Mechanisms of E2F2-mediated transcriptional repression

In this work we wanted to study the mechanism of E2F2-mediated repression. Our hypothesis is that E2F2 activates the expression of one or more E2F members of the “repressor” subset of the family through the E2F motifs present in their promoters, and those repressor E2F(s) would subsequently repress the target promoters. To address this hypothesis, we focused on E2F7. E2F7 is a repressor that lacks the Rb binding domain, and associates with DNA through E2F binding sites (de Bruin et al., 2003). Furthermore, E2F7 itself is also regulated by E2F motifs on its own promoter, and it has been shown to repress DNA metabolism and replication genes in late S-phase (de Bruin et al., 2003; Westendorp et al., 2012). E2F7, together with E2F8 has been found to form heterodimers, being critical on cell proliferation and development, and both seem to have similar functions (Li et al., 2008). Preliminary results from Zubiaga’s group have indicated that E2F2 activates E2F7 transcription in U2OS cells, suggesting that E2F2’s repressor function could be mediated by E2F7. For this purpose, we focused on studying E2F7’s role on the target genes previously known to be repressed by E2F2: Chk1 and Mcm5. The specific aims for this work were the following: - Confirm that E2F2 induces E2F7 in HEK-293T cells - Assess whether E2F7 acts as a transcriptional repressor on E2F sites - Evaluate the role of E2F7 on E2F2-mediated transcriptional repression of Chk1 and Mcm5.

Museu de Favela: pensando turismo e patrimônio no Pavão, Pavãozinho e Cantagalo

Esta dissertação versa sobre o Museu de Favela (MUF), organização não-governamental fundada em 2008 durante as obras do Programa de Aceleração do Crescimento (PAC) nas favelas do Pavão, Pavãozinho e Cantagalo, na Zona Sul da cidade do Rio de Janeiro. As principais propostas do MUF são a patrimonialização da favela/seus elementos culturais, bem como o desenvolvimento turístico dessas favelas. A partir de um trabalho de campo, do tipo observação participante junto à diretoria do MUF, analiso a construção de sua proposta de turismo em oposição à favela da Rocinha, caso emblemático do turismo em favelas. Mostro a presença de duas ideias de turismo a propósito do caso estudado: o que chamo de turismo vilão, representado pelo contexto da Rocinha na visão dos promotores do MUF; e o que chamo de turismo solução, representado na mesma visão pelo turismo organizado pelos moradores para benefício da população local e solução dos problemas das favelas. Analiso ainda a proposta do MUF, sua organização como ONG e seus diretores. Por fim, focalizo o Museu de Favela e o Estado como atores sociais básicos envolvidos na questão analisada, mostrando como turismo e patrimônio aparecem como ferramentas para ambas as instituições, que guardam semelhanças e diferenças no que se refere a dois processos em andamento nas favelas do Pavão, Pavãozinho e Cantagalo: o processo de patrimonialização e o processo de turismização.

Promoção de Segurança Alimentar e Nutricional na Estratégia Saúde da Família: uma aproximação a partir da escuta de diferentes atores sociais

