BRPI0610654B1 - molécula de ácido nucléico do evento mon89788, produto primário de soja, seu método de produção, pares de moléculas iniciadoras, kit de detecção de dna, métodos de produzir uma planta de soja tolerante ao herbicida glifosato, detectar a presença de dna e controlar o crescimento de ervas daninhas - Google Patents
molécula de ácido nucléico do evento mon89788, produto primário de soja, seu método de produção, pares de moléculas iniciadoras, kit de detecção de dna, métodos de produzir uma planta de soja tolerante ao herbicida glifosato, detectar a presença de dna e controlar o crescimento de ervas daninhas Download PDFInfo
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Abstract
patente de invenção: ocorrência de soja m0n8978 e métodos para detecção da mesma. a presente invenção refere-se a planta e semente de soja que compreendem o evento de transformação m0n89788 e moléculas de dna únicas para esses eventos. a invenção também fornece métodos para de- tectar a presença dessas moléculas de dna em uma amostra.
Description
Relatório Descritivo da Patente de Invenção para "MOLÉCULA DE ÁCIDO NUCLÉICO DO EVENTO MON89788, PRODUTO PRIMÁRIO DE SOJA, SEU MÉTODO DE PRODUÇÃO, PARES DE MOLÉCULAS INI-ClADORAS, KIT DE DETECÇÃO DE DNA, MÉTODOS DE PRODUZIR UMA PLANTA DE SOJA TOLERANTE AO HERBICIDA GLIFOSATO, DETECTAR A PRESENÇA DE DNA E CONTROLAR O CRESCIMENTO DE ERVAS DANINHAS".
Antecedentes da Invenção Esse pedido de patente reivindica prioridade do Pedido de patente Provisório U.S. N° 60/685.584, depositado em 27 de maio de 2005, cuja descrição completa está incorporada aqui por referência. 1. Campo da Invenção A presente invenção refere-se a um novo e característico evento de transformação de soja transgênico, chamado MON89788, um cultivar de soja derivado dele, e partes de planta, semente e produtos desse. A invenção também refere-se a testes para detectar a presença de uma molécula de DNA específica para MON89788 em um extrato de parte de planta ou extrato de semente. 2. Descrição da Técnica Relacionada Soja (Glycine max) é um cultivo importante em várias áreas do mundo. Os métodos de biotecnologia têm sido aplicados à soja para melhora das características agronômicas e da qualidade do produto. Uma tal característica agronômica importante na produção de soja é a tolerância à herbicida, em particular, tolerância herbicida glifosato. Um evento de 1 a soja tolerante à herbicida seria uma uai acterístíca útil para controlar plantas daninhas. N-fosfonometilglicina, também conhecido como glifosato, é um herbicida bem-conhecido que tem atividade em um amplo espectro de espécies de planta. O glifosato é o ingrediente ativo de Roundup® (Monsanto Co., St. Louis, MO), um herbicida seguro que tem meia vida desejavelmente curta no meio ambiente. Quando aplicado à superfície de uma planta, o glifosato se move sistemicamente através da planta. O gli-setjue-se página 1 a fosato é fitotóxico devido a sua inibição da via do ácido chiquímico, que fornece um precursor para a síntese de aminoãcidos aromáticos. O glifo-sato inibe a enzima 5-enolpíruνίί-3-fosfochiquimato sintase (EPSPS) encontrada em plantas. A tolerância ao glifosato pode ser alcançada pela expressão de variantes de EPSPS que têm menor afinidade por glifosato e portanto retêm sua atividade catalítica na presença de glifosato (Patente U.S. NF, 5.633.435; 5.094.945: 4.535.060. e 6.040.497). Enzimas que degradam o glifosato em tecidos de planta (Patente U.S. N- 5.463.175) também são capazes de com ferir tolerância celular ao glifosato. Tais genes são usados para a produção de cultivos transgênicos que são tolerantes ao glifosato, assim permitindo que o glifosato seja usado para controle eficaz de plantas daninhas com preocupação mínima de danos ao cultivo. Por exemplo, tolerância ao glifosato foi manipulada geneticamente em milho (Patente U.S. 5.554.798), trigo (Patente U.S. 6.689.880), algodão (Patente U.S. 6.740.488), soja (WO 9200377) e canola (Pedido de Patente U.S. 20040018518). Os transgenes para tolerância ao glifosato e os transgenes para tolerância a outros herbicidas, por exemplo, o gene bar, (Toki et al., 1992; Thompson et ai, 1987; fosfinotricina acetiltransferase (DeBlock et ai, 1987), para tolerância ao herbicida glufosi-nato) também são úteis como marcadores selecionáveis e marcadores classificáveis e pode fornecer um fenótipo útil para seleção de plantas ligadas com outras características agronomicamente úteis. A expressão de genes estranhos em plantas é conhecida por ser influenciada por sua posição cromossômica, talvez devido à estrutura da cromatina (por exemplo, heterocromatina) ou a proximidade de elementos de regulação transcricional (por exemplo, intensificadores) próximos ao sítio de integração (Weising et al., 1988). Por essa razão, é freqüentemente necessário rastrear um grande número de eventos a fim de identificar um evento caracterizado por expressão ótima de um gene de interesse introduzido. Por exemplo, foi observado em plantas e em outros organismos que pode haver uma grande variação nos níveis de expressão de um gene introduzido entre eventos. Também pode haver diferenças nos padrões espaciais ou temporais de expressão, por exemplo, diferenças na expressão relativa de um transgene em vários tecidos de planta, que pode não corresponder aos padrões esperados a partir de elementos regulatórios transcricionais presentes no construto gênico introduzido. Por essa razão é comum produzir centenas a milhares de eventos diferentes e rastrear esses eventos por um único vento que tenha os níveis e padrões de expressão do transgene para fins comerciais desejados. Um evento que tem os níveis ou padrões de expressão de transgene desejados é útil para introgredir o transgene em outro perfil genético por cruzamento sexual usando métodos de reprodução convencionais. A progênie de tais cruzamentos mantém as características de expressão do transgene do transformante original. Essa estratégia é usada para garantir expressão gênica confiável em diversas variedades que estão bem adaptadas às condições de crescimento locais.
Seria vantajoso ser capaz de detectar a presença de um evento particular a fim de determinar se a progênie de um cruzamento sexual contém um transgene de interesse. Além disso, um método para detectar um evento particular seria útil para cumprir os regulamentos que requerem a pré-aprovação do mercado e rotulagem de alimentos derivados de plantas de cultivos transgênicos, por exemplo. É possível detectar a presença de um transgene por qualquer método de detecção de ácido polinucíéico bem-conhecido tal como a reação em cadeia da polimerase (PCR) ou hibridiza-ção de DNA usando sondas de ácido polinucíéico. Esses métodos de detecção geralmente focam em elementos genéticos usados freqüentemente, tais como promotores, terminadores, genes marcadores, etc. Como um resultado, tais métodos podem não ser úteis para discriminar entre eventos diferentes, particularmente aqueles produzidos usando o mesmo construto de DNA a menos que a seqüência de DNA cromossômico (“DNA flanqueador”) adjacente ao DNA do transgene inserido seja conhecida. Um teste de PCR even-to-específico é discutido, por exemplo, por Windels et ai. (1999), que identificou o evento de soja tolerante ao glifosato 40-3-2 por PCR usando um conjunto de iniciadores que ocupa a junção entre o inserto do transgene e um DNA flanqueador, especificamente um iniciador que incluía a seqüência do inserto e um segundo iniciador que incluía seqüência do DNA flanqueador. Métodos de detecção de DNA específico de evento de planta transgênico também foram descritos nas Patentes U.S. Nos. 6.893.826; 6.825.400; 6.740.488; 6.733.974 e 6.689.880; 6.900.014 and 6.818.807, aqui incorporadas por referência nas suas totalidades. A invenção refere-se ao evento de soja tolerante ao glifosato MON89788 (também referido como MON19788 ou GM_A19788) e às molé- cuias de DNA contidas nessas plantas de soja que são úteis em métodos de detecção para a planta e progênie dessa e tecidos de planta derivados de MON89788.
Sumário da Invenção A presente invenção fornece um evento transgênico de soja chamado MON89788 (também referido como MON19788) e progênie desse que tem semente representante depositada com a American Type Culture Collection (ATCC) com N2 de acesso PTA-6708. Outro aspecto da invenção são as células de planta ou partes regeneráveis da planta e sementes do evento de soja MON89788. A invenção também inclui partes de planta do evento de soja MON89788 que incluem, mas não estão limitadas a uma célula, pólen, óvulo, flores, ramos, folhas, e produtos derivados de MON89788, por exemplo, farelo, farinha e óleo de soja.
