Construction of Donor Plasmid in the Gene Integration Platform System for Oryza sativa L.

提高水稻产量，改良稻米品质是育种学家广泛研究的课题.随着现代生物技术的发展，水稻已成为植物基因工程的重要研究对象.许多实验室已成功地建立了一系列供外源基因转化水稻的系统.但是这些转化系统主要应用TI质粒衍生的载体，通过T-dnA左右两端的序列将目的基...Many laboratories have set up several foreign gene transformation systems for rice.But these transformation systems were all used T DNA system of Ti plasmid, then the foreign gene integrated into plant genomes DNA by means of the insertion of both ends of T DNA.The integrated target of T DNA in plant genome DNA is random, while the thoroughly random integration would interfere with self stable system of plant genome, which could lead to pernicious mutation.In this study, according to the mechanism of homologous recombination, we had constructed a new donor plasmid for indica rice.It would cause the foreign gene intergated into chromsomal rDNA locus. According to the known sequence of ribosomal DNA, we cloned the 2.5kb fragment of it by PCR, and used the fragment as the integrated homologous sequence.After subcloning foreign DNA including nptII gene and metallothionein gene at the end of homologous fragment, the donor plasmid pURKMT which will integrate into the genomes DNA of indica rice was constructed.Then, donor plasmid pURKMT was introduced into calli of indica rice (Jiahe NO.7) by electroporation.Screened by kanamycin, the transformant calli which was integrated the foreign DNA were selected.Dot blotting data showed that the foreign DNA including nptII gene and MT gene has integrated into rice genome DNA

A study on antioxidation of synechococcus sp. PCC 7942 withTrans-thymosin α1-gene in Mice

本试验室已在蓝藻聚球藻中高效表达了人源胸腺素α1(thymosinα1,Tα1)基因,为研究转Tα1基因聚球藻口服后的生物活性,本研究给小鼠灌服转Tα1基因聚球藻14d,研究其对小鼠谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(Cat)和超氧化物歧化酶(SOD)活力以及丙二醛(MDA)含量的影响,结果表明:转胸腺素α1基因聚球藻可显著提高小鼠心、肝与肾中GSH-Px活力(P<0.01);明显提高心脏Cat活性(P<0.01);显著降低肝脏中MDA的含量(P<0.01);但对SOD活力无明显作用。提示转胸腺素α1基因聚球藻较强的抗氧化作用。Human thymosin α1 gene was expressed effectively in Synechococcus sp. PCC 7942 and antioxidant effect of Synechococcus sp. PCC 7942 with trans-thymosin α1-gene in mice were investigated. Synechococcus sp. PCC 7942 with trans-thymosin al-gene were administrated orally 14d,Laters the results showed that the activities of glutathione peroxidase (GSH-Px) in heart、liver and kidney were increased significantly (P<0. 01);the activity of catalase (Cat) in heart was increased markedly;the content of malondialdehyde (MDA) in liver was decreased obviously (P<0.01). But no significant change in the activity of super-oxide dismutase (SOD) was observed. It indicated that Synechococcus sp. PCC 7942 with trans-thymosin α1-gene had obvious antioxidation in vivo.国家海洋863课题(项目编号:819-04-03);福建省自然科学基金项目(项目编号:c0010002

Preparation and determination of polyclonal antibody to thymosin α1

目的 :制备用于检测转胸腺素基因蓝藻表达产物的特异性抗体。方法 :用提纯的小肽 (2 8个氨基酸 )胸腺素α1与牛血清白蛋白偶联后作为抗原 ,采用皮下多点注射免疫的方法免疫大鼠。经过 3个月的免疫 ,获得抗Tα的多克隆抗体。结果 :用间接酶联免疫吸附 (ELISA)方法 ,测得抗体效价超过 40 96。蛋白印迹 (Westernblot)检测结果显示 ,该抗体能特异性地与胸腺素α1抗原产生明显免疫亲和反应。结论 :所制备的抗体具有很好的灵敏性和特异Objective:To prepare specific antibody against Thymosin α1 Methods:Thymosin α1(Tα1,28 peptide) was conjuncted with BSA as immune antigen,rats were immunized and the polycolonal antibody to Tα1 was obtained.Results:With ELISA detection,the titer of antibody was more than 1:4 096;Western blot analysis showed that the antibody can bind with Tα1 specifically.Conclusion:The prepared antibody against Tα1 has good specificity and reactivity and can be used to detect the expression products of transgenic cyanobacteria which expressed Tα1 gene.国家“863”课题资助!(No 819 0 4 0 3

