Sarcocystis neurona is an apicomplexan parasite that is a major cause of equine protozoal myeloencephalitis (EPM). During intracellular development of S. neurona, many genes are temporally regulated. To better understand gene regulation, it is important to identify and characterize regulatory elements controlling gene expression in S. neurona. To perform this study, it was essential to establish transfection system for this parasite. Hence, the 5 flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules -galactosidase (-gal) and yellow fluorescent protein (YFP) were expressed by electroporated S. neurona, thereby confirming the feasibility of performing molecular genetic experiments in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of T. gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express -gal and YFP. These transgenic clones were shown to be useful for analyzing growth rate of parasites in-vitro and for assessing drug sensitivities. To uncover possible sequence elements involved in promoter activity, the 5 flanking regions of five S. neurona genes were subjected to comparative analysis. This revealed the presence of a 7-base conserved motif GCGTCTC. Using a dual luciferase assay system, the SnSAG1 promoter was subjected to functional analysis. The motif GAGACGC located between -136 and -129 upstream of the transcription start site was found to be essential for SnSAG1 expression. This motif functions in an orientation dependent manner and was shown to play a role in binding nuclear proteins of S. neurona.

Unusual DNA secondary structures, especially G-quadruplexes and i-motifs, play important roles in gene transcriptional regulation and have been identified as novel drug targets. In this dissertation, I explored their formation in the human VEGF and RET promoters and their roles in gene transcriptional regulation. VEGF is a key regulator of angiogenesis and is up-regulated in many types of tumors. A poly-guanine/poly-cytosine (polyG/polyC) tract in its proximal promoter (-85 to -50 base pairs relative to the transcription starting site) is essential for both basal and inducible VEGF expression. I demonstrated that the guanine-rich (G-rich) and cytosine-rich (C-rich) strands in the VEGF proximal promoter are able to form G-quadruplex and i-motif structures, respectively. The major G-quadruplex formed by the VEGF G-rich sequence is an intramolecular parallel G-quadruplex containing three G-tetrads and a 1:4:1 arrangement of three double-chain-reversal loops (two single-base loops and one loop with four bases). The complementary C-rich sequence in the same region forms an intramolecular i-motif containing six semiprotonated cytosine-cytosine⁺ base pairs and a 2:3:2 loop configuration (two double-base loops and one loop with three bases). The Gquadruplexes formed by the native VEGF G-rich and its derivative sequences were also confirmed by NMR. In addition, various transcription factors including Sp1, hnRNP K, CNBP and nucleolin, which recognize different DNA structural elements including single-stranded, double-stranded or G-quadruplex/i-motif DNA in the VEGF proximal promoter, have been confirmed by EMSA, siRNA and chromatin immunoprecipitation (ChIP) assay, suggesting that the DNA in the VEGF proximal promoter region is capable of undergoing transitions between those three structures. Based on my studies, I have proposed a model to describe how various transcription factors recognize different DNA structures in the VEGF proximal promoter to regulate transcription. In the proximal promoter of another important oncogene RET, I demonstrated that the guanine-rich strand forms an intramolecular parallel G-quadruplex containing three G-tetrads and a 1:3:1 arrangement of three double-chain-reversal loops. The complementary cytosine-rich strand forms an i-motif structure containing six semiprotonated cytosine-cytosine⁺ base pairs and a 2:3:2 loop configuration. Moreover, G-quadruplex-interactive compounds TMPyP4 and telomestatin were shown to further stabilize the RET G-quadruplex structure.