Um aspecto da invenção fornece composições e métodos para detectar a presença de uma região de junção de DNA de transgene/genômico de uma planta ou semente de evento de soja MON89788 ou produtos derivados das partes de planta ou semente. São fornecidas moléculas de DNA que compreendem pelo menos uma molécula de DNA da junção transgene/ ge-nômico selecionada a partir do grupo que consiste em SEQ ID NO: 1 e SEQ ID NO: 2, e complementos dessas, em que a molécula de junção ocupa o sítio de inserção que compreende um DNA heterólogo inserido no genoma da célula de soja e o DNA genômico da célula de soja flanqueando o sítio de inserção do evento de soja MON89788. Tais seqüências de junção podem, em um aspecto da invenção, ser definidas como compreendendo os nucieo-tídeos 1093-1113 ou 5396-5416 de SEQ ID NO: 9, respectivamente. Em outros aspectos da invenção, as junções podem ser definidas como incluindo porções adicionais do genoma flanqueador e do transgene, por exemplo, e podem ser definidas como compreendendo uma ou mais seqüências como dadas pelos nucleotídeos 1073-1113, 1043-1113, 1093-1133, 1093-1163, 1043-1163, 5376-5416, 5346-5416, 5396-5436, 5396-5416, 5396-5466, ou 5346-5466 de SEQ ID NO: 9. Tais seqüências e plantas e sementes que compreendem essas seqüências formam, portanto um aspecto da invenção. É fornecida uma nova molécula de DNA que é uma região de DNA de transgene/genômico SEQ ID NO: 3 ou o complemento dessa, do evento de soja MON89788. Uma planta de soja e semente que compreende SEQ ID NO: 3 no seu genoma é um aspecto dessa invenção, SEQ ID NO: 3 ainda compreende SEQ ID NO: 1 na sua totalidade.
De acordo com outro aspecto da invenção, é fornecida uma molécula de DNA que é uma região de DNA de transgene/genômico SEQ ID NO: 4, ou o complemento dessa, em que essa molécula de DNA é nova no evento de soja MON89788. Uma planta de soja e semente que compreende SEQ ID NO: 4 no seu genoma é um aspecto dessa invenção. SEQ ID NO: 4 ainda compreende SEQ ID NO: 2 na sua totalidade.
De acordo com outro aspecto da invenção, duas moléculas de ácido nucleíco são fornecidas para uso em um método de detecção de DNA, em que a primeira molécula de ácido nucleíco compreende pelo menos 11 ou mais polinucleotídeos contíguos de qualquer porção da região do transgene da molécula de DNA de SEQ ID NO: 3 e o segundo ácido nucleíco é uma molécula de extensão similar de qualquer porção de uma região de DNA genômico de soja flanqueadora 5’ de SEQ ID NO: 3, em que essas moléculas de ácido nucleíco quando usadas juntas são úteis como iniciadores em um método de amplificação de DNA que produz um amplicon. O ampli-con produzido usando esses iniciadores no método de amplificação de DNA é diagnóstico para o DNA do evento de soja MON89788. O amplicon produzido pelos iniciadores descritos que é homólogo ou complementar a uma porção de SEQ ID NO: 3 compreendendo SEQ ID NO: 1 é um aspecto da invenção.
De acordo com outro aspecto da invenção, duas moléculas de ácido nucleíco são fornecidas para uso em um método de detecção de DNA, em que a primeira molécula de ácido nucleíco compreende pelo menos 11 ou mais polinucleotídeos contíguos de qualquer porção da região do transgene da molécula de DNA de SEQ ID NO: 4 e uma segunda molécula de ácido nucleíco de extensão similar de qualquer porção de um DNA genômico de soja flanqueador 3’ de SEQ ID NO: 4, em que essas moléculas de ácido nucleíco quando usadas juntas são úteis como iniciadores em um método de amplificação de DNA que produz um amplicon. O amplicon produzido usando esses iniciadores no método de amplificação de DNA é diagnóstico para o DNA do evento de soja MON89788. O amplicon produzido pelos iniciadores descritos que é homólogo ou complementar a uma porção de SEQ ID NO: 4 que compreende SEQ ID NO: 2 é um aspecto da invenção.
Qualquer par de iniciadores de ácido nucleíco derivado de SEQ ID NO: 3 ou SEQ ID NO: 4, ou SEQ ID NO: 9 ou os complementos desses, que quando usados em uma reação de amplificação de DNA produz um amplicon diagnóstico para tecido derivado do evento de soja MON89788 tal como um amplicon que compreende SEQ ID NO: 1 ou SEQ ID NO: 2 ou qualquer porção de SEQ ID NO: 9, respectivamente, é outra modalidade da invenção. Em uma modalidade particular, o par de iniciadores pode consistir em um iniciador A (SEQ ID NO: 5) e iniciador D (SEQ ID NO: 8).
Outro aspecto da invenção é uma planta de soja ou semente, ou produto derivado de uma planta ou semente que compreende o evento MON89788, no qual o DNA genômico quando isolado da planta de soja, ou semente, ou produto, produz um amplicon em um método de amplificação de DNA que compreende SEQ ID NO: 1 ou SEQ ID NO: 2.
Ainda outro aspecto da invenção é uma planta de soja, ou semente, ou produto derivado de uma planta ou semente que compreende MON89788, no qual o DNA genômico quando isolado da planta de soja, ou semente, ou produto produz um amplicon em um método de amplificação de DNA, em que as moléculas íntciadoras de DNA SEQ ID NO: 5 e SEQ ID NO: 6 são usadas no método de amplificação de DNA.
Ainda outro aspecto da invenção é uma planta de soja, semente, produto ou mercadoria derivada da planta ou semente, que compreende MON89788, no qual o DNA genômico quando isolado da planta de soja, ou semente, ou produto produz um amplicon em um método de amplificação de DNA, em que as moléculas iniciadoras de DNA SEQ ID NO: 7 e SEQ ID NO: 8 são usadas no método de amplificação de DNA. O produto ou mercadoria pode compreender, sem limitação, um alimento ou produto alimentício que compreende ou é derivado de um ou mais dos seguintes produtos de uma planta de soja que compreende o evento MON89788: lecitina, ácidos graxos, giicerol, esterol, óleo comestível, flocos de soja desengordurados. farelos de soja incluindo farelos de soja desengordurados e tostados, coalhada de leite de soja, tofú, farinha de soja, concentrado de proteína de soja, proteína de soja isolada, proteína de soja hidrolisada, proteína de soja texturizada, e fibra de proteína de soja.
De acordo com outro aspecto da invenção, um método de detectar a presença de DNA que corresponde especificamente ao DNA do evento de soja MON89788 em uma amostra é fornecido. Tal método compreendendo: (a) colocar uma amostra compreendendo DNA em contato com um par de iniciadores de DNA (b) realizar uma reação de amplificação de ácido nu-cleíco, assim produzindo o amplicon; e (c) detectar o amplicon, em que o dito amplicon compreende SEQ ID NO: 1 ou SEQ ID NO: 2. Um kit que compreende moléculas de iniciadores de DNA que quando usadas em um método de amplificação de DNA produz um amplicon que compreende SEQ ID NO: 1 ou SEQ ID NO: 2 é um aspecto adicional da invenção.
De acordo com outro aspecto da invenção, um método de detectar a presença de DNA que corresponde especificamente ao DNA do evento de soja MON89788 em uma amostra é fornecido. Tal método compreendendo: (a) colocar uma amostra compreendendo DNA em contato com uma sonda que hibridiza sob condições estringentes de hibridização com um DNA de planta de soja de controle; (b) submeter a amostra e a sonda a condições de hibridização estringentes; e (c) detectar a hibridização da sonda ao DNA do evento de soja MON89788, em que a dita sonda compreende SEQ ID NO: 1 ou SEQ ID NO: 2. A amostra pode compreender uma semente, planta, ou parte de planta de uma progênie que compreende o evento de soja MON89788, ou qualquer parte dos seguintes produtos derivados de uma planta que compreende MON89788: lecitina, ácidos graxos, giicerol, esterol, óleo comestível, flocos de soja desengordurados, farelos de soja incluindo farelos de soja desengordurados e tostados, coalhada de leite de soja, tofú, farinha de soja, concentrado de proteína de soja, proteína de soja isolada, proteína de soja hidroiisada. proteína de soja texturizada e fibra de proteína de soja. Um kit compreendendo uma sonda que compreende uma molécula de DNA que é homóloga ou complementar a SEQ ID NO: 1 ou SEQ ID NO: 2 é um aspecto da invenção. Um kit que compreende uma molécula de DNA que compreende SEQ ID NO: 18, SEQ ID NO: 19, ou SEQ ID NO: 20, ou seus complementos, também é um aspecto da invenção.
