摘要：根据棉花纤维特异表达cDNA文库分析得到的咖啡酰辅酶A-O-甲基转移酶(CCoAOMT)基因EST序列设计引物,采用RT-PCR技术首次从棉花中克隆了一个CCoAOMT基因,命名为GhCCoAOMT1(GenBank登录号为FJ848871)。研究结果表明:GhCCoAOMT1基因cDNA全长960bp,具有一个753bp的开放阅读框,5′非编码区为9bp,3′非编码区为198bp,编码250个氨基酸,预测分子量约为28.306kDa,等电点为5.39。利用PCR方法克隆了GhCCoAOMT1基因的基因组序列,长度为1311bp,包含5个外显子和4个内含子。氨基酸同源性分析发现,GhCCoAOMT1与来自毛白杨、烟草和苎麻的CCoAOMT同源性较高。半定量RT-PCR检测表明,GhC-CoAOMT1基因在棉花各个组织中都有表达,其中茎部的表达量最高,其次表达量依次为根>花瓣>子叶>10d纤维>雄蕊>胚珠>叶。

摘要：【目的】棉蚜(Aphis gossypii)是棉花主要害虫之一,严重危害棉花的生产。利用靶标昆虫重要基因的双链RNA进行干涉从而防治农业害虫是当今发展的1种新技术。通过转基因的方法,在棉花中表达棉蚜靶标基因的双链RNA(Double stranded RNA,dsRNA),特异性地抑制棉蚜内源基因的表达,从而控制蚜虫对棉花的危害。【方法】本研究利用棉蚜腺苷三磷酸酶E亚基(Adenosine triphosphatase subunit E,ATPase E)基因,构建其RNA干扰载体,并通过农杆菌介导转化棉花。PCR技术初步筛查45个独立转化植株,对PCR阳性植株进行DNA印迹(Southern blot)分析,确认外源基因插入的拷贝数。同时利用RT-PCR (Reverse transcription polymerase chain reaction)方法检测dsRNA的表达情况。最后结合网棚和大田抗蚜性鉴定分析转基因株系的抗蚜效果。【结果】从45个转基因再生植株中获得22株阳性转基因株,其中12株为单拷贝插入。根据农艺性状和dsRNA表达量分析结果,筛选出4个转基因株系,分别为CZ1、CZ2、CZ3和CZ4。2年的网棚和大田的蚜虫抗性鉴定结果表明:转基因棉花CZ1、CZ2和CZ3具有较高的抗蚜性,2015年和2016年的蚜害减退率分别达70%和60%以上。【结论】利用RNAi技术将蚜虫腺苷三磷酸酶E亚基基因片段转化棉花,可以提高棉花的抗蚜性,为棉花抗蚜育种提供了新种质和新方法。

摘要：In cultivated rice ( Oryza sativa L.), F-1 pollen sterility is controlled by at least 6 loci of the F, pollen sterility genes. To map S-b, one of loci, rice variety Taichung 65 (T65) carrying S-b(j)/S-b(j) and its near-isogenic line TIST2 carrying S-b(i)/S-b(i) were used to develop the mapping population. One hundred and fifty-eight microsatellite markers were selected to survey T65 and TISL2. RM13 on chromosome 5 was found to be polymorphic between them. Cosegregation indicated that RM13 was closely linked with locus S-b. Eleven RFLP markers were selected on the corresponding region from the genetic map of Rice Genome Research Program (RGP) of Japan to convert into sequence-tagged site (STS) markers. Amplicon length polymorphism (ALP) was carried out, but none of them was found to be polymorphic between T65 and TISL2. Then PCR-based RFLP (PBR) was done using six 4-nucleotide recognizing restriction endonucleases. Polymorphism was detected when PCR products of R830STS and R2213SSTS were digested with Taq I. Genetic analysis indicated that the distance between locus S-b and markers, R830STS, RM13 and R2213SSTS were 3.3 cM (centi-Morgan), 5.2 cM and 5.5 cM, respectively. These PCR-based markers could be directly used in marker-assisted selection. The technical system combining genetic mapping and PCR-based marker-assisted selection will facilitate the development of molecular breeding.

摘要：5-烯醇丙酮莽草酸-3-磷酸（EPSP）合酶是芳香族氨基酸合成途径中的一个关键酶,该基因在植物抗除草剂基因工程中具有重要的应用价值.根据水稻EPSP合酶基因的EST序列设计探针,在水稻TAC基因组文库中筛选到16个阳性克隆.对阳性克隆E11进行亚克隆,由此获得了由3661核苷酸组成的水稻EPSP合酶基因全序列.序列分析和同源性比较揭示,该基因由8个外显子和7个内含子组成.以窄叶青8号/京系17组合构建的DH群体和分子图谱将水稻EPSP合酶基因定位于水稻第6条染色体的上端.