All organisms require rapid and flexible signaling mechanisms in order to effectively respond to biotic and abiotic stress. Calcium ions (Ca2+) have proven to be important components of signaling networks. Observations of stimulus-specific oscillations of cytosolic Ca2+ during signal transduction suggest that Ca2+ signals directly encode information. These stimulus-specific oscillations, known as Ca2+ signatures, can be interpreted by an array of Ca2+-binding sensors and effectors, which subsequently regulate appropriate cellular responses. While progress has been made regarding the classic Ca2+-sensor calmodulin (CaM), less research has been directed towards the CaM-like family of Ca2+-sensors (CMLs). This family – unique to plants – is suspected to regulate a multitude of stress and developmental pathways; however, to date very few members of this family have had their functions elucidated by the identification of downstream targets and upstream regulators. In the present study, I investigate the regulation of CML39, which has previously been shown to strongly respond to the stress hormone jasmonic acid (JA) in Arabidopsis. Bioinformatic tools predict a large number of putative JA-responsive cis-elements within the CML39 promoter. Deletion analysis of CML39 promoter fragments in planta reveals that some cis-elements respond in a tissue-specific manner. Analysis of transgenic MYC2 loss-of-function (myc2) mutants demonstrates that MYC2 – the preeminent JA-responsive transcription factor – is not necessary for CML39 promoter activity. Collectively, these data reveal a complex tissue-specific pattern of CML39 regulation and provide a foundation for the future identification of relevant transcription factors. / Thesis (Master, Biology) -- Queen's University, 2013-09-24 21:06:30.592

Microbial metabolism can be tailored to meet human specifications, but the degree to which these living systems can be repurposed is still unknown. Artificial biological control strategies are being developed with the goal of enabling the predictable implementation of novel biological functions (e.g., engineered metabolism). This dissertation project contributes genetic tools useful for modulating gene expression levels (extending promoters with UP elements) and isolating transcription and translation of engineered DNA from the endogenous cellular network (expression by orthogonal cellular machinery), which have been demonstrated in Escherichia coli for the production of lycopene, a 40-carbon tetraterpene carotenoid with antioxidant activity and a number of other desirable properties.

Transcriptomics is the large-scale study of RNA molecules produced by the genome, in single cells or population of cells using high-throughput methods. With the advances in transcriptomic analysis, the monitoring of genome-wide gene expression provides a powerful approach for determining the action of drugs. In this study, we analyzed the transcriptional responses of cells treated with drugs either alone or in combinations to explore their effects in two different applications: breast cancer therapy and cell conversion. In the first part of this thesis, we aim at modeling the relationship between single and multidrug breast therapy at the transcriptome level. We monitored the effects of three drugs, and their combinations in human breast cancer MCF-7 cells using the cap analysis of gene expression method. We are the first to explore the impact of single and combinatorial treatment on promoter and enhancer expression on a genome-wide scale. After applying and customizing a broad spectrum of regression algorithms, we showed that the transcriptional response to combinatorial drug treatment at both promoters and enhancers is accurately described by a linear combination of the responses to the individual drugs. Our analysis is promising for eliciting the transcriptional reaction to multidrug therapies in an unbiased genome-wide way, which may minimize the need for exhaustive combinatorial screens. Following the drug combination analysis, we explored the possibility to systematically identify drugs that either alone or in combinations facilitate cell conversion. To date, no computational approach prioritizes or suggests chemical compounds promoting cell reprogramming. Using transcriptomic data of human primary cells and drug response expression profiles, we developed a computational framework which accurately predicts single drugs or drug cocktails driving any source cell type towards the desired lineage. Experimental and in-silico validation on human pluripotent stem cells confirms the ability of the top predicted drugs to enhance reprogramming. The introduced method has countless applications in regenerative medicine and can significantly speed up the research in this field.