De acordo com outro aspecto da invenção, é fornecido um método de produzir uma planta de soja que tolera uma aplicação de glifosato que compreende as etapas de: (a) cruzar sexualmente uma primeira planta de soja parental tolerante ao glifosato que compreende o evento MON98788, e uma segunda planta de soja parental que não tem a tolerância ao glifosato, assim produzindo uma pluralidade de plantas de progênie; (b) selecionar uma planta da progênie que tolera aplicação de glifosato. Métodos de reprodução podem compreender adicionalmente as etapas de cruzar a planta parental que compreende o evento de soja MON89788 a uma segunda planta de soja parental que também é tolerante ao glifosato e selecionar a progênie tolerante ao glifosato por DNA marcador molecular geneticamente ligado ao fenótipo tolerante ao glifosato encontrado em cada parente.
Outro aspecto da invenção é um método de controlar plantas daninhas em um campo de plantas de soja que compreendem MON89788, em que o dito método compreende plantar um campo com semente de soja que compreende o evento MON89788 a dita semente representante depositada como N2 de acesso da ATCC PTA-6708, permitir que a dita semente germine e tratar as ditas plantas com uma dose eficaz de glifosato para controlar o crescimento de planta daninha no dito campo.
Os aspectos anteriores e outros aspectos da invenção se tornarão mais claros a partir da seguinte descrição detalhada e desenhos anexos. Breve Descrição dos Desenhos Figura 1. Organização da inserção do transgene o genoma de uma planta de soja que compreende o evento MON89788.
Figura 2A- 2B. Processamento de produtos de consumo de soja.
Descrição Detalhada A presente invenção refere-se a um novo evento de transformação de soja chamado MON89788 que fornece tolerância ao glifosato, e as partes de planta e sementes e produtos produzidos a partir das plantas, partes de plantas, semente e produtos compreendendo o evento. A invenção fornece moléculas de DNA que são novas no genoma de células de soja compreendendo MON89788 e moléculas de DNA que podem ser usadas em vários métodos de detecção de DNA para identificar DNA de MON89788 em uma amostra. A invenção fornece um método para controlar plantas daninhas em um campo de plantas que contem MON89788 pelo tratamento das plantas daninhas no campo compreendendo plantas que compreendem o evento MON89788 com um herbicida de glifosato.
As seguintes definições e métodos são fornecidos para melhor definir a presente invenção e para guiar os versados na técnica na prática da presente invenção. A menos que observado de outra maneira, os termos devem ser entendidos de acordo com o uso convencional por aqueles versados na técnica relevante. Definições de termos comuns em biologia molecular também podem ser encontrados em Rieger et ai. (1991) e Lewin (1994). A nomenclatura para bases de DNA conforme descrita em 37 CFR § 1.822 é usada.
Como usado aqui, o termo “soja” significa Glycine Max e inclui todas as variedades de planta que podem ser geradas com soja.
Como usado aqui, o termo “compreendendo” significa “incluindo, mas não limitado a". “Glifosato" refere-se a N-fosfonometilglicina e seus sais, Glifosato é o ingrediente ativo do herbicida Roundup® (Monsanto Co.). Tratamentos com o “herbicida glifosato" referem-se a tratamentos com os herbicidas Roundup®, Roundup Ultra®, Roundup Pro® ou qualquer outra formulação de herbicida contendo glifosato. Exemplos de formulações comerciais de glifosato incluem, sem restrição, aqueles comercializados por Monsanto Company como os herbicidas ROUNDUP®, ROUNDUP® ULTRA, ROUNDUP® ULTRAMAX, ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP® BIACTIVE, ROUNDUP® BIOFORCE, RO DECT. POLARIS" SPARK® θ ACCORD® todos os quais contêm glifosato como seu sal de isopropilamônio; ROUNDUP® WEATHERMAX (sal de potássio de glifosato), aqueles comercializados por Monsanto Company como os herbicidas ROUNDUP® DRY e RIVAL®, que contêm glifosato como seu sal de amônio; aqueles comercializados por Monsanto Company, como ROUNDUP® GEOFORCE, que contêm glifosato como seu sal de sódio: e aqueles comercializados por Syngenta Crop Protection como o herbicida TOUCFIDOWN®, que contêm glifosato como seu sal de trimetilsulfônio. Tratamento de um campo compreendendo plantas de soja tolerantes ao glifosato compreendendo o evento MON89788 com qualquer uma dessas formulações de herbicida de glifosato controlará o crescimento de plantas daninhas no campo e não afetará o cresicmento ou produção das plantas de soja compreeendendo MON89788.
Um “evento” transgênico é produzido por transformação de células de planta com DNA heterólogo, por exemplo, um construto de ácido nucleíco que inclui um transgene de interesse, regeneração de uma população de plantas que resulta da inserção do transgene no genoma da planta, e seleção de uma planta particular caracterizada pela inserção em uma localização particular do genoma. O termo “evento” refere-se ao trans-formante original e progênie do transformante que incluem o DNA heterólogo. O termo “evento” também refere-se a progênie produzida por um cruzamento sexual entre o transformante e outra variedade que inclui o DNA do transgene heterólogo e o DNA genômico flanqueador. O termo “evento” também refere-se ao DNA do transformante original compreendendo o DNA inserido e a seqüência genômica flanqueadora imediatamente adjacente ao DNA inserido que seria esperado ser transferida para uma progênie que recebe DNA inserido incluindo o transgene de interesse como o resultado de um cruzamento sexual de uma linhagem parental que inclui o DNA inserido (por exemplo, o transformante original e a progênie que resulta do “selfing”) e uma linhagem parental que não contém o DNA inserido.
Uma planta de soja tolerante ao glifosato pode ser gerada por primeiramente cruzar sexualmente uma primeira planta de soja parental que consiste em uma planta de soja cultivada a partir de uma planta de soja tolerante ao glifosato compreendendo MON89788 ou uma planta de soja que é uma progênie do cruzamento de tal planta que expressa o fenótipo toleram te ao glifosato, e uma segunda planta de soja parental que não tem a tolerância ao glifosato, assim produzindo uma pluralidade de plantas de primeira progênie; e então selecionar uma planta da progênie que é tolerante à aplicação do herbicida glifosato. Essas etapas podem ainda incluir o retrocru-zamento da planta da progênie tolerante ao glifosato à segunda planta de soja parental ou uma terceira planta de soja parental, e então selecionar a progênie por aplicação com glifosato ou pela identificação com marcadores moleculares associados com a característica produzindo assim uma planta de soja que tolera a aplicação do herbicida glifosato. Marcadores moleculares que podem ser usados compreendem as moléculas de DNA de junção identificadas nos sítios 5’ e 3’ da inserção do transgene no evento MON89788.
Também deve ser entendido que duas plantas transgênicas diferentes também podem ser acasaladas para produzir descendência que contém dois transgenes exógenos de segregação independente. O retrocruza-mento com uma planta parental e cruzamento com uma planta não-transgê-nica como descrito anteriormente também é contemplado, como é a propagação vegetativa. Descrições de outros métodos de reprodução que são comumente usados para diferentes características e cultivos podem ser encontradas em uma de várias referências, por exemplo, Fehr, (1987).
Uma “sonda” é um ácido nucleíco isolado ao qual está ligado um marcador detectável convencional ou uma molécula repórter, por exemplo, um isótopo radioativo, um ligante, um agente quimioluminescente, ou uma enzima. Tal sonda é complementar a um filamento de um ácido nucleíco alvo, no caso da presente invenção, a um filamento de DNA genômico de uma planta de soja que compreende o evento MON89788 seja de uma planta de soja ou semente ou de uma amostra ou extrato da planta ou semente que inclui DNA do evento. Sondas de acordo com a presente invenção incluem não apenas ácidos desoxirribonucléicos ou ribonucléicos, mas também poli-amidas e outros materiais de sonda que se ligam especificamente a uma seqüência de DNA-alvo e podem ser usados para detectar a presença de uma seqüência de DNA-alvo. “Iniciadores” são ácidos polinucléicos isolados que são anelados a um filamento de ácido polinuciéico complementar por hibridização de ácido nucleíco para formar um híbrido entre o iniciador e o filamento de ácido poli-nucléico-alvo, então estendidos ao longo do filamento de ácido polinucléico-alvo por uma polimerase, por exemplo, DNA polimerase. Pares de iniciadores da presente invenção referem-se ao seu uso para amplificação de uma molécula de ácido polinucléico-alvo, por exemplo, pela reação em cadeia da polimerase (PCR) ou outros métodos de amplificação de ácido nucleíco convencionais.