A garantia de Segurança Alimentar e Nutricional (SAN) remete à necessidade de ações intersetoriais que articulem as dimensões alimentar e nutricional, além da questão contemporânea da sustentabilidade e da perspectiva do direito humano à alimentação adequada. O setor saúde tem funções específicas e importantes que contribuem para o conjunto das políticas de governo voltadas para a garantia da SAN a população. Desta forma, ações promotoras de SAN devem ser desenvolvidas em todos os níveis de atenção do Sistema Único de Saúde, sendo a Atenção Básica à Saúde, por meio da Estratégia Saúde da Família (ESF), um campo privilegiado de implementação dessas ações, uma vez que está configurada como a porta preferencial de entrada dos usuários no sistema de saúde e como o centro norteador da rede de assistência. Este é um estudo exploratório e descritivo de abordagem qualitativa que teve como objetivo conhecer o que profissionais de equipes de saúde da Família, gestores dos âmbitos federal e municipal ligados à ESF, além de representantes de organizações da sociedade civil atuantes no campo da SAN entendem sobre SAN e sobre práticas promotoras de SAN na ESF. A construção das informações ocorreu por meio de entrevistas semi-estruturadas e grupos focais. Os profissionais referiram-se a SAN como a garantia de uma alimentação que atenda às necessidades nutricionais e que seja segura para o consumo, enquanto que entre os representantes da sociedade civil organizada e gestores predominou uma compreensão mais ampla da SAN. Os diferentes atores identificaram a ESF com um espaço promotor de SAN a partir do levantamento de ações já desenvolvidas ou que possam vir a ser desenvolvidas, porém as ações citadas encontram-se majoritariamente ligadas à dimensão nutricional da SAN. Os atores referiram um conjunto de problemas estruturais que desencadeiam dificuldades no cotidiano da organização dos serviços e das práticas dos profissionais e consequentemente na execução de ações promotoras de SAN nessa estratégia. Este trabalho levantou a necessidade de difundir a interdependência entre saúde e SAN entre gestores e profissionais ligados à ESF para que estes possam identificar melhor nas ações dos serviços de saúde elementos promotores da SAN, e desta forma compreender seu papel de agentes promotores de saúde e SAN.

Mães adolescentes: estudo das vivências de amamentação dos seus filhos

No presente trabalho analisamos a amamentação desde a óptica de um grupo de mães-nutrizes adolescentes de baixa renda, residentes no Estado do Rio de Janeiro. Objetivamos compreender como as mulheres estudadas vivenciavam o processo de amamentação de seus filhos e identificar os fatores que contribuíam para a construção de tais vivências. Partimos do pressuposto de que as mães adolescentes vivenciavam o ser nutriz como uma ação cotidiana que se consolidava ou se desfazia em função de fatores da ordem da natureza e/ou da cultura que permeavam essa prática. Constitui-se de uma pesquisa qualitativa. Participaram 12 mães adolescentes, entre 15 e 19 anos de idade, que estavam amamentando seus filhos durante o primeiro trimestre pós-parto. Os depoimentos das entrevistadas foram analisados com base na técnica de análise de conteúdo, modalidade temática e interpretados à luz do referencial teórico da Pesquisa Qualitativa em Saúde. Para estas mulheres, a essência da prática do aleitamento materno se constrói ao redor de sua preocupação em relação ao sucesso/insucesso da amamentação e do exercício de deveres inerentes ao ser mãe adolescente. Expressam sua preocupação em produzir leite em quantidade e qualidade suficiente, ter mamas aptas para amamentar e conseguir que o bebê mamasse efetivamente. Assim mesmo, manifestam sentir o dever de ser responsáveis e amamentar, vencer o cansaço e a dor, pensar no bem-estar do filho e aceitar as mudanças que a maternidade e a amamentação imprimiram nos seus corpos. Reafirmam o conceito de amamentação como prática histórico-social aprendida. Grande ênfase é dada ao papel do profissional de saúde e do meio social como mediadores da aprendizagem e rede de suporte à amamentação. A partir dos resultados deste estudo sugerimos a necessidade de refletir sobre nossa função assistencial na promoção, proteção e apoio ao aleitamento materno neste grupo etário.

