Cloning and prokaryotic expression of the multi-copies of the human proinsulin-like gene

根据大肠杆菌密码子偏好性,优化人胰岛素原基因序列,同时用2个赖氨酸取代C链。重叠PCr扩增法克隆优化的类人胰岛素原基因,PCr扩增引物中引入胰蛋白酶酶切位点和核酸限制性内切酶识别位点。经酶切、拼接获得类人胰岛素原多拷贝基因,并构建人胰岛素原基因多拷贝原核表达载体,转化感受态大肠杆菌诱导表达融合蛋白。表达产物经SdS-PAgE电泳,考马斯亮兰染色和融合蛋白染色条带光密度分析表明,含有4拷贝的类人胰岛素原重复基因序列原核表达载体的诱导表达类人胰岛素原效率最高,是单拷贝类人胰岛素基因原核表达载体表达效率的3倍左右。表达产物经质谱鉴定,确定为优化设计的类人胰岛素原,表明构建的多拷贝类人胰岛素原基因表达载体可应用于后续人胰岛素原的高效表达生产研究。The human pre-insulin gene was redesigned for optimal expression in Escherichia coli through the alteration of its codon bias to reflect were redesigned according to the Codon Preference Parameter of Escherichia coli codon preference,and the C peptide of human preinsulin was replaced with Lys-Lys.Then the redesigned human pre-insulin gene(hINS)was cloned using Polymerase Chain Reaction(PCR),and further performed as a template on which based the multi-copy genes of hINS(hINS-M)was cloned by PCR with several couples of special primers.Besides,sequences of both the Restriction Enzyme and Trypsin cutting sites were included in the primers.Accordingly,restriction Endonucleases were employed to cut the PCR products,and T4DNA Ligase was then employed to tape the cut PCR produces to the differently designed multi-copies of the hINS-M.The prokaryotic expression vectors of these multi-copies genes were constructed and transformed into host Escherichia coli strains for expression.Furthermore,dodecyl sufate,sodium salt-polyacrylamide gel electrophoresis(SDS-PAGE),coomassie brilliant blue staining,photodensitometry,and mass spectrometry were employed to analyze the the expression products of the hINS-M.Our results indicated that the expression efficiency of the 4-copies gene of hINS-M was significantly higher than that of the others.The ratio of the expression product of the 4-copies gene hINS-Mthe human preinsulin to the total expression proteins of the host strains was over 28.0%.The ratio was about 3times of that of the expression product of the 1-copy gene of hINS-M.Moreover,the expression products from the different mutil-copes genes of hINS-M were all indentified to be the re-designed human preinsulin.Therefore,the prokaryotic expression vectors of the multi-copies genes of hINS-M could be used to improve the expression efficiency of the recombinant human preinsulin.国家自然科学基金资助项目(31201969); 厦门市科技计划项目(3502Z20093041

Comparative research of the structures of plant functional groups in different successional stages of lowland secondary forests in Zhejiang Province(浙江省不同演替阶段的低海拔次生林植物功能群结构的比较研究)

选取浙江省低海拔地区的16个次生林样地并调查其中胸径(DBH)≥1 cm的木本植物.通过观察群落外貌，结合数量分类方法(TWINSPAN)和排序方法(DCA)，将这些样地划分为4种演替阶段的森林类型（马尾松林，马尾松针阔叶混交林，演替早期常绿阔叶林，演替中期常绿阔叶林）.分析了不同演替阶段的次生林群落物种多样性和植物功能群结构组成变化.结果显示：(1)适度择伐进入采伐期的马尾松可加速整个群落向常绿阔叶林演替；(2)随着演替的进行，物种丰富度、Shannon-Wiener指数、Simpson指数和Pielou均匀度指数均呈现增加趋势；(3)按生活型、生长型、叶的生活期和对光的耐受性这4种功能群划分，所有样地分别以小高位芽植物和中高位芽植物、乔木和灌木或小乔木、常绿植物和耐阴植物占优；(4)随着演替的进行，功能群结构比例呈现先降后升再降的是矮高位芽植物，先降后升的为小高位芽植物，呈现先升后降的为大高位芽植物、灌木、乔木、小乔木、常绿植物、阴性植物呈现上升趋势，落叶植物和阳性植物呈现下降趋势，而中高位芽植物、灌木或小乔木、耐阴植物则维持相对恒定的比例

Analysis of woody flora based on quadrat method in eleven natural reserves of Zhejiang Province(基于样方法的浙江省11个自然保护区木本植物区系成分分析)

为了探讨样方法在木本植物区系研究中的应用前景，通过设置固定样地进行实地调查，收集到了浙江省11个自然保护区样地内木本植物名录，应用Czechanowski系数对11个自然保护区的科、属相似性系数进行分析.结果表明：11个自然保护区的66个30 m×30 m的样方内，共计木本植物435种，隶属于169属70科；科、属的亚热带性质显著，其热带与温带类型占比分别为57.89%，41.07%和40.72%，53.89%，从属级区系成分看，样地内木本植物区系来源于多种地理成分，有13个大的分布类型；11个自然保护区可以大致划分为3个类型.各保护区的区系相似性数值分析结果与其所处的地理位置及山脉分支相关性较大.研究表明，样方调查法对植物区系的研究具有一定的借鉴意义

荒漠地区公路建设环境保护与生态恢复技术

《荒漠地区公路建设环境保护与生态恢复技术》项目组通过资料收集、理论分析、室内外试验和工程实践,系统开展了荒漠地区公路建设与自然环境相互影响、荒漠地区公路建设的生态环境敏感性、荒漠地区公路建设环境保护与生态恢复技术集成以及典型区域公路建设环保与生态建设示范四方面研究,取得了如下主要创新性成果： 1．分析揭示了荒漠地区公路与环境的相互影响关系,建立了公路建设环境保护与生态恢复的基础平台。 2. 提出了荒漠地区公路路域生态功能重要性、环境敏感性和景观类型区划的原则与方法,建立了相应的区划体系。 3．构建了荒漠地区公路路域生态修复技术评价指标体系和评价数学模型。 4．提出了荒漠..