Sondas e iniciadores geraimente têm 11 polinucleotídeos ou mais de extensão, preferivelmente 18 polinucleotídeos ou mais, mais preferivelmente 24 polinucleotídeos ou mais. Tais sondas e iniciadores hibridizam especificamente a uma molécula-alvo sob condições de alta estringência de hibridização. Preferivelmente, sondas e iniciadores de acordo com a presente invenção têm identidade de seqüência completa com a molécula-alvo, embora sondas que diferem da seqüência-alvo e que retêm a habilidade de hibridizar a seqüências-alvo sob condições de alta estringência podem ser desenhadas por métodos convencionais. Métodos para preparar e usar sondas e iniciadores são descritos, por exemplo, em Sambrook etal. (1989); Ausubel et ai. (1992); e Innis et al. (1990). Pares de iniciadores de PCR (um conjunto de iniciadores) podem ser derivados de uma seqüência conhecida, por exemplo, pelo uso de programas planejados para esse fim tal como Primer (Versão 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).
Iniciadores e sondas baseadas nas seqüências do DNA genômico flanqueador e do inserto descritas aqui (SEQ ID NOs: 1-4 e 9) podem ser usadas para confirmar e, se necessário, corrigir as seqüências descritas por métodos convencionais, por exemplo, por isolar a molécula de DNA correspondente a partir de uma depósito de semente compreendendo MON89788, e determinar a seqüência de ácido nucleíco de tais moléculas. Moléculas de DNA associadas adicionais podem ser isoladas do genoma de uma célula compreendendo MON89788 que compreende as regiões de inserto do transgene e flanqueadora genômica, e fragmentos dessas moléculas podem ser usados como iniciadores ou sondas.
As sondas e iniciadores de ácido nucleíco da presente invenção hibridizam sob condições estringentes a uma seqüência de DNA-alvo. Qualquer método de hibridização ou amplificação de ácido nucleíco convencional pode ser usado para identificar a presença de DNA do evento MON89788 em uma amostra. Moléculas de ácido nucleíco ou fragmentos dessas são capazes de hibridizar especificamente a outras moléculas de ácido nucleíco sob certas circunstâncias. Como usado aqui, duas moléculas de ácido nucleíco são ditas serem capazes de hibridizar especificamente uma a outra se as duas moléculas forem capazes de formar uma estrutura de ácido nucleíco de duplo filamento, antiparalelo e tiverem extensão suficiente para manter essa estrutura sob condições de alta estringência. Uma molécula de ácido nucleíco é dita ser o “complemento” de outra molécula de ácido nucleíco se ela exibir complementaridade completa. Como usado aqui, moléculas são ditas exibir “complementaridade completa” quando cada nucleotídeo de uma das moléculas for complementar a um nucleotídeo da outra. Duas moléculas são ditas serem “minimamente complementares” se elas puderem hibridizar uma a outra com estabilidade suficiente para permitir que elas permaneçam ane-ladas uma a outra sob pelo menos condições de “baixa estringência" convencionais. Similarmente, as moléculas são ditas serem “complementares” se elas puderem hibridar uma a outra com estabilidade suficiente para permitir que elas permaneçam aneladas uma a outra sob condições de “alta estringência”. Condições de estringência convencionais são descritas por Sambrook et al., 1989, e por Haymes et ai (1985). Desvios da complementaridade completa são portanto permissíveis, contando que tais desvios não impossibilitem completamente a capacidade das moléculas formarem uma estrutura de duplo-filamento. A fim de uma molécula de ácido nucleíco servir como um iniciador ou sonda ela precisa ser suficientemente complementar em seqüência para ser capaz de formar uma estrutura de duplo-filamento estável sob o solvente particular e as concentrações de sal empregadas.
Como usado aqui, uma seqüência substancialmente homóloga é uma seqüência de ácido nucleíco que hibridizará especificamente ao complemento da seqüência de ácido nucleíco a qual ela está sendo comparada sob condições de alta estringência. Condições de estringência apropriadas que promovem hibridização de DNA, por exemplo. 6,0 x cloreto de sódio/ citrato de sódio (SSC) a cerca de 45°C, seguido por uma lavagem de 2,0 x SSC a 50°C, são conhecidas daqueles versados na técnica ou podem ser encontradas em Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Por exemplo, a concentração de sal na etapa de lavagem pode ser selecionada desde uma estringência baixa de cerca de 2,0 x SSC a 50°C até uma estringência alta de 0,2 x SSC a 50°C. Além disso, a temperatura na etapa de lavagem pode ser aumentada de condições de baixa estringência em temperatura ambiente, cerca de 22°C, para condições de alta estringência a cerca de 65°C. Tanto-a temperatura como o sal podem ser variados, ou temperatura ou a concentração de sal pode ser mantida constante enquanto a outra variável é alterada. Em uma modalidade preferida, um ácido nucleíco da presente invenção hibridizará especificamente a uma ou mais das moléculas de ácido nucleíco descritas em SEQ ID NOs:1-4, e 9 e complementos dessas ou fragmentos de qualquer uma dessas sob condições moderadamente estringentes, por exemplo a cerca de 2,0 x SSC e cerca de 65°C. Em uma modalidade particularmente preferida, um ácido nucleíco da presente invenção hibridizará especificamente a uma ou mais moléculas de ácido nucleíco descritas em SEQ ID NOs: 1-4, e 9 e complementos dessas ou fragmentos de qualquer uma dessas sob condições de alta estringência. Em um aspecto da presente invenção, uma molécula de ácido nucleíco marcadora preferida da presente invenção compreende a seqüência de ácido nucleíco descrita em SEQ ID NO: 1 ou SEQ ID NO: 2 ou complementos dessas ou fragmentos de qualquer uma dessas. Em outro aspecto da presente invenção, uma molécula de ácido nucleíco marcadora preferida da presente invenção compartilha entre 80% e 100% ou 90% e 100% de identidade de seqüência com a seqüência de ácido nucleíco descrita em SEQ ID NO: 1 ou SEQ ID NO: 2 ou complementos desses ou fragmentos de qualquer uma dessas. Moléculas de DNA marcadoras moleculares que compreendem SEQ ID NO: 1, ou SEQ ID NO: 2, ou complementos dessas ou fragmentos de qualquer uma dessas podem ser usados como marcadores em métodos de reprodução de plantas para identificar a progê-nie de cruzamentos genéticos similares aos métodos descritos para análise de marcador de DNA de repetição de seqüência simples em Cregan et ai (1997); todos os quais estão aqui incorporados por referência nas suas tota-lidades. A hibridização da sonda à molécula de DNA-alvo pode ser detectada por qualquer número de vários métodos conhecidos daqueles versados na técnica, esses podem incluir, mas não estão limitados a, sinalizadores fluorescentes, sinalizadores radioativos, sinalizadores a base de anticorpos, e sinalizadores quimioluminescentes.
Com relação à amplificação de uma seqüência de ácido nucléi-co-alvo (por exemplo, por PCR) usando um par de iniciadores de amplificação particular, “condições estringentes” são condições que permitem que o par de iniciadores hibridize apenas à seqüência de ácido nucléico-aivo a qual um iniciador que tem a seqüência selvagem correspondente (ou seu complemento) podería se ligar e preferivelmente produzir um produto de amplificação único, o amplicon, em uma reação de amplificação térmica de DNA. O termo “específico para (para uma seqüência-alvo)” indica que uma sonda ou iniciador hibridiza sob condições de hibridização estringentes apenas à seqüência-alvo em uma amostra que compreende a seqüência-alvo.
Como usado aqui, “DNA amplificado” ou “amplicon” refere-se ao produto de amplificação de ácido nucleíco de uma seqüência de ácido nucle-íco alvo que é parte de um molde de ácido nucleíco. Por exemplo, para determinar se a planta de soja que resulta de um cruzamento sexual contém o evento transgênico MON89788 ou se uma planta de soja coletada de um campo compreende MON89788, ou um extrato de soja, tal como um farelo, farinha ou óleo compreende MON89788. DNA extraído de uma amostra de tecido ou extrato de planta de soja pode ser submetido a um método de amplificação de ácido nucleíco usando um par de iniciadores que inclui um ini-ciador derivado da região genômica adjacente ao sítio de inserção do DNA do transgene heterólogo inserido, e um segundo iniciador derivado do DNA do transgene heterólogo inserido para produzir um amplicon que é diagnóstico para a presença do DNA do evento. O amplicon tem um tamanho e tem uma seqüência que também é diagnostica para o evento. O amplicon pode variar em tamanho desde o tamanho combinado dos pares de iniciadores mais um par de base de nucleotídeo, ou mais cerca de cinqüenta pares de base de nucleotídeo, ou mais cerca de duzentos e cinqüenta pares de base de nucleotídeo, ou mais cerca de trezentos e cinqüenta pares de base de nucleotídeo ou mais.