Isolation of a Thermostable Restriction Endonuclease From Thermophilic Synechococcus Elongatus T3

从喜温蓝藻SynECHOCOCuSElOngATuST3分离出一种限制性内切核酸酶，命名为SElI.此酶是限制性内切核酸酶SAu96I的同切酶，它们的识别序列ggnCC.但是SElI的反应条件与SAu96I不同.SElI是一种热稳定的限制性内切核酸酶，最适反应温度的范围是50~57℃A restriction endonuclease was isolated From thermophilic cyanobacterium Synechococcus elongatus T3, which was named For SelI.This restriction endonuclease was characterized as an isoschizomer of Sau96I, its recognition sequences is GGNCC.But the reaction conditions of SelI are diFFerent From that of Sau 96I.Sal I is a thermostable restriction endonuclease with optimal reaction temperature 50￣70℃

Advances of research of Cyanobacterium genome

本文对近年来蓝藻基因组研究进展进行了综述.介绍了聚胞藻PCC6803基因组研究方法和成果,包括最佳分析软件的选择,所获得的基因组基本信息,以及后基因组研究的部分成果.蓝藻基因组间差异很大,文中介绍了其它蓝藻基因组的基本信息和在各个藻种里的重要发现.文章最后探讨了蓝藻基因组的研究意义和前景.The recent research advances of Cyanobacteria genome were reviewed in this paper. As the representative of genome of Cyanobacteria, the research　methods and results of genome of Synechocystis sp.strain PCC6803 wereintroduced, including the selection about most suitable analyzing soft, the genome message and some results of its post-genome research. For the much difference among cyanobacteria, the basic message and significant discovery of others Cyanobacteriagenome were described. Furthermore, the research meaning and future develoment of the Cyanobacteria genome were discussed

Spheroplast Isolation and Regeneration in the Filamentous Cyanobacterium Calothrix sp.PCC7601

采用溶菌酶、纤维素酶和果胶酶混合处理法 ,首次较好地分离具活力的丝状蓝藻 Calothrixsp.PCC76 0 1的原生质球 .通过再生培养 ,跟踪显微观察了原生质球的再生过程 ,并且获得了再生藻株 .为丝状蓝藻的转基因工作提供了一种潜在的受体细胞 .Viable spheroplasts were first isolated enzymatically form the filamentous cyanobacterium Calothrix sp.PCC76 0 1 ,by using a mixture of lysozyme,cellulase and pectolyase.Developmentof spheroplast in culture of was observed periodically by microscope and whole filaments were obtained.This study offers a potential hostcell for gene transfer of foreign DNA into filamentous cyanobacteria.:国家自然科学基金!(39570 4 0 7);; 86 3计划资助项目!(819- 0 4 - 0 3

A New Trangenic Plasmid Vector For Oryza Sativa L.

根据同源重组机制,以水稻17S-5.8SrdnA 2.5 kb 同源片段和含有MT基因、kM r 基因的外源dnA片段构建了质粒载体PurkMT,并用电激法转化水稻愈伤组织,经kM 筛选,获得了对kM 抗性明显高于基础抗性的愈伤组织.经dnA斑点杂交检测,证实外源基因已整合到水稻基因组dnA中.According to them echanism ofhom ologous recom bination, a new plasm id vector pURKMT was constructed, w hich contained Kanam ycin resistance gene, Metalloth- ionein geneand 2.5 kb hom ologous fragm entofOryza Sativa L.including 17S-5.8Sribosom al DNA.Then, donor plasm id pURKMT was introduced into intactrice calliby electrporation. By selection by kanam ycin, the transform antcalli, w hose kanam ycin resistance w as obviously higher than basic resistance, w ere obtained.Dot blotting data show ed that the foreign DNA had been integrated into rice genom e DNA.The results provided a new plasm id vector for transform ation offoreign gene in Oryza Sativa L.