Briefly, some findings in my research are as follows: (1) The genetic variants of the CA repeats in IGF1 promoter 1 can affect the activity of promoter 1, and the CA repeat showed a suppressive effect on the activity of the promoter 1 of IGF1 gene. EMSA results have shown that the CA repeats could bind to certain nuclear protein. (2) The SNPs T/C (rs5742612) and T/A (rs2288377) can also affect the activity of the promoter 1 in IGF1 gene, and the activity of C-A haplotype is significantly higher than that of T-T haplotype. EMSA results have shown that the SNP T/A (rs2288377) could bind to certain nuclear protein. (3) I developed the new dual reporter assay method to investigate the transactivation interaction between the SNP T/G (rs2071430) and C/A (rs17000900) in the MxA promorer. This new method can not only improve the detection limit for small difference between haplotypes, but also calculate the model of transactivation effect between these two SNPs. The results were better than those of traditional method, and it gave a clear-cut demonstration of the effect of interaction between these two SNPs on the activity of MxA promoter. / In addition, in the IGF1 study, the core promoter region of promoter 2 was identified through 5' deletion mutagenesis methods. Moreover, a cell-type specific mechanism of bidirectional activation of promoter was found. / Recently, more and more studies focus on gene function with the completion of the Human Genome Project. It is well known that polymorphism of human genome sequence is a common phenomenon in the human population. Specially, a lot of genetic polymorphisms, including single nucleotide polymorphisms (SNPs) and microsatellites, have been reported in the regulatory region of many genes. However, the effects of most of these genetic polymorphisms on gene expression are still unknown. The polymorphisms in the promoter can play an important role in the gene regulation. For example, some SNPs located in the transcription factor binding site (TFBS) can affect gene transcription. So, it is very necessary to directly study the effect of genetic variants on promoter transactivation activities. In this study, we studied the effect of genetic polymorphisms on gene expression through reporter gene assay, electrophoretic mobility shift assay (EMSA), and so on. And the candidate genes include insulin-like growth factor 1 (IGF1) and myxovirus resistence 1 (MxA). Some SNPs and microsatellites have been reported in the promoters of these genes. In our previous researches, we focused on the study of the association between these polymorphisms and some diseases, and it was found that a few SNPs significantly associated with relevant diseases. Based on the previous results, in my project, I developed new functional assays and also improved existing methods to analyse the functional effect of these genetic variants of promoters on transactivation activities. / by Huang, Wei. / "March 2008." / Adviser: Nelson Leung Sang Tang. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1483. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 139-145). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Lau Suk Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 128-150). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Table of content --- p.ii / List of Figures --- p.x / List of Tables --- p.xiv / Abbreviation --- p.xv / Abstract --- p.xviii / 論文摘要 --- p.xx / Chapter 1 --- Chapter 1 Introduction --- p.1 / Chapter 1.1 --- Tetraodon nigroviridis --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Genomic Sequencing Project --- p.3 / Chapter 1.1.3 --- Tetraodon nigroviridis as Study Model --- p.4 / Chapter 1.1.3.1 --- Genomic Comparison --- p.4 / Chapter 1.1.3.2 --- Gene Order and Structural Studies --- p.5 / Chapter 1.1.3.3 --- Genomic Evolution --- p.6 / Chapter 1.2 --- Transcriptional Regulation and Transcription Factors Binding Sites Prediction --- p.7 / Chapter 1.2.1 --- Transcriptional Regulation --- p.7 / Chapter 1.2.1.1 --- Chromatin Remodeling --- p.7 / Chapter 1.2.1.2 --- Locus Control Regions (LCR) and Boundary Elements --- p.8 / Chapter 1.2.1.3 --- Promoter Structure --- p.9 / Chapter 1.2.1.4 --- Transcriptional Machinery Assembly --- p.10 / Chapter 1.2.2 --- Transcription Factors and Their Binding Sites --- p.11 / Chapter 1.2.3 --- Transcription Factor Binding Site Prediction --- p.12 / Chapter 1.3 --- Acetylcholinesterase --- p.15 / Chapter 1.3.1 --- Background --- p.15 / Chapter 1.3.2 --- Regulation ofAChE --- p.17 / Chapter 1.3.2.1 --- Transcriptional Level --- p.17 / Chapter 1.3.2.2 --- Post-transcriptional Level --- p.19 / Chapter 1.3.2.3 --- Post-translational Level --- p.20 / Chapter 1.3.2.3.1 --- Oligomerization --- p.20 / Chapter 1.3.2.3.2 --- Glycosylation --- p.21 / Chapter 1.3.2.3.3 --- Phosphroylation --- p.22 / Chapter 1.3.3 --- Functions of AChE --- p.23 / Chapter 1.3.3.1 --- Hydrolysis Acetylcholine --- p.23 / Chapter 1.3.3.2 --- Embryonic Development --- p.23 / Chapter 1.3.3.3 --- Haemotopotesis and Thrombopsiesis --- p.24 / Chapter 1.3.3.4 --- Neuritogensis --- p.24 / Chapter 1.3.3.5 --- Amyloid Fibre Assembly --- p.24 / Chapter 1.3.3.6 --- Apoptosis --- p.25 / Chapter 1.3.4 --- AChE and Alzheimer's disease --- p.25 / Chapter 1.3.4.1 --- Treatment for AD Patients --- p.27 / Chapter 1.4 --- Inducible Cell Expression Systems --- p.28 / Chapter 1.5 --- Objectives --- p.32 / Chapter 2 --- Chapter 2 Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Primer Design --- p.34 / Chapter 2.2.2 --- Cell Culture --- p.34 / Chapter 2.2.3 --- Transformation --- p.35 / Chapter 2.2.4 --- Plasmids Preparation --- p.35 / Chapter 2.2.5 --- Plasmids Screening --- p.36 / Chapter 2.2.6 --- RNA Extraction --- p.36 / Chapter 2.2.7 --- Reverse Transcriptase Polymerase Chain Reaction and Construction tnAChE/pCR4 vector --- p.37 / Chapter 2.2.8 --- Genomic Analysis --- p.37 / Chapter 2.2.9 --- Protein Sequence Analysis --- p.38 / Chapter 2.2.10 --- Genomic DNA Extraction --- p.39 / Chapter 2.2.11 --- Construction of Reporter Vectors ptnAChE_565/pGL3 and ptnAChK1143/pGL3 --- p.39 / Chapter 2.2.12 --- Luciferase Assay --- p.40 / Chapter 2.2.13 --- Transcription Factors and Promoter Prediction --- p.40 / Chapter 2.2.14 --- Protein Assay --- p.41 / Chapter 2.2.15 --- AChE Activity Determined by Ellman's Method --- p.41 / Chapter 2.2.16 --- Histochemistry --- p.42 / Chapter 2.2.17 --- Protein Extraction from Tissues --- p.42 / Chapter 2.2.18 --- Construction of Bacterial Expression Vector His-MBP-tnAChEAC/pHISMAL --- p.43 / Chapter 2.2.19 --- Protein Expression in Bacterial Expression System --- p.43 / Chapter 2.2.20 --- Purification and Thrombin Cleavage of His-MBP- tnAChEAC --- p.44 / Chapter 2.2.21 --- SDS Electrophoresis --- p.44 / Chapter 2.2.22 --- Western Blotting --- p.45 / Chapter 2.2.23 --- Construction of Tet-Off Expression Vector --- p.45 / Chapter 2.2.24 --- Transient Expression of tnAChEAC --- p.46 / Chapter 2.2.25 --- Establishment of Stable Tet-Off CHO Cell Lines Overexpressing tnAChEAC --- p.47 / Chapter 2.2.26 --- MTT Assay --- p.47 / Chapter 2.2.27 --- Partial Purification of tnAChEΔC --- p.48 / Chapter 3 --- Chapter 3 Sequence Analysis of AChE Gene of Tetraodon nigroviridis --- p.49 / Chapter 3.1 --- Results --- p.49 / Chapter 3.1.1 --- Cloning of tnAChE from Tetraodon nigroviridis Brain --- p.49 / Chapter 3.1.2 --- "Comparative genomic analysis of tnAChE with Human, Rat, Mouse, Takifugu rubripes, ZebrafishAChE" --- p.49 / Chapter 3.1.3 --- Primary Sequence Analysis --- p.52 / Chapter 3.1.4 --- Promoter and Transcriptional