Alternativamente, um par de iniciadores pode ser derivado da seqüência genômica flanqueadora em ambos os lados do DNA inserido para produzir um amplicon que inclui toda a seqüência de nucleotídeo do inserto. “ Uirrmemfcro de unrparde iniciadores derivado da seqüência genômica da planta pode estar localizado a distância da molécula de DNA do transgene inserida, essa distância pode variar desde um par de base de nucleotídeo até cerca de vinte mil pares de base. O uso do termo “amplicon” exclui especificamente dímeros de iniciadores que podem ser formados na reação de amplificação térmica de DNA.
Amplificação de ácido nucleíco pode ser realizada por qualquer um dos vários métodos de amplificação de nucleíco conhecidos na técnica, incluindo a reação em cadeia da polimerase (PCR). Uma variedade de métodos de amplificação são conhecidos na técnica e são descritos, inter alia, nas Patentes U.S. Nos. 4.683.195 e 4.683.202 e em Innis et ai (1990). Métodos de amplificação por PCR foram desenvolvidos para amplificar até 22 kb de DNA genômico e até 42 kb de DNA de bacteriófago (Cheng et ai, 1994). Esses métodos assim como outros métodos conhecidos na técnica de amplificação de DNA podem ser usados na prática da presente invenção. A seqüência do inserto de DNA heterólogo ou seqüência flanqueadora do evento de soja MON89788 pode ser verificada, e corrigida, se necessário, pela amplificação de tais moléculas a partir do genoma do evento usando iniciadores derivados das seqüências fornecidas aqui seguido por métodos de seqüenciamento de DNA padronizados aplicados ao amplicon de PCR ou ao DNA do transgene/genômico clonado isolado. O amplicon produzido por esses métodos pode ser detectado por uma pluralidade de técnicas. Um de tais métodos é Genetic Bit Analysis (Nikiforov, et ai, 1994) onde é desenhado um oligonucleotídeo de DNA que coincide tanto com a seqüência de DNA genômico flanqueadora adjacente como com a seqüência do transgene de DNA inserida. O oligonucleotídeo é imobilizado em poços de uma placa de micropoços. Após PCR da região de interesse (usando um iniciador na seqüência inserida e um na seqüência genômica flanqueadora adjacente), um produto de PCR de filamento simples pode ser hibridizado ao oligonucleotídeo imobilizado e servir como um molde para uma reação de extensão de base individual usando uma DNA polime-rase e ddNTPs marcados específicos para a próxima base esperada. A leitura pode ser à base de fluorescência ou ELISA. Um sinal indica a presença da seqüência genômica do inserto/flanqueadora devido a amplificação, hibri-dização e extensão de base individual bem-sucedidas.
Outro método é a técnica de pirosseqüenciamento como descrito por Winge (2000). Nesse método é desenhado um oligonucleotídeo que coincide com a junção do DNA genômico adjacente e DNA do inserto. O oligonucleotídeo é hibridizado a um produto de PCR de filamento simples a partir da região de interesse (um iniciador na seqüência inserida e um na seqüência genômica flanqueadora) e incubado na presença de uma DNA polimerase, ATP, sulfurilase, luciferase, apirase, adenosina 5’ fosfossulfato e luciferina. DNTPs são adicionadas individualmente e a incorporação resulta em um sinal de luz que é medido. Um sinal de luz indica a presença da seqüência do inserto do transgene/flanqueadora devido a amplificação, hibridi-zação e extensão de múltiplas bases ou de base individual bem-sucedidas.
Polarização de Fluorescência como descrito por Chen et al. (1999) é um método que pode ser usado para detectar o amplicon da presente invenção. Usando esse método é desenhado um oligonucleotídeo que coincide com a junção de DNA inserido e genómico flanqueador. O oligonu-cleotídeo é hibridizado a um produto de PCR de filamento simples a partir da região de interesse (um iniciador no DNA inserido e um na seqüência de DNA genómico flanqueadora) e incubado na presença de uma DNA polime-rase e uma ddNTP marcada com fluorescência. Extensão de base individual resulta na incorporação da ddNTP. A incorporação pode ser medida como uma alteração na polarização usando um fluorímetro. Uma alteração na polarização indica a presença da seqüência do inserto do transgene/ flanqueadora genômica devido a amplificação, hibridização e extensão de base individual bem-sucedida.
Taqman® (PE Applied Biosystems, Foster City, CA) é descrito como um método de detectar a quantificar a presença de uma seqüência de DNA e é completamente entendido nas instruções fornecidas pelo fabricante. Resumidamente, é desenhada uma sonda de oligonucleotídeo FRET que coincide com a junção de DNA do inserto e flanqueadora genômica. A sonda FRET e iniciadores de PCR (um iniciador na seqüência de DNA do inserto e um na seqüência genômica flanqueadora) são ciclados na presença de uma polimerase termoestável e dNTPs. A hibridização da sonda FRET resulta na divagem e liberação da porção fluorescente para longe da porção inibidora na sonda FRET. Um sinal fluorescente indica a presença da seqüência genômica flanqueadora/ inserto do transgene devido a amplificação e hibridização bem-sucedidas. “Molecular Beacons” foram descritos para uso em detecção de seqüência como descrito por Tyangi et ai. (1996). Resumidamente, é desenhada uma sonda de oligonucleotídeo FRET que coincide com a junção do DNA do inserto e genômica flanqueadora. A estrutura única da sonda FRET resulta nela conter uma estrutura secundária que mantém as porções fluorescente e inibidora em íntima proximidade. A sonda FRET e iniciadores de PCR (um iniciador na seqüência do DNA do inserto e um na seqüência genômica flanqueadora) são ciclados na presença de uma polimerase termoestável e dNTPs. Após amplificação por PCR bem-sucedida, a hibridização da sonda FRET à seqüência-alvo resulta na remoção da estrutura secundária da sonda e separação espacial das porções fluorescente e inibidora. Resulta um sinal fluorescente. Um sinal fluorescente indica a presença da seqüência do inserto do transgene/genômica flanqueadora devido a amplificação e hl· bridização bem-sucedidas.
Outros métodos descritos, tais como, microfluídica (Pub Patente U.S. 2006068398, Patente US N- 6.544.734) fornecem métodos e aparelhos para separar e amplificar amostras de DNA. Corantes ópticos usados para detectar e quantificar moléculas de DNA específicas (WO/05017181). Aparelhos de nanotubo (WO/06024023) que compreendem um sensor eletrônico para a detecção de moléculas de DNA ou nanoesferas que ligam-se a moléculas de DNA específicas e podem então ser detectadas são úteis para detectar moléculas de DNA da presente invenção.
Kits de detecção de DNA podem ser desenvolvidos usando as composições descritas aqui e os métodos descritos ou conhecidos na técnica de detecção de DNA. Os kits são úteis para a identificação de DNA de evento de soja em uma amostra e podem ser aplicados a métodos para reproduzir plantas de soja contendo DNA. Os kits podem conter iniciadores ou sondas de DNA que são homólogos ou complementares a SEQ ID NOs: 1-4 e 9 ou iniciadores ou sondas de DNA homólogos ou complementares ao DNA contido nos elementos genéticos do transgene de DNA, essas seqüên-cias de DNA podem ser usadas em reações de amplificação de DNA ou como sondas em um método de hibridização de DNA. A estrutura do DNA dos elementos genéticos do transgene contidos no genoma de soja e ilustrados na figura 1 compreende uma região genômica 5’ do genoma de A3244 de soja que flanqueia o inserto do transgene, o inserto compreendendo uma porção da região da borda direita (RB) de Agrobacterium tumefaciens, o promotor quimérico FMV/Tsfl e elementos ligados relacionados (Patente U.S. 6.660.911; também referido como FMV/EIFta) é conectado operativamente a uma seqüência codificante de peptídeo de trânsito cloroplástico de EPSPS de Arabidopsis (aqui referido como CTP2 ou TS-AtEPSPS CTP2, Patente US 5.633.435) operativamente conectado a uma EPSPS resistente a glifosato (aqui referida como CP4 EPSPS ou aroA:CP4, isolada da cepa CP4 de Agrobacterium tumefaciens e seqüência codificaníe modificada para expressão aumentada em células de planta, Patente U.S. 5.633.435), operativamente conectada à região de terminação 3’ de ribulose 1,5-bisfosfato carboxilase de ervilha (aqui referida como E9 3’ ou T-Ps.RbcS:E9, Coruzzi et ai, (1984), uma porção da região da borda esquerda (LB) de Agrobacterium tumefaciens, e a região genômica 3’ do genoma A3244 de soja que flan-queia o inserto de trangene. Moléculas de DNA úteis como iniciadores em métodos de amplificação de DNA podem ser derivadas das sequências dos elementos genéticos do inserto do transgene contidos no evento de soja MON89788. Essas moléculas de iniciadores podem ser usadas como parte de um conjunto de iniciadores que também inclui uma molécula de iniciador de DNA derivada do genoma de soja que flanqueia do inserto do transgene. O evento de soja MON89788 foi produzido por transformação da linhagem de soja A3244 (Patente US 5.659.114) por um método mediado por Agrobacterium, por exemplo, métodos descritos nas Patentes U.S. 6.384.301 e 7.002.058 (aqui incorporados por referência nas suas totalidades).