Getúlio Damado e Roger Mello: conexões entre o campo da arte e o universo lúdico infantil

A ideia central da pesquisa foi promover uma aproximação das narrativas visuais de Getúlio Damado e Roger Mello e a partir daí evidenciar os processos cognitivos e as estratégias que envolveram a estruturação de suas identidades sociais e linguagens artísticas contemporâneas, bem como destacar o ludismo e o acesso ao universo da infância e a cultura popular que ambos ressignificam em suas obras. Além disso, buscou-se comentar e avaliar momentos específicos de desprestígio e repúdio versus consagração ocorridos com os artistas. Para tal, essas produções foram contextualizadas como produções culturais em constante processo de mutação, do qual participam tanto eventos históricos como de inovação tecnológica, em contínua construção. Essas linguagens estão submetidas a uma grande velocidade e a superposição de influências. Outro ponto destacado nas analises foi o caráter de subjetividade dessas produções, identificadas como práticas estéticas promotoras de transformação política; de mobilização; ou também por sua capacidade de operar com o uso simbólico dos brinquedos e livros infantojuvenis recuperando-lhes o uso original. Os devaneios, o ludismo infantil e a cultura popular presentes nos objetos feitos de sucata e livros ilustrados foram confrontados com práticas consoantes, ou de discursos abrangentes, correntes no mundo da arte ou outras vezes aproximados de enfoques locais. Além da possibilidade de convergência entre as produções foram destacadas também muitas diferenças, e, sobretudo evidenciadas as potencialidades que podem advir da cultura popular como campo exploratório de pesquisa, bem como de sua ressignificação

Controlled adhesion and growth of long term glial and neuronal cultures on Parylene-c

This paper explores the long term development of networks of glia and neurons on patterns of Parylene-C on a SiO 2 substrate. We harvested glia and neurons from the Sprague-Dawley (P1-P7) rat hippocampus and utilized an established cell patterning technique in order to investigate cellular migration, over the course of 3 weeks. This work demonstrates that uncontrolled glial mitosis gradually disrupts cellular patterns that are established early during culture. This effect is not attributed to a loss of protein from the Parylene-C surface, as nitrogen levels on the substrate remain stable over 3 weeks. The inclusion of the anti-mitotic cytarabine (Ara-C) in the culture medium moderates glial division and thus, adequately preserves initial glial and neuronal conformity to underlying patterns. Neuronal apoptosis, often associated with the use of Ara-C, is mitigated by the addition of brain derived neurotrophic factor (BDNF). We believe that with the right combination of glial inhibitors and neuronal promoters, the Parylene-C based cell patterning method can generate structured, active neural networks that can be sustained and investigated over extended periods of time. To our knowledge this is the first report on the concurrent application of Ara-C and BDNF on patterned cell cultures. © 2011 Delivopoulos, Murray.

Single base-resolution methylome of the silkworm reveals a sparse epigenomic map

Epigenetic regulation in insects may have effects on diverse biological processes. Here we survey the methylome of a model insect, the silkworm Bombyx mori, at single-base resolution using Illumina high-throughput bisulfite sequencing (MethylC-Seq). We conservatively estimate that 0.11% of genomic cytosines are methylcytosines, all of which probably occur in CG dinucleotides. CG methylation is substantially enriched in gene bodies and is positively correlated with gene expression levels, suggesting it has a positive role in gene transcription. We find that transposable elements, promoters and ribosomal DNAs are hypomethylated, but in contrast, genomic loci matching small RNAs in gene bodies are densely methylated. This work contributes to our understanding of epigenetics in insects, and in contrast to previous studies of the highly methylated genomes of Arabidopsis(1) and human(2), demonstrates a strategy for sequencing the epigenomes of organisms such as insects that have low levels of methylation.

Ecological impacts of coastal aquaculture developments

The study presents the type and scale of any ecological change associated with coastal aquaculture development. These are enrichment, interaction with the food web, oxygen consumption, disturbance of wildlife and habitat destruction, interaction between escaped farmed stock and wild species, introduction and transfers, bioactive compounds (including pesticides and antibiotics), chemicals introduced via construction materials, and hormones and growth promoters.

