin.

将MCP、DAF两个基因以富含Gly和Ser的10aa编码序列作铰链串接，并接入表达载体pcDNA 3.1中，构成含hMCP-DAF的真核表达质粒(pcDNA-MD3)；以脂质体转染猪内皮细胞，人血清处理阳性克隆细胞，鉴定抑制人补体溶破的功能；应用显微注射将线性基因导入猪受精卵；PCR检测导入基因整合率。

GFP gene eukaryotic express plasmids were transfected into ES cells. The cells were observed under the inverted fluorescent microscope. The clones which expressed the powerful green fluorescence were chosen to be named ES-GFP cells and cultured continuously. The examination of ES cell totipotent including Alkine phosphatase staining, embryonic body formation in vitro and teratomas formation in nude mice was implemented.

1。采用脂质体方法将GFP质粒转染ES-D3细胞株，经筛选后倒置荧光显微镜观察细胞形态及荧光状态，挑取荧光最强的克隆，命名为ES-GFP细胞，进一步扩增培养，观察细胞生长及荧光表达情况，行细胞分化全能性鉴定包括碱性磷酸酶（alkine Phosphtase，AKP）染色、体外胚胎体（embryonic body，EB）形成实验及裸鼠体内成瘤实验。2。

The method will involve a sequence of ammonification of glass surface, functionalization of carbon nanotube surface, and bonding between the nanotubes.

将奈米碳管与其它材料以一定的比例复合而制备，进而形成一种具有奈米碳管之复合吸附材料所组成之固相萃取柱介质，其合成步骤包括：奈米碳管表面改质官能化，玻璃粒体之载体氨化及最后将两者经缩合剂作用而产生化学键结使奈米碳管批覆在玻璃粒体之载体上。

The constructed pcDNA3-IL-15 plasmid was identified by Bgl Ⅱ digestion,PCR amplification and sequenceanalysis respectively,to ensure that the hIL-15 cDNA was properly inserted into thevector and the sequence was correct.Then the pcDNA3-IL-15 plasmid was instantlyexpressed in COS-7 cells by means of lipofectamine transfection,and followed by astable expression in Chinese hamster oval cells.8 positive cell colonies wereobtained after the G418 selection,designated CHOA~H cells.

经过BglⅡ酶解分析，PCR扩增鉴定和DNA测序分析，证明IL-15 cDNA已正确插入克隆载体且序列正确后，采用脂质体法，首先将pcDNA3-IL-15重组质粒转染至非洲绿猴肾细胞（COS-7）做瞬时表达，再转染至中国仓鼠卵巢细胞进行稳定表达。

By the electropositive Liposome 2000, in the 6-plate for cell culture, pEGFP-P80 above is transfected into the PK-15 cells from the kidney of pigs and expressed.

利用阳性脂质体L2000 介导，在六孔细胞培养板中，将已经构建好的pEGFP-P80重组表达质粒转染到PK-15 细胞中进行表达。

The expression of CD59 on the cell membrane was tested by cell immunohistochemistry and flow cytometry. Results pALTER plasmid containing CD59 was cut with restriction enzymes and a 496 bp fragment obtained by electrophoresis, which was complete conformity with the insert.

将两种含有不同突变体的人CD59全长cDNA序列重组pALTER质粒，应用阳离子脂质体导入法与pcDNA共转染CHO细胞，用G418筛选阳性克隆，应用细胞免疫组化及流式细胞仪检测CD59在CHO细胞表面的表达。

Inaddition,we extract total RNA from cultural U251 cell of humanglioma,make coding gene extron amplification of TfR through ReverseTranscription-Polymerase Chain Reaction,construct expressionplasmid of pCDNA3.1-TfR after digestion of enzyme andconjunction,do transfection to U251 cell by liposome afteridentification,cause excess expression,detect the efficiency of transientexpression of TfR protein by pEGFPN1-TfR, sieve positive cell ofpCDNA3.1-TfR through G418, choose positive cell clone, detect theexpression of TfR in anteroposterior period through Western blot,FCM,cell immunochemistry and mRNA through RT-PCR, identify the effect of transfection.