Os inventores da presente invenção descobriram que uma linhagem de soja que compreende a região genômica tipo-T de MON89788 (tipo-T é combinação de um transgene e a região de haplótipo associada de um genoma de planta) no seu genoma tem um rendimento melhorado em relação a uma linhagem que compreende a região genômica tipo-T de 40-32 anterior. Isso foi demonstrado em testes de campo replicados que incluem dados de rendimento coletados de múltiplas localidades nos Estados Unidos (pedido de patente U.S. 60/685584).
Os seguintes exemplos estão incluídos para demonstrar exemplos de certas modalidades preferidas da invenção. Deve ser observado por aqueles versados na técnica que certas técnicas descritas nos exemplos que seguem representam abordagens que os inventores descobriram que funcionam bem na prática da invenção, e assim podem ser considerados por constituir exemplos de modos preferidos para sua prática. Entretanto, aqueles versados na técnica, em vista da presente descrição, observam que várias alterações podem ser feitas nas modalidades especificas que são des- critas e ainaa obtem um resultado semelhante ou similar sem desconsiderar o espírito e escopo da invenção.
Exemplos Exemplo 1 Produção de Ampiicon Diagnóstico para DNA Genômico de MON89788 DNA de evento de soja transgênico MON89788 é extraído de tecido compreendendo sementes de soja, tecido vegetativo, ou farelo. O DNA é isolado do tecido usando o kit Qiagen’s DNeasy Plant Miniprep de acordo com as instruções do fabricante (Qiagen Corp. Valencia, CA). É produzido um produto de PCR que compreende uma porção do DNA genômico que flanqueia a terminação 5’ da inserção de T-DNA (DNA de transferência que compreende o transgene) no genoma de uma planta compreendendo MON89788. Esse produto de DNA compreende SEQ ID NO: 3. A PCR pode ser realizada usando um iniciador desenhado para hibri-dizar às seqüências de DNA genômico que flanqueiam a terminação 5’ do inserto do transgene (iniciador de DNA A, SEQ ID NO: 5; veja figura 1) pare-ado com um segundo iniciador (iniciador de DNA B, SEQ ID NO: 6) localizado na região promotora do transgene (Patente US 6.660.911, SEQ ID NO: 28, aqui incorporada por referência e encontrada dentro de SEQ ID NO: 9).
Um produto de PCR é produzido a partir da terminação 3’ do inserto do transgene que compreende uma porção do DNA genômico que flanqueia a terminação 3’ da inserção de T-DNA no genoma de uma planta que compreende MON89788. Esse produto de DNA compreende SEQ ID NO: 4. PCR pode ser realizada usando um iniciador desenhado para hibridi-zar às seqüências de DNA genômico que flanqueiam a terminação 3’ do inserto de cada evento (iniciador de DNA D, SEQ ID NO: 8) e pareado com um segundo iniciador (iniciador de DNA C, SEQ ID NO: 7) localizado na se-qüência de terminação de transcrição T-Ps.RbcS:E9 3' na terminação 3’ do inserto.
O molde de PCR inclui -50 ng de DNA genômico. Como um controle negativo -50 ng de DNA genômico DNA do cultivar de soja não transgênico é utilizado. Cada reação de PCR contém 5 μ! de Tampão 10 X para Mistura de REDAccuTaq® LA DNA Polimerase (Sigma-Aldrich, St Louis, MO), 200 μΜ de cada dNTP (Sigma-Aldrich), 0,4 μΜ de cada iniciador, e 2,5 unidades de JumpStart® REDTaq® DNA Polimerase (Sigma-Aldrich) em um volume total de reação de 50 μΙ. As reações de PCR são realizadas sob as seguintes condições de ciclagem: 1 ciclo a 94°C por 3 minutos (min); 32 ou 35 ciclos a 94°C por 30 segundos (s), 58°C por 30 s. 72°C por 30 s ou 1 min: 1 ciclo a 72°C por 10 min.
Os pares de iniciadores do evento de DNA são usados para produzir um amplicon diagnóstico para DNA genômico de MON89788. Esses pares de iniciadores do evento incluem, mas não estão limitados aos iniciadores A e B (SEQ ID NO: 5 e 6) e pares de iniciadores de evento C e D (SEQ ID NO: 7 e 8), que são usados no método de amplificação de DNA descrito. Além desses pares de iniciadores, qualquer par de iniciadores derivado de SEQ ID NO: 3 ou SEQ ID NO: 4, ou os complementos dessas, que quando usados em uma reação de amplificação de DNA produz um amplicon compreendendo SEQ ID NO: 1 ou SEQ ID NO: 2 diagnóstico para tecido derivado de evento MON89788 de soja, respectivamente, pode ser utilizado. Condições de amplificação de DNA ilustradas na Tabela 1 e Tabela 2 podem ser usadas para produzir um amplicon diagnóstico para MON89788 usando os pares de iniciadores de evento apropriados. Qualquer modificação desses métodos usados para produzir um amplicon diagnóstico para MON89788 está dentro da habilidade da técnica. Um extrato supostamente contendo DNA de uma planta ou semente de soja compreendendo MON89788, ou um produto derivado de uma planta compreendendo MON89788 que quando testado em um método de amplificação DNA produz um amplicon diagnóstico para o evento de soja MON89788 pode ser utilizado como um molde para amplificação para determinar se MON89788 está presente. O amplicon é produzido pelo uso de pelo menos uma seqüência de iniciador derivada de SEQ ID NO: 3 ou SEQ ID NO: 4 que quando usada em um método de PCR produz um amplicon diagnóstico para o evento MON89788. Por exemplo, a produção dos amplicons de MON89788 pode ser realizada usando um termociclador Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700: ou Eppendorf Mastercycler Gradient como mostrado na Tabela 2, ou por métodos e aparelhos conhecidos daqueles versados na técnica.
Tabela 1. Procedimento de PCR e condições de mistura de reação para a identificação da região de junção do inserto de transgene/genômica 5’de MON89788.
Etapa Reagente__________________Quantidade_________Comentários______ Etapa Reagente___________________Quantidade__________Comentários______ Misture gentilmente, e se necessário (não houver tampa aquecida no termo-ciclador), adicione 1-2 gotas de óleo mineral sobre cada reação. Prossiga com a PCR em um termociclador Stratagene Robocycler (Stratagene, La Jolla, CA), MJ Engine (MJR-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA), ou Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Alemanha) usando os seguintes parâmetros de ciclagem (Tabela 2). O termociclador MJ Engine ou Eppendorf Mastercycler Gradient deve ser executado no modo calculado. Execute o termociclador Perkin-Elmer 9700 com a velocidade da rampa ajustada no máximo.
Tabela 2. Condições do termociclador Ciclo N2 Ajustes: Stratagene Robocycler 1 94°C 3 minutos 34 94°C 1 minuto 64°C 1 minuto 72°C 1 minuto e 30 segundos 1 72°C 10 minutos Exemplo 2 Determinação da seaüência da região transaene/genômica e Análise de Southern Seqüenciamento de DNA dos produtos de PCR fornece DNA que pode ser usados para desenhar moléculas de DNA adicionais como ini-ciadores e sondas para a identificação de plantas ou semente de soja compreendendo MON89788. Produtos de PCR dos tamanhos esperados representando as sequências transgene/genômica 5’ e 3’ foram isolados por separação dos produtos de PCR em um gel de agarose 2,0% por eletroforese. Os produtos de PCR que são isolados incluem as regiões de DNA 5’ e 3’ que ocupam a junção do inserto entre a inserção do transgene dentro do genoma de soja. Os produtos de PCR 5’ e 3’ para MON89788 são purificados por eletroforese em gel de agarose seguido pelo isolamento da matriz de agarose usando o kit QIAquick Gel Extraction (n2 catálogo 28704, Qiagen Inc., Valência, CA). Os produtos de PCR purificados são então seqüencia-dos (por exemplo, ABI Prism® 377, PE Biosystems, Foster City, CA) e analisados (por exemplo programa de análise de seqüência DNASTAR, DNAS-TAR Inc., Madison, Wl).