A possible mode of the specific recognition of nucleic acids by proteins

Seven sets of protein target sites, which occur in several gene promoters, have been analyzed. The results suggest that there is a possible mode of specific recognition of double-helical nucleic acids by proteins, This recognition mode is related to a spe

Expression pattern, cellular localization and promoter activity analysis of ovarian aromatase (Cyp19a1a) in protogynous hermaphrodite red-spotted grouper

Aromatase plays a key role in sex differentiation of gonads. In this study, we cloned the full-length cDNA of ovarian aromatase from protogynous hermaphrodite red-spotted grouper (Epinephelus akaara), and prepared the corresponding anti-EaCyp19a1a antiserum. Western blot and immunofluorescence studies revealed ovary-specific expression pattern of EaCyp19a1a in adults and its dynamic expression change during artificial sex reversal. EaCyp19a1a was expressed by follicular cells of follicular layer around oocytes because strong EaCyp19a1a immunofluorescence was observed in the cells of ovaries. During artificial sex reversal, EaCyp19a1a expression dropped significantly from female to male, and almost no any positive EaCyp19a1a signal was observed in testicular tissues. Then, we cloned and sequenced a total of 1967 bp T-flanking sequence of EaCyp19a1a promoter, and showed a number of potential binding sites for some transcriptional factors, such as SOX5, GATA gene family, CREB, AP1, FOXL1, C/EBP, ARE and SF-1. Moreover, we prepared a series of 5' deletion promoter constructs and performed in vitro luciferase assays of EaCyp19a1a promoter activities. The data indicated that the CREB regulation region from -1010 to -898 might be a major cis-acting element to EaCyp19a1a promoter, whereas the elements GATA and SOX5 in the region from -1216 to -1010 might be suppression elements. Significantly, we found a common conserved sequence region in the fish ovary-type aromatase promoters with identities from 93% to 34%. And, the motifs of TATA box, SF-1, SOX5, and CREB existed in the region and were conserved among the most of fish species. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Hybrid cytomegalovirus-U6 promoter-based plasmid vectors improve efficiency of RNA interference in zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

Gene structures and promoter characteristics of interferon regulatory factor 1 (IRF-1), IRF-2 and IRF-7 from snakehead Channa argus

Three interferon regulatory factor (IRF) genes, CaIRF-1, CaIRF-2 and CaIRF-7, and their promoters of snakehead (Channa argus) were cloned and characterized. The CaIRF-1 gene consists of ten exons, spans 4.3 kb and encodes a putative peptide of 299 aa. The CaIRF-2 gene consists of nine exons, spans 8 kb and encodes a putative peptide of 328 aa. The gene organizations of CaIRF-1 and CaIRF-2 are very similar to that of human IRF-1 and IRF-2 except more compact. Comparison of exon-intron organization of the two genes indicated a common evolutionary structure, notably within the exons encoding the DNA binding domain (DBD) of the two factors. The CaIRF-7 gene spans 4.1 kb and encodes a putative peptide of 437 aa. However, the gene organization of CaIRF-7 consisting of ten exons is different to human IRF-7a gene which has an intron in 5' UTR. Three CaIRFs share homology in N-terminal encompassing the DBD that contains a characteristic repeat of tryptophan residues. The promoters of CaIRF-1 and CaIRF-2 genes contain the conserved sites for NF-kappa B and Sp1. The gamma-IFN activation sites (GAS) were found in the promoters of CaIRF-1 and CaIRF-7. The promoter of CaIRF-7 contains conserved interferon stimulating response element (ISRE) which is characteristic of IFN-induced gene promoter, and suggests that there also exist intracellular amplifier circuit in fish IFN signal pathway. Moreover, the element GAAANN oriented in both directions is repeated in CaIRF promoter regions, which confers to further inducibility by IFN. The constitutive expression of CaIRF genes were found to increase obviously in response to induction by the known IFN-inducer poly I:C. (c) 2008 Published by Elsevier Ltd.

Mechanics of biological networks: from the cell cytoskeleton to connective tissue.