STUDIES ON TRANSFORMATION OF SALT-TOLERANT-RELATED GENES TO CYANOBACTERIUM Synechococcus sp. PCC 7942

将从极端耐盐的假单胞杆菌中克隆得到的3个基因 ,即转录调控因子基因、NADH脱氢酶Ⅱ基因和磷酸甘油磷酸酯酶基因转入淡水蓝藻Synechococcussp.PCC7942中 ,使受体藻对NaCl的耐受性从低于2.5%显著提高到4.5% ,进一步证明了这3个基因确实与生物的耐盐性密切相关。本实验为进一步培育耐盐经济作物奠定了基础。Three salt-tolerant-related genes, transcriptional regulator gene, NADH dehydrogenase II gene and phosphoglycolate phosphatase gene,which are cloned from Pseudomonas xiamenensis, a species of halophilic bacterium, have been transferred as a gene cluster to cyanobacterium Synechococcus sp. PCC 7942. As a result, the NaCl resistant level of the microorganism has been obviously raised from less than 2.5 % to 4.5 %. It shows that the three genes are close correlative to the ability of salt-resistance. This experiment provides a potential possibility for cultivation of salt-resistant economic plants.厦门市科委科技发展计划资助项目350222000104号

EXPRESSION OF THYMOSIN α_1 IN Synechococcus sp.PCC7942 BY HOMOLOGY INTEGRATION DONOR PLASMID pUTK

采用本实验室构建的丝状蓝藻Calothrixsp.PCC7601基因整合平台系统供体质粒pUTK转化单胞蓝藻Synechococcussp.PCC7942,通过抗性筛选获得了具卡那霉素抗性的转化藻株。经Southern杂交证实 pUTK部分序列已整合到Synechococcussp.7942染色体DNA上 ;红光诱导后通过蛋白质SDS PAGE电泳 ,Westernblot证实 pUTK的胸腺素α1 基因得以有效表达,表达量达到总蛋白的4.8 %。结果表明,基因整合平台系统供体质粒 pUTK不仅可转化丝状蓝藻Calothrixsp.PCC7601,还可转化单胞蓝藻Synechococcussp.PCC7942。Donor plasmid pUTK constructed for transferring Calothrix sp.PCC7601 was introduced into Synechococcus sp. PCC7942 by natural transformation. Screened by kanamycin and detected by Southern blot analysis, the transformants which occurred by plasmid homology integration were obtained. Thymosin α1 was expressed up to 4.8% of total protein mass by red light inducing, which was tested by SDS PAGE and confirmed by Western blot. The results showed that the donor plasmid pUTK can be used to transfer not only filament blue green algae Calothrix sp.PCC7601, but also single cell blue green algae Synechococcus sp.PCC7942?国家863计划资助项目819 04 03

A Comparative Study on the Extration of β-carotene and its Content of the Ten Species of Dunaliella (Chlorophyceae)

建立了从杜氏藻鲜藻中直接提取、纯化β-胡萝卜素及快速检测其含量的新方法，发现在十种（株）杜氏藻dunAlIEllAbArdAuIl中生成β-胡萝卜素的量最高，达藻干重的4.19%，在最佳条件下可达13%，经动物急性毒性实验测得小鼠对杜氏藻β-胡萝卜素的最大耐受量＞240Mg·kg￣（-1）.A new method has been used For extraction,puriFication and determination of thecontent level of β-carotene From the green alga Dunaliella.It has been Found that D .bardauil can accu-mulate 4.19%β-carotene of its dried weight.The content of β-carotene is the highest level in the tenspecies(strains) of Dunaliella tested.Morever under a Favourable condition,D.bardauil is able to pro-duce β-carotene as much as 13% of its dried weight.According to the tolerance determination,thetolerance For rats to the β-carotene extracted From Dunaliella was more than 240 mg·kg￣(-1)