Uma sequência de DNA foi determinada para o segmento de par de base de nucleotídeo que representa a região transgene/genômica do evento MON89788 como ilustrado na FIG 1 e identificado como SEQ ID NO: 9. Os elementos de transgene e genômico que estão contidos em SEQ ID NO: 9 são descritos na Tabela 3. As regiões flanqueadoras 5’ e 3’ estão incluídas em SEQ ID NO: 9 e dadas em SEQ ID NOs: 21 e 22.
As seqüências de junção são moléculas de polinucleotídeo relativamente curtas que são novas seqüências de DNA e são diagnosticas para DNA de MON89788 quando detectadas em um teste de detecção de ácido polinucléico. As seqüências de junção em SEQ ID NO: 1 e SEQ ID NO: 2 representam 10 polinucleotídeos em cada lado de um sitio de inserção do fragmento do transgene e DNA genômico de soja em MON89788. Seqüências de junção de polinucleotídeo mais longas ou mais curtas podem ser selecionadas a partir de SEQ ID NO: 3 ou SEQ ID NO: 4. As moléculas de junção (região de junção 5’ SEQ ID NO: 1, e região de junção 3' SEQ ID NO: 2) são úteis como sondas de DNA ou como moléculas iniciadoras de DNA em métodos para detecção de DNA.
Iniciadores e sondas usados em um método Taqman® (Roche Molecular Systems, Inc., Pleasanton, CA) para detecção de uma molécula de DNA evento-específica foram desenvolvidos para o evento MON89788. As moléculas de iniciadores são referidas como SQ2824 (SEQ ID NO: 10), SQ2826 (SEQ ID NO: 11), SQ1141 (SEQ ID NO: 12), SQ1142 (SEQ ID NO: 13), SQ5543 (SEQ ID NO: 14) e as moléculas de sonda são referidas como PB871-6FAM (SEQ ID NO: 15), PB2191-VIC (SEQ ID NO: 16), e PB57-VIC (SEQ ID NO: 17). Os iniciadores e sondas foram usados no método Taqman® de acordo com as instruções do fabricante para fornecer DNA compreendendo MON89788. Tecidos de soja incluindo produtos processados, por exemplo, farelo, são fontes úteis de DNA para esse método. Iniciadores adicionais usados para produzir um amplicon de farelo de soja incluem SEQ ID NOs: 18-20.
Tabela 3. Anotação do genoma e elemento genético do fragmento de DNA de transgene/genômico (SEQ ID NO: 9) contido no genoma de soja compreendendo MON89788.
Análise de Southern Blot DNA genômico de uma planta compreendendo MON89788 e DNA genômico de soja de controle (—15pg de cada) é digerido com várias enzimas de restrição (140U) em um volume total de 150 μ! incluindo 15μΙ do tampão do fabricante correspondente (NEB, Beverly, MA). Endonucleases de restrição, por exemplo Bgl11, BamH1, Nco1, Hindl 11, e Bell, são usadas na análise de Southern de MON89788. Digestões de endonuclease são realizadas em uma temperatura apropriada por pelo menos 6 horas. Após incubação, o DNA é precipitado com acetato de sódio 3M e 2,5 volumes de eta- nol. Subseqüentemente, ο DNA é lavado com etanol 70%, seco, e ressus-penso em 40μΙ de TBE. Tampão de aplicação (0,2X) é adicionado às amostras e então submetido a eletroforese em géis de agarose (0,8%) por 16-18 horas a 30 volts. Os géis são corados com brometo de etídeo, e então tratados com uma solução de depurinação (HCL 0,125N) por 10 minutos, com uma solução desnaturante (hidróxido de sódio 0,5M, cloreto de sódio 1.5M) por 30 minutos, e finalmente com uma solução neutralizante (Trizma base 0,5M, cloreto de sódio 1,5M) por 30 minutos. O DNA é transferido para membrana Hybond-N (Amersham Pharmacia Biotech, Buckingamshire, Inglaterra) usando um Turboblotter (Schleicher & Schuell, Dassel, Alemanha) por 4-6 horas e então fixado à membrana usando luz UV.
As membranas são pré-hibridizadas com 20 ml de solução DIG Easy Hyb (Roche Molecular Biochemicals, Indianapolis, IN; n- cat. 1603558) por 2-4 horas a 45°C. Sondas de DNA radioativo (32P dCTP) homólogas ou complementares a SEQ ID NO: 1, ou SEQ ID NO: 2, ou SEQ ID NO: 3, ou SEQ ID NO: 4, ou uma porção dessas são feitas usando kit Radprime DNA Labeling (Invitrogen, Carlsbad, CA; n2 cat. 18428-011). Nucleotídeos não incorporados são removidos usando colunas SEPHADEX G-50 (Invitrogen). A solução de pré-hibridização é substituída com 10 ml de solução DIG Easy Hyb pré-aquecida contendo a sonda desnaturada para uma concentração final de 1 milhão por ml. Os blots são hibridizados a 45°C por 16-18 horas.
Os blots são lavados com uma solução de baixa estringência (SSC 5X, SDS 0,1X) a 45°C e então lavados repetidamente com uma solução de estringência maior (SSC 0,1X, SDS 0,1%) a 65°C. Os blots são expostos a uma tela de fósforo (Amersham Biosciences, Piscataway, NJ) por >2 horas e a exposição lida usando uma máquina Data Storm 860 (Amersham Biosciences). Esses métodos e condições exemplificados podem ser modificados por aqueles versados na técnica de detectar DA em uma amostra.
Exemplo 3 Controle de Plantas Daninhas Controlar o crescimento de plantas daninhas em um campo de sojas compreendendo MON89788, Um campo é plantado com sementes de soja compreendendo MON89788, as sementes são deixadas germinar em plantas e o campo de plantas é tratado com uma formulação de herbicida contendo glifosato. Uma dose eficaz de uma formulação de glifosato em taxas de tratamento de cerca de 0.11 kg (0,25 Ib) ae/A (libras de equivalente de ácido de glifosato /acre) até 1,36 kg ar/A (3 Ib ou mais) é aplicada ao campo. As taxas geralmente aplicadas variam de cerca de 0,34 kg (0,75 Ib) ae/A até 0,68 kg (1,5 Ib) ae/A em uma freqüência de um ou mais tratamentos durante a época de crescimento conforme necessário para controlar o crescimento de plantas daninhas em um campo. As sementes das plantas compreendendo MON89788 são colhidas das plantas tratadas.
Um depósito da Monsanto Technology LLC, semente de soja de representativa do evento MON89788 descrito aqui acima e referido nas reivindicações foi feito sob o Tratado de Budapeste com a American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110. O número de acesso da ATCC para o deposito compreendendo o evento MON89788 (também conhecido como MON19788 ou GM_A19788) é PTA-6708, depositado em 11 de maio de 2005. O depósito será mantido no depositário por um período de 30 anos, ou 5 anos após a última solicitação, ou pela vida eficaz da patente, seja qual for maior, e será substituído conforme necessário durante esse período.
Tendo ilustrado e descrito os princípios da presente invenção, deve ser claro para pessoas versadas na técnica que a invenção pode ser modificada em combinação e detalhe sem desconsiderar tais princípios. Reivindicam-se todas as modificações que estão dentro do espírito e escopo das reivindicações anexas.
Todas as publicações e documentos de patente publicados citados nesse relatório descritivo estão incorporados aqui por referência na mesma extensão que se cada publicação ou pedido de patente individual fosse especificamente e individualmente indicado por ser incorporado por referência.
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REIVINDICAÇÕES
Claims (17)
1. Molécula de ácido nucléico, caracterizada pelo fato de que consiste na sequência de SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 ou SEQ ID NO:4 e uma sequência que codifica uma 5-enol-piruvilchiquimato-3-fosfato sintase (EPSPS) tolerante a glifosato.
2. Molécula de ácido nucléico de acordo com a reivindicação 1, caracterizada pelo fato de que é inserida no DNA genômico de uma planta, semente ou célula de soja.
3. Produto primário de soja, caracterizado pelo fato de que é produzido a partir de uma semente de uma planta compreendendo o evento MON89788, em que o evento MON89788 compreende SEQ ID NO:3 e SEQ ID NO:4, e uma amostra representativa de sementes compreendendo o evento MON89788 foi depositada sob o número de acesso da ATCC PTA-6708.