From the cell cytoskeleton to connective tissues, fibrous networks are ubiquitous in metazoan life as the key promoters of mechanical strength, support and integrity. In recent decades, the application of physics to biological systems has made substantial strides in elucidating the striking mechanical phenomena observed in such networks, explaining strain stiffening, power law rheology and cytoskeletal fluidisation - all key to the biological function of individual cells and tissues. In this review we focus on the current progress in the field, with a primer into the basic physics of individual filaments and the networks they form. This is followed by a discussion of biological networks in the context of a broad spread of recent in vitro and in vivo experiments.

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

转座子Minos介导的果蝇转基因平台构建及水稻高表达启动子的克隆 任娟 指

本实验室果蝇研究工作，主要集中在黑腹果蝇的新基因起源的研究。新基因起源的分子机制主要包括：外显子重排、基因复制、基因逆转座、移动元件介导、基因水平转移、基因从头起源、基因的断裂融合。为了阐述这些新基因的产生和它们所带来的物种适应性，我们对这些新近起源的基因进行了功能研究。但是，仅仅限于新基因所在物种的功能研究并不能完全解释新基因产生的进化原因，我们需要了解它是否能够给没有该基因的果蝇物种带来一定的适应性。例如一些生殖相关新基因，如果我们将它们转入没有该基因的果蝇，那是否能够给该果蝇带来生殖能力的提高？无论结果如何，这都为我们研究新基因的起源提供一个重要线索。由此，黑腹果蝇以外的其它果蝇物种中实现转基因成为该研究的重要技术环节。但是，实验室目前的转基因系统仅限于P转座子介导的黑腹果蝇转基因系统，因而我们需要建立一种新的转基因平台。而转座子Minos打破物种范围的转基因特性，以及它的转座特点为我们提供了选择。转座子Minos是从果蝇D. hydei中克隆出来长约1.8kb的Ⅱ型转座子，Tc1家族转座元件成员。Minos的转座机制与大部分转座子一样，在宿主基因组里面实行着剪切和粘贴的运作机制。Minos在转座时，偏向插入TA位点并且主要集中于内含子区域，这样可以减少对插入位置基因的影响。此外，Minos在黑腹果蝇中的转座效率约30%，并且拥有一套成熟的选择标记。因此，Minos成为我们解决非黑腹果蝇转基因技术难题的首选。 在本文的工作中，我们采用由希腊Savakis教授（希腊分子生物学与生物技术研究所）提供的Minos转基因系统，完成果蝇的转基因实验。在这套转基因系统中，非自主的转座子Minos和转座酶基因被克隆到了不同载体当中。其中Minos转座子序列中插入了由3xP3眼睛特异表达的启动子介导表达的eGFP报告基因，而转座酶基因则由热激蛋白hsp70启动子调控表达。实验过程中，我们在果蝇D. melanogaster 和D. yakuba的胚胎中分别同时显微注射入含有转座子和转座酶本实验室果蝇研究工作，主要集中在黑腹果蝇的新基因起源的研究。新基因起源的分子机制主要包括：外显子重排、基因复制、基因逆转座、移动元件介导、基因水平转移、基因从头起源、基因的断裂融合。