Factors Predictedin tnAChE Promoter Region --- p.60 / Chapter 3.1.4.1 --- Promoter Region Analysis In Silico --- p.60 / Chapter 3.1.4.2 --- Promoter Activity Analysis --- p.76 / Chapter 3.2 --- Discussion --- p.78 / Chapter 4 --- Characterization of tnAChE in Prokaryotic and Eukaryotic Tet-Off Inducible Expression System --- p.91 / Chapter 4.1 --- Results --- p.91 / Chapter 4.1.1 --- AChE Expresses in Tetraodon nigroviridis --- p.91 / Chapter 4.1.2 --- Expression of recombinant tnAChE in Bacterial Expression System --- p.94 / Chapter 4.1.2.1 --- Construction of His-MBP-tnAChEΔC/pHISMAL Construct --- p.94 / Chapter 4.1.2.2 --- His-MBP-tnAChEAC Expression in E. coli Strains BL21 (DE) and C41 --- p.94 / Chapter 4.1.3 --- Expression of tnAChEAC in Mammalian Expression System --- p.99 / Chapter 4.1.3.1 --- Construction of tnAChEAC/pTRE2hgyo Mammalian Expression Vector --- p.99 / Chapter 4.1.3.2 --- Transient Expression of tnAChEAC --- p.99 / Chapter 4.1.3.3 --- Establishment of Tet-Off CHO Cells Stably Expressing the Inducible tnAChEAC --- p.101 / Chapter 4.1.3.4 --- Characterization of Tet-Off tnAChEAC Stably Transfected Cell Clones --- p.103 / Chapter 4.1.3.5 --- Effect of Over Expressed tnAChEAC on cell viability --- p.103 / Chapter 4.1.3.6 --- Partial Purification of tnAChEAC from Stably Transfected Cells --- p.107 / Chapter 4.1.3.7 --- tnAChE and tnAChEAC in Different pH Values --- p.112 / Chapter 4.1.3.8 --- Kinetic Study of tnAChEAC --- p.112 / Chapter 4.1.3.9 --- Inhibition of AChE Activity of Partial Purified tnAChEAC by Huperzine --- p.112 / Chapter 4.2 --- Discussion --- p.116 / Chapter 4.2.1 --- Bacterial Expression System --- p.116 / Chapter 4.2.2 --- Expression of tnAChEΔC in Mammalian System --- p.119 / Chapter 5 --- General Discussion --- p.124 / Chapter 5.1 --- Summaries --- p.124 / Chapter 5.2 --- Further works --- p.126 / Chapter 6 --- References --- p.128 / Appendix 1 internet software and database used in this project --- p.151 / Appendix 2 tnAChE mRNA sequence --- p.152 / Appendix 3 ptnAChE-1143 sequence --- p.154 / Appendix 4 Six open reading frame translation of ptnAChE-1143 --- p.156

Ho, Sui Fan Tong. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 68-71). / LIST OF TABLES / LIST OF FIGURES / ACKNOWLEDGEMENTS / ABSTRACT / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 2. --- OBJECTIVES --- p.12 / Chapter 3. --- MATERIALS AND METHODS --- p.13 / Chapter 3.1 --- Materials --- p.13 / Chapter 3.1.1 --- Enzymes --- p.13 / Chapter 3.1.2 --- DNA Markers --- p.13 / Chapter 3.1.3 --- General Reagents --- p.13 / Chapter 3.2 --- General Methods --- p.15 / Chapter 3.2.1 --- Phenol and Phenol/Chloroform (1:1) Preparation --- p.15 / Chapter 3.2.2 --- Buffer Preparation --- p.15 / Chapter 3.2.3 --- Agarose Gel Electrophoresis --- p.18 / Chapter 3.2.4 --- Polyacrylamide Gel Electrophoresis --- p.18 / Chapter 3.3 --- DNA Study --- p.19 / Chapter 3.3.1 --- Haemophilia B Patient --- p.19 / Chapter 3.3.2 --- Blood Collection --- p.20 / Chapter 3.3.3 --- DNA Extraction --- p.20 / Chapter 3.3.4 --- DNA Quantitation --- p.21 / Chapter 3.3.5 --- Polymerase Chain Reaction --- p.22 / Chapter 3.3.6 --- Purification of PCR Products --- p.28 / Chapter 3.3.7 --- Sequencing --- p.32 / Chapter 3.3.8 --- Cloning --- p.37 / Chapter 4. --- RESULTS --- p.40 / Chapter 4.1 --- DNA Extraction --- p.40 / Chapter 4.2 --- Calibration of the Coy TempCycler --- p.42 / Chapter 4.3 --- Optimization of PCR --- p.44 / Chapter 4.3.1 --- PCR-1 --- p.44 / Chapter 4.3.2 --- PCR-2 --- p.46 / Chapter 4.3.3 --- PCR-3 --- p.46 / Chapter 4.3.4 --- PCR-4 --- p.48 / Chapter 4.3.5 --- PCR-5 --- p.49 / Chapter 4.3.6 --- PCR-6 --- p.50 / Chapter 4.3.7 --- PCR-7 --- p.51 / Chapter 4.4 --- Purification of PCR Product --- p.52 / Chapter 4.4.1 --- GC-1 --- p.52 / Chapter 4.4.2 --- GC-2 --- p.52 / Chapter 4.4.3 --- GC-3 --- p.53 / Chapter 4.4.4 --- PAGE-1 --- p.54 / Chapter 4.4.5 --- PAGE-2 --- p.54 / Chapter 4.4.6 --- Agarose Gel Extraction with Glasswool Exclusion --- p.55 / Chapter 4.5 --- Direct Sequencing of PCR Products --- p.55 / Chapter 4.6 --- Cloning --- p.55 / Chapter 5. --- DISCUSSION --- p.57 / Chapter 5.1 --- DNA Extraction --- p.57 / Chapter 5.2 --- Polymerase Chain Reaction --- p.57 / Chapter 5.3 --- Purification of PCR Products --- p.58 / Chapter 5.4 --- Sequencing --- p.61 / Chapter 5.5 --- Cloning --- p.61 / Chapter 6. --- CONCLUSION --- p.67 / Chapter 7. --- PHOTOGRAPHS --- p.64 / Chapter 8. --- REFERENCES --- p.68

Orientador: Marco Eustáquio de Sá / Resumo: O tratamento químico de sementes é uma prática tradicional na agricultura, utilizado no controle de agentes fitopatogênicos presentes nas sementes e no solo. No tratamento biológico o uso de rizobactérias tem sido uma alternativa interessante, como a espécie Bacillus subtilis, resistente a condições adversas de calor e baixa umidade. Assim, objetivou-se avaliar a ação de dois produtos biológicos comerciais feito à base de Bacillus subtilis FMT001 e Serenade®, e um produto químico Vitavax Thiram® 200 SC, aplicados via tratamento de sementes, como promotores de crescimento de plantas e protetores das sementes, visando verificar o desempenho germinativo das sementes e os efeitos da inoculação de doses sob diferentes espaços de tempo de exposição aos tratamentos, em caupi (Vigna unguiculata) e em feijoeiro comum (Phaseolus vulgaris). O experimento foi conduzido no LAS da UNESP. utilizando cinco lotes de sementes de feijão, quatro lotes de Vigna unguiculata e um de Phaseolus vulgaris. As sementes foram submetidas aos tratamentos biológico e químico, foram estabelecidos seis tratamentos e quatro repetições, com quatro doses de inoculantes à base de Bacillus subtilis: FMT001 com doses 100 ml, 200 ml, 400 ml e 200 ml produto Serenade®, e 100 ml VitavaxThiram® 200 SC, para 50 kg-1 sementes. Foram avaliados condutividade elétrica, teste de germinação, comprimento de raiz e plântulas, envelhecimento acelerado, teste de frio, sob os períodos de 00, 24, 48, 72 e 96 horas após a inoculação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The seed chemical treatment is a traditional practice in agriculture, used for controlling pathogenic agents present in the seeds and soil. In the biological treatment using rhizobacteria it has been an interesting alternative as a Bacillus subtilis species, resistant to adverse conditions of heat and low humidity. The objective of this study was to evaluate the action of two commercial biological products based on Bacillus subtilis FMT001 and Serenade® and a chemical product Vitavax Thiram® 200 SC, applied through seed treatment, as plant growth promoters and seed protectors, to verify the germination performance of the seeds and the effects of the inoculation of doses under different time periods of exposure to the treatments, in Vigna unguiculata cowpea and common bean Phaseolus vulgaris. The test was conducted in the LAS of UNESP. using five lots of bean seeds four lots of Vigna unguiculata and one of Phaseolus vulgaris. The seeds were submitted to biological and chemical treatments. Six treatments and four replications were established with four doses of Bacillus. subtilis inoculants: FMT001 with doses of 100 ml, 200 ml, 400 ml and 200 ml Serenade® product, e 100 ml VitavaxThiram® 200 SC, for 50 kg-1 seeds. Electrical conductivity were evaluated, the test germination, seedling root length and, accelerated aging, cold test, in periods of 00, 24, 48, 72 and 96 hours after inoculation. Additional chemical and biological treatments, particularly the chemical, are generally s... (Complete abstract click electronic access below) / Doutor

