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Work plan for Scientific Initiation Fellowship #A

Title: Selection of transformation events to evaluate PK promoter activity

The Sugarcane Regulatory Network project will study gene regulation in sugarcane. The main goal of this project is todesign tools that will allow a System Biology approach in sugarcane and to identify regulatory networks in thissystem. As a starting point, we will characterize two agronomic traits of interest in this feedstock: sucroseconcentration and drought resistance. Furthermore, we will analyze gene categories with known regulatory role(transcription factors, protein kinases and phosphatases), further the study of the sugarcane transcriptome,generate plants that are transgenic for genes of interest and develop bioinformatics tools and databases that willintegrate all the information levels. Finally, we will identify transcription factor targets by using the ChIP-HTS methodand by cloning new promoters. As part of this Scientific Initiation Fellowship, an undergraduate student willcharacterize one promoter from a SnRK protein kinase family that is expressed in sugarcane internodes. About 50transgenic plants independently transformed with a GUS reporter under a protein kinase promoter will be examinedfor transgene insertion, uidA gene expression levels and GUS activity in tissues.

Aims:

The main goal of this project is to confirm the promoter activity of protein kinase promoter fragments

associated to soluble sugar contents.

Activities:

In this project we will:

1- Extract DNA samples from 50 independent transformation events with the pUSPProG::PRO(SnRK-X)construct.

2- Confirm the construction insertion in the genome by PCR using specific primers. At least 20 lines will beselected.

3- Analyze the uidA gene expression levels in the plants selected above by real-time PCR.4- Search for tissue specific expression (internodes, root or leaves) of the GUS reporter by histochemical

assays.

Methods:

Detection of transformed sugarcane will be done by PCR. At first, total DNA will be isolate from resistant buds bythe CTAB method [1]. PCR reactions will be done using Taq polimerase (Invitrogen), using primers that are specificto neo (or bar, if we choose this selection marker). Real time PCR will be performed as described [2]. uidA geneexpression will be estimated by histochemical assays. In these assays, internodes, leaf and root samples fromtransgenic plants will be incubated in reaction buffer (100 mM NaH2PO4.H2O, 0,5 mM K4Fe(CN)6.3H2O, 10 mMNa2EDTA.2H2O, 0,1% Triton X-100 e 1 mM X-Gluc) in the dark at 37

oC for 16 hours. After the incubation, thereaction buffer will be replaced by 70% ethanol in order to stop the reaction. The tissues will be analyzed using a

stereomicroscope and pictures will be taken.

Relevance:

This project will introduce the undergraduate student to the academic research in molecular biology andbioinformatics at the same time that may result in patentable data with great biotechnological potential.

Observao: O plano de atividades de IC anexado, contm o plano de atividades (#A ),que o mesmo plano

relativo a bolsa e aprovado no auxlio do projeto temtico 2008/52146-0. Este plano (#A ) ser substitudo pelo

plano (#B), que est abaixo do primeiro.

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Work plan for Scientific Initiation Fellowship #B

REDES REGULATRIAS DA CANA-DE-ACAR - Processo Temtico: 2008/52146-0

TTULO DO PROJETOEstudo estrutural gnico de BACs de cana-de-acar pelo mtodo de pirosequenciamento

Candidata Iniciao CientificaSideny Lima Nunes

OrientadoraGlaucia Mendes Souza

INSTITUIO SEDEDepartamento de BioqumicaInstituto de Qumica - Universidade de So Paulo

RESUMO

O sequenciamento do transcriptoma da cana-de-acar, atravs do Projeto SUCEST gerou por voltade 43 mil genes e proporcionou um aprofundamento e aprimoramento nos estudos bioqumicos,moleculares e computacionais de uma das plantas mais importantes economicamente no mundo, a cana-de-acar. A identificao de genes alvo, bem como localizao de suas respectivas regies promotores,nos permite estudos da funo de cada protena, como por exemplo, quinases e fosfatases (PKs e PPases),para gerao de plantas transgnicas, resistentes a seca ou com alto teor de fibras.

O objetivo deste projeto o pirosequenciamento de 30 BACs de cana, identificao e anotaoestrutural gnica dos dados gerados.

PLANO DE ATIVIDADES

Sequenciamento de cidos nuclicos em equipamento de ultima gerao 454, identificao e anotaoestrutural gnica dos dados gerados.

CRONOGRAMA DE ATIVIDADES

Trimestre 1 2 3 4Extrao e purificao de DNA de cana-de-acar X

Pirosequenciamento de 30 BACs X

Identificao e anotao estrutural gnica dos dados gerados X X

JUSTIFICATIVA

O projeto SUCEST-FUN, proporcionou o desenvolvimento de ferramentas para estudos e anlisesdos dados de microarray, aplicao de mtodos estatsticos e relacionamento com informaes de genesenvolvidos com vias de transduo de sinal e metabolismo de cana-de-acar. Visando buscar uma coleode ferramentas biotecnolgicas para o descobrimento de regies ricas em genes no genoma de cana e umaumento da eficincia de sequenciamento, para o desenvolvimento desse projeto, optamos pelaconstruo de uma coleo de 30 BACs que sero sequenciados do cultivar R570 de cana-de-acar. O

sequenciamento desses BACs ser feito com a tecnologia 454 Pyrosequencing High-throughput(Roche), noequipamento do Laboratrio do Centro Avanado de Tecnologias em Genmica (CATG), no Departamentode Bioqumica do Instituto de Qumica da Universidade de So Paulo (IQ-USP).