为了阐述这些新基因的产生和它们所带来的物种适应性，我们对这些新近起源的基因进行了功能研究。但是，仅仅限于新基因所在物种的功能研究并不能完全解释新基因产生的进化原因，我们需要了解它是否能够给没有该基因的果蝇物种带来一定的适应性。例如一些生殖相关新基因，如果我们将它们转入没有该基因的果蝇，那是否能够给该果蝇带来生殖能力的提高？无论结果如何，这都为我们研究新基因的起源提供一个重要线索。由此，黑腹果蝇以外的其它果蝇物种中实现转基因成为该研究的重要技术环节。但是，实验室目前的转基因系统仅限于P转座子介导的黑腹果蝇转基因系统，因而我们需要建立一种新的转基因平台。而转座子Minos打破物种范围的转基因特性，以及它的转座特点为我们提供了选择。转座子Minos是从果蝇D. hydei中克隆出来长约1.8kb的Ⅱ型转座子，Tc1家族转座元件成员。Minos的转座机制与大部分转座子一样，在宿主基因组里面实行着剪切和粘贴的运作机制。Minos在转座时，偏向插入TA位点并且主要集中于内含子区域，这样可以减少对插入位置基因的影响。此外，Minos在黑腹果蝇中的转座效率约30%，并且拥有一套成熟的选择标记。因此，Minos成为我们解决非黑腹果蝇转基因技术难题的首选。 在本文的工作中，我们采用由希腊Savakis教授（希腊分子生物学与生物技术研究所）提供的Minos转基因系统，完成果蝇的转基因实验。在这套转基因系统中，非自主的转座子Minos和转座酶基因被克隆到了不同载体当中。其中Minos转座子序列中插入了由3xP3眼睛特异表达的启动子介导表达的eGFP报告基因，而转座酶基因则由热激蛋白hsp70启动子调控表达。实验过程中，我们在果蝇D. melanogaster 和D. yakuba的胚胎中分别同时显微注射入含有转座子和转座酶所在的质粒。转座酶在37度条件诱导下进行表达，协助Minos完成转座过程。在转基因果蝇的阳性筛选中，我们利用眼睛特异表达的绿色荧光蛋作为选择标记。并且，我们通过PCR实验进一步验证了转基因果蝇的真实性。本研究中，我们对转基因实验条件进行了初步优化。我们通过对黑腹果蝇白眼突变品系W1118和D. yakuba注射后胚胎进行保湿，对D. yakuba注射胚胎进行非退壳处理。在改进条件下W1118和D. yakuba的存活率分别为10%和3%左右。通过筛选转基因阳性果蝇，我们得出Minos在W1118和D. yakuba中的转座效率分别在32%和20%左右。我们的实验结果再一次证实了Minos在果蝇D. melanogaster中可行性。同时，该工作也初步完成了在果蝇D. yakuba 中的第一次Minos介导的转基因实验，为新基因的跨物种功能研究奠定了实验基础。在未来的工作计划中，我们将采用Minos转基因系统，把实验室目前研究的黑腹果蝇新基因导入其它物种果蝇进行功能研究。 水稻是一种重要的世界粮食作物，世界上过半的人口以水稻为主食。水稻相对别的粮食作物来讲具有较小的基因组，并且拥有较好的基因组注释，是一种理想的单子叶模式生物。植物转基因技术的发展推动着水稻功能基因组学的研究，目前水稻的转基因技术主要依赖于土壤细菌农杆菌（Agrobacterium tumefaciens）T-DNA介导的外源基因染色体插入。在自然状态下，农杆菌的T-DNA位于Ti致瘤质粒当中。它包括了一些转座元件和一些帮助T-DNA转座的毒性蛋白基因和调节基因。由于Ti质粒上的T-DNA太长，并且没有太多的酶切位点，因此自然状态的T-DNA不适合进行转基因实验。为了方便T-DNA的实际应用，研究人员创立了双载体转基因系统。T-DNA转座区被分离到出Ti载体，并且装载到另外一个适合实验操作的质粒当中，而毒性蛋白表达基因等则保留在Ti质粒上。因此，在进行T-DNA介导的转基因实验时，需要同时存在T-DNA载体和Ti质粒。 本文以“水稻注释计划数据库RAP-DB”的表达数据为参考，选择了60个高表达基因的启动子区域进行克隆。通过对T-DNA载体pCAMBIA1301 进行改造，去掉其原来的35S启动子，将预测的基因启动子克隆到该载体中并与报告基 摘要 因GUS 基因融合。通过分子克隆实验，我们得到了45个高表达基因的启动子载体。最终，为了测试这45个启动子的启动效率，我们会将它们转化到水稻愈伤组织中通过启动子融合的GUS基于表达情况来判断我们启动子的启动效率。