Atualmente o Brasil ocupa lugar de destaque entre os produtores de citros sendo o maior produtor de laranja doce do mundo. Entretanto, essa produção vem sendo gravemente afetada, por doenças como o huanglongbing (HLB), que tem causado perdas significativas para toda cadeia citrícola. O HLB está associado a bactérias Gram-negativas, restritas ao floema das plantas, denominadas Candidatus Liberibacter spp., que além de reduzir a produção de frutos, podem, em casos mais severos da doença, ocasionar a morte da planta. Uma importante estratégia para controle desta doença é a produção de plantas transgênicas expressando genes, especificamente no local de colonização do patógeno. O objetivo deste trabalho foi a obtenção de plantas transgênicas de Citrus sinensis cvs. \'Pera\' e \'Natal\', contendo o gene atacina A (attA) que codifica um peptídeo antibacteriano, dirigido por promotores preferencialmente expressos no floema: AtSuc2 (transportador de sacarose) ou AtPP2 (proteína de floema 2), clonados de Arabidopsis thaliana, ou CsPP2 (proteína de floema 2) clonado de Citrus sinensis. A identificação de 65 plantas transgênicas foi realizada, por meio da análise de PCR. Foi verificado um número menor de eventos transgênicos, utilizando-se a construção gênica pCAtSuc2/attA em relação ao número de eventos transgênicos obtidos com as construções gênicas pCCsPP2/attA e pCAtPP2/attA. Plantas identificadas como transgênicas pela análise de PCR, foram aclimatizadas e transferidas para casa-devegetação certificada para o cultivo de plantas transgênicas. Análises de Southern blot foram realizadas em plantas aclimatizadas que apresentaram desenvolvimento suficiente, confirmando-se a integração do gene attA. A expressão do gene attA foi confirmada pela análise de RT-qPCR. As plantas transgênicas obtidas neste trabalho, contendo o gene attA dirigido por promotores preferencialmente expressos no floema, serão avaliadas em uma etapa futura para resistência a Candidatus Liberibacter spp. / Currently, Brazil is a major citrus producer and the world\'s largest producer of sweet oranges. However, diseases, such as huanglongbing (HLB) have seriously affected this production, causing significant losses in citrus production chain. HLB is associated with Gram-negative bacterias, restricted to the phloem of plants, called Candidatus Liberibacter spp., which besides reducing fruit production, can lead to plant death. An important strategy to control this disease is the production of transgenic plants expressing genes, specifically at the region of pathogen colonization. The aim of this study was to obtain transgenic plants of Citrus sinensis cv. \'Natal\' and \'Pera\', containing the gene attacin A (attA) that encodes an antibacterial peptide, driven by preferentially expressed in phloem promoters: AtSuc2 (sucrose transporter) or AtPP2 (phloem protein 2), cloned from Arabidopsis thaliana, or CsPP2 (phloem protein 2) cloned from Citrus sinensis. The identification of 65 transgenic plants was performed by PCR analysis. A lower number of transgenic events were verified using the gene construct pCAtSuc2/attA in relation events obtained with the gene constructs pCCsPP2/attA and pCAtPP2/attA. Plants identified as transgenic by PCR analysis were acclimatized and transferred to a greenhouse certified for growing transgenic plants. The Southern blot analyses were performed in acclimatized plants, which had sufficiently developed, confirming the integration of attA gene. The expression of attA gene was confirmed by RT-qPCR analysis. The transgenic plants obtained in this work, containing the gene attA directed by preferentially expressed in phloem promoters, will be further evaluated for resistance to Candidatus Liberibacter spp.