摘要：从小麦品种中国春中克隆了TaPhyB在染色体4D上的cDNA的全长编码序列,所得序列定位于4D染色体长臂上,被命名为TaPhyB3。通过中国春1D-4D-6D BAC文库的筛选,设计引物亚克隆得到光敏色素基因B的基因序列,其含有4个外显子和3个内含子。通过生物信息学软件的预测,该基因编码蛋白的功能结构域有6个:DAF DOMAIN,PHYTOCHROME REGION,PAS-ADOMAIN,PAS-B DOMAIN,HISTIDINE KINASE RELATED DOMAIN 1和HIETIDINE KINASE RELATED DOMAIN 2。编码序列与染色体倍数关系的分析结果表明TaPhyB3基因的表达并不随染色体组的加倍而出现相应增长的趋势,推测在小麦中存在TaPhyB3的表达的调控与平衡问题。

摘要：构建和筛选对PID1(phosphotyrosine interaction domain containing 1,PID1)基因有RNA干扰作用的PID1-shRNA表达载体。据小鼠PID1 cDNA序列,优化设计了4条shRNA及1条阴性干扰序列,插入pGPU6/GFP/Neo载体中,得到pGPU6/GFP/Neo-PID1-1、pGPU6/GFP/Neo-PID1-2、pGPU6/GFP/Neo-PID1-3、pGPU6/GFP/Neo-PID1-4和pGPU6/GFP/Neo-PID1-NC。干扰载体转染C2C12细胞,以RT-PCR和Western blot技术检测shRNA对C2C12细胞中PID1 mRNA和蛋白表达的下调作用。结果表明:靶向PID1基因的4个shRNA重组质粒载体经测序分析,其shRNA编码序列与预期设计的完全一致,经酶切鉴定和测序分析证实,靶向PID1基因的shRNA重组质粒载体构建成功。进一步将构建的4个表达载体分别转染C2C12细胞,24 h后细胞中PID1基因mRNA表达水平依次下调(23.58±1.87)%、(75.44±0.77)%、(70.52±0.41)%和(56.60±3.13)%。48 h后细胞中PID1蛋白表达水平依次降低(30.15±5.05)%、(71.86±4.85)%、(67.93±2.28)%和(56.81±2.01)%。所筛选出的pGPU6/GFP/Neo-PID1-2、pGPU6/GFP/Neo-PID1-3和pGPU6/GFP/Neo-PID1-4三个表达载体均能高效地抑制转染细胞PID1 mRNA和蛋白的表达,为进一步研究PID1基因的功能奠定了基础。

摘要：根据棉花GhCCR1基因的cDNA序列设计引物,采用PCR技术从棉花中克隆了GhCCR1基因的DNA序列,并采用半定量RT-PCR方法分析了GhCCR1基因在不同发育阶段棉纤维中的表达情况.结果表明:GhCCR1编码区DNA序列长度为1 161 bp,包含4个外显子和3个内含子,内含子富含AT,所有外显子/内含子交接点都遵从gt/ag剪接规则.半定量RT-PCR检测表明,GhCCR1基因在不同发育阶段的棉纤维中均有表达,在开花后20 d的棉纤维中表达量最高,说明该基因可能参与调控棉纤维细胞的伸长和次生壁的增厚过程.

摘要：9-顺式环氧类胡萝卜素双加氧酶（NCED）是高等植物ABA生物合成途径中的关键酶，本研究以3种豆科植物NCED的同源序列设计简并引物，用RT—PCR结合RACE以及嵌套PCR的方法。从柱花草叶片中克隆了一个编码NCED的基因，命名为SgNCED1。其cDNA全长2241bp，开放阅读框架为1809bp，编码602个氨基酸。Southern杂交结果表明，该基因在柱花草基因组中以单拷贝形式存在。氨基酸序列同源比对和系统进化分析表明SgNCED1与花生的AhNCED1高度同源（92％），与另外3种豆科植物的NCED也有很高的同源性（72％～75％）。亚细胞定位预测表明SgNCED1蛋白的N-末端具有叶绿体转运肽。叶片脱水诱导的SgNCED1基因的表达与内源ABA的积累、变化同步。

摘要：Two pairs of degenerate primers were designed based on nucleotide-binding site (NBS) and serine/threonine kinase domain. PCR was performed with the primers and cDNA from the Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL. Amplified products were cloned and sequenced. Nine clones with NBS and one with serine/threonine kinase domain were obtained. The NBS clones were classified to six groups according to their nucleotide sequence identities (90% or higher). These resistance gene analogs (RGAs) all have open reading frames (ORF), and their amino acid sequences show high similarity to Yr10 in wheat, Mla1 and Mla6 in barley, RPS2 in Arabidopsis and other resistance (R) genes with conserved motifs. They were preliminarily mapped on the chromosomes of homoeologous groups 1, 2 and 5 of common wheat by nulli-tetrasomic analysis. The 5'-end sequence of an RGA N5 was obtained by 5'-RACE PCR. It encodes six leucine zipper (LZ) and has high sequence similarity to RPS2.

摘要：中国的养老金计划由政府进行设计并以合约的形式提供,成员按照政府给出的统一缴费率和退休年龄标准进行缴费并领取养老金,我们称这种在相同合约下的统一养老金计划1为"一刀切2",与其相对的即是针对"不同成员特点"的"差异化3"养老金计划。一刀切式的养老金计划虽然可以带来推广上的简便,但由于参与人群的差异性,对其道德风险进行监管的成本很高,已经对我国目前养老金计划的可持续性和有效性构成了威胁。这促使我们思考差异化的养老金计划能否成为一个更为可行的方案,通过设计一组让"不同"成员都"乐于主动"参加的合约,从而在根本上解决道德风险的问题。本文从合约设计的视角,就中国养老金计划的可持续性,养老金计划的差异化和统一化之间孰优孰劣进行了比较,得出了一个反直觉的结论:总体来看,养老金计划中固有的道德风险难以通过差异化的合约设计得到改善,一刀切模式的合约反而能够通过让社会成员选择唯一合约提高资源配置效率。