另外，培养人脑胶质瘤U251细胞，进行细胞总RNA抽提，运用RT-PCR方法进行TfR基因的完整编码区外显子的扩增，经过酶切、连接后构建pCDNA3.1-TfR的表达质粒，鉴定正确后按脂质体转染方法转染人胶质瘤U251细胞，使其过量表达，通过pEGFPN1-TfR检测其瞬时转染的效率；用G418对稳定表达pCDNA3.1-TfR的细胞进行筛选，挑选阳性细胞克隆，运用Western blot、流式细胞检测技术和细胞免疫化学以及mRNA半定量的方法检测TfR在转染前后的表达水平，鉴定转染的效果。

The main results are as following:(1) Arabidopsis in planta transformation were made by Agrobacterium tumefaciens carrying the activation tagging vector pSKI015 with herbicide-resistant Bar gene as a selective marker. About 12 000 independent transformant lines were acquired by the above method, from which two mutants related to plant defense response and development, asr1 (auxin and .salicylic acid responsive 1.) and dai1 (brought tolerant and obscisic acid insensitive 1) were isolated.

本文以拟南芥为实验材料，以激发标签为技术平台，构建了拟南芥的功能获得突变体库，从中筛选具有抗病、抗逆表型的突变体，克隆基因并进行基因的功能研究，取得的主要结果如下：(1)利用含激发标签质粒pSK1015的农杆菌直接转化拟南芥植株，以抗除草剂的Bar基因为筛选标记，获得了约有12000个独立转化株系的突变体库，并从中筛选到2个与抗病、抗逆和发育相关的突变体asr1(auxin and salicylic acid responsive 1)和dai1(drought tolerant and abscisic acid insensitive 1)。

The main work of this dissertation is constructing Arabidopsis gain of function mutants pool, screening mutants related to plant defense response, cloning the genes and studying their functions. The main results are as following:(1) Arabidopsis in planta transformation were made by Agrobacterium tumefaciens carrying the activation tagging vector pSKI015 with herbicide-resistant Bar gene as a selective marker. About 12000 independent transformant lines were acquired by the above method from which two mutants related to plant defense response and development, asr1 (auxin and salicylic acid responsive 1) and dai1 (drought tolerant and abscisic acid insensitive 1) were isolated.

本文以拟南芥为实验材料，以激发标签为技术平台，构建了拟南芥的功能获得突变体库，从中筛选具有抗病、抗逆表型的突变体，克隆基因并进行基因的功能研究，取得的主要结果如下：(1)利用含激发标签质粒pSKI015的农杆菌直接转化拟南芥植株，以抗除草剂的Bar基因为筛选标记，获得了约有12000个独立转化株系的突变体库，并从中筛选到2个与抗病、抗逆和发育相关的突变体asrl(auxin and salicylic acid responsive l)和dail(dought tolerant and abscisic acidinsensitive l)。

A transposon mutagenesis system applicable for Xanthomonas campestrispv.campestrishas been developed by transferring the transposonTn5gusA5,which contains the gus reporter gene,to the broad host rangvector pLAFR1 and taking the advantage of incompatibility between pLAFR1and pPH1JI plasmids.Five different types of exopolysaccharidedeficient mutants related respectively to eight different mutated sites wereisolated by applying the system to mutagenize the Xcc wild type strain.

本研究将含gus报告基因的转座子Tn5gusA5转座到广谱宿主载体质粒pLAFR1后，利用pLAFR1与pPH1JI质粒的不相容性，建立了一套适合于甘蓝黑腐病菌的转座子诱变体系，应用该体系诱变甘蓝黑腐病菌野生型菌株，成功地分离到5种不同类型、分别涉及8个不同突变位点的胞外多糖突变体。

She gently rebuff ed him, but agreed that they could be friends

她婉言拒绝了，但同意作为朋友相处。

If in the penal farm, you were sure to be criticized.

要是在劳改农场，你等着挨绳子吧！