4. Produto primário de soja como de acordo com a reivindicação 3, caracterizado pelo fato de que é ainda definido como alimento, farinha, flocos, ou óleo.
5. Alimento, farinha, flocos, ou óleo, como definido na reivindicação 5, caracterizado pelo fato de que compreende um ácido nucléico que produz um amplicon diagnóstico para o evento MON89788 quando testado em um método de amplificação de DNA, o dito amplicon compreendendo SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 ou SEQ ID NO:4, em que o evento MON89788 compreende SEQ ID NO:3 e SEQ ID NO:4, e uma amostra representativa de sementes compreendendo o evento MON89788 foi depositada sob o número de acesso da ATCC PTA-6708.
6. Par de moléculas iniciadoras de polinucleotídeo de DNA, caracterizado pelo fato de que compreende uma primeira molécula iniciadora de polinucleotídeo de DNA compreendendo pelo menos 11 nucleotídeos contíguos da região de transgene de SEQ ID NO:3, ou seu complemento completo, e uma segunda molécula iniciadora de polinucleotídeo de DNA compreendendo pelo menos 11 nucleotídeos contíguos da região flanquea-dora genômica de soja de SEQ ID NO:3, ou seu complemento completo, em que o par de moléculas iniciadoras de polinucleotídeo de DNA produz um amplicon diagnóstico para o evento MON89788, em que o evento MON89788 compreende SEQ ID NO:3 e SEQ ID NO:4, e uma amostra representativa de sementes compreendendo o evento MON89788 foi depositada sob o número de acesso da ATCC PTA-6708.
7. Par de moléculas iniciadoras de polinucleotídeo de DNA, caracterizado pelo fato de que compreende uma primeira molécula iniciadora de polinucleotídeo de DNA compreendendo pelo menos 11 nucleotídeos contíguos da região de transgene de SEQ ID NO:4, ou seu complemento completo, e uma segunda molécula iniciadora de polinucleotídeo de DNA compreendendo pelo menos 11 nucleotídeos contíguos da região flanquea-dora genômica de soja de SEQ ID NO:4, ou seu complemento completo, em que o par de moléculas iniciadoras de polinucleotídeo de DNA produz um amplicon diagnóstico para o evento MON89788, em que o evento MON89788 compreende SEQ ID NO:3 e SEQ ID NO:4, e uma amostra representativa de sementes compreendendo o evento MON89788 foi depositada sob o número de acesso da ATCC PTA-6708.
8. Kit de detecção de DNA específico para o evento MON89788, caracterizado pelo fato de que compreende: (a) um par de moléculas iniciadoras de polinucleotídeo de DNA compreendendo uma primeira molécula iniciadora de polinucleotídeo de DNA compreendendo pelo menos 11 ou mais nucleotídeos contíguos da região de transgene de SEQ ID NO:3 ou SEQ ID NO:4, ou seu complemento completo, e uma segunda molécula iniciadora de polinucleotídeo de DNA compreendendo pelo menos 11 ou mais nucleotídeos contíguos da região flanqueadora genômica de soja de SEQ ID NO:3 ou 4, ou seu complemento completo, em que o par de moléculas iniciadoras de polinucleotídeo de DNA produz um amplicon diagnóstico para o evento MON89788; ou (b) uma sonda de DNA compreendendo uma molécula de ácido nucleico compreendendo SEQ ID NO:1 ou SEQ ID NO:2, em que o evento MON89788 compreende SEQ ID NO:3 e SEQ ID NO:4, e uma amostra representativa de sementes compreendendo o refe- rido evento foi depositada sob o número de acesso da ATCC PTA-6708.
9. Método de produzir uma planta de soja tolerante ao herbicida glifosato, caracterizado pelo fato de que compreende introduzir no genoma da dita planta o evento MON89788, em que o evento MON89788 compreende SEQ ID NO:3 e SEQ ID NO:4, e uma amostra representativa de sementes compreendendo o evento MON89788 foi depositada sob o número de acesso da ATCC PTA-6708.
10. Método de acordo com a reivindicação 9, caracterizado pelo fato de que compreende as etapas de: (a) cruzar uma primeira planta de soja que compreende o evento MON89788 com uma segunda planta de soja que não tem o evento MON89788 para produzir plantas de progênie; e (b) selecionar pelo menos uma planta de primeira progênie que compreende o dito evento MON89788 e é tolerante ao glifosato, em que o evento MON89788 compreende SEQ ID NO:3 e SEQ ID NO:4, e uma amostra representativa de sementes compreendendo o evento MON89788 foi depositada sob o número de acesso da ATCC PTA-6708.
11. Método de acordo com a reivindicação 10, caracterizado pelo fato de que compreende ainda autocruzar a dita planta de primeira progênie para produzir plantas de progênie de segunda geração e selecionar pelo menos uma primeira planta homozigótica para o dito evento MON89788, em que o evento MON89788 compreende SEQ ID NO:3 e SEQ ID NO:4, e uma amostra representativa de sementes compreendendo o evento MON89788 foi depositada sob o número de acesso da ATCC PTA-6708.
12. Método de detectar a presença de DNA que corresponde ao evento de soja MON89788 em uma amostra, caracterizado pelo fato de que compreende as etapas de: (a) colocar uma amostra que compreende DNA de soja em contato com o par de moléculas iniciadoras de polinucleotídeo de DNA como definido na reivindicação 6 ou 7; e (b) realizar uma reação de amplificação de ácido nucléico, assim produzindo o amplicon de diagnóstico; e (c) detectar o amplicon de diagnóstico em que o evento MON89788 compreende SEQ ID NO:3 e SEQ ID NO:4, e uma amostra representativa de sementes compreendendo o evento MON89788 foi depositada sob o número de acesso da ATCC PTA-6708.
13. Método de detectar a presença de um ácido nucléico que corresponde ao evento MON89788 em uma amostra, caracterizado pelo fato de que compreende as etapas de: (a) obter uma amostra de DNA de soja; e (b) testar a amostra para a presença de uma sequência de ácido nucléico de pelo menos um de SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 ou complementos completos das mesmas; em que o evento MON89788 compreende SEQ ID NO:3 e SEQ ID NO:4, e uma amostra representativa de sementes compreendendo o evento MON89788 foi depositada sob o número de acesso da ATCC PTA-6708.
14. Método de produzir um produto primário de soja, caracterizado pelo fato de que compreende as etapas de: (a) obter uma planta de soja ou parte da mesma compreendendo o evento MON89788, em que o evento MON89788 compreende SEQ ID NO:3 e SEQ ID NO:4, e uma amostra representativa de sementes compreendendo o evento MON89788 foi depositada sob o número de acesso da ATCC PTA-6708; e (b) produzir um produto primário de soja a partir da planta de soja ou parte dessa.
15. Método de acordo com a reivindicação 14, caracterizado pelo fato de que o produto primário de soja é definido como alimento, farinha, flocos, isolado de proteína, ou óleo.
16. Método para controlar o crescimento de ervas daninhas em um campo que compreende plantas de soja que compreendem o evento MON89788, caracterizado pelo fato de que compreende tratar o campo com uma quantidade de glifosato eficaz para controlar o crescimento de ervas daninhas, em que as plantas de soja exibem tolerância ao glifosato, em que o evento MON89788 compreende SEQ ID NO:3 e SEQ ID NO:4, e uma amostra representativa de sementes compreendendo o evento MON89788 foi depositada sob o número de acesso da ATCC PTA-6708.
17. Método de acordo com a reivindicação 16, caracterizado pelo fato de que o tratamento do campo é realizado do estágio de crescimento V1 ao R4.
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US8309810B2 (en) | 2010-07-08 | 2012-11-13 | Monsanto Technology Llc | Soybean variety A1019993 |
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US8404938B2 (en) | 2010-07-15 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1022842 |
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US8507762B2 (en) | 2010-07-15 | 2013-08-13 | Monsanto Technology Llc | Soybean variety A1024315 |
US8293997B2 (en) | 2010-07-15 | 2012-10-23 | Monsanto Technology Llc | Soybean variety A1024318 |
US8299333B2 (en) | 2010-07-15 | 2012-10-30 | Monsanto Technology Llc | Soybean variety A1024289 |
US8395025B2 (en) | 2010-07-15 | 2013-03-12 | Monsanto Technology Llc | Soybean variety A1024249 |
US8293998B2 (en) | 2010-07-15 | 2012-10-23 | Monsanto Technology Llc | Soybean variety A1024338 |
US8420897B2 (en) | 2010-07-15 | 2013-04-16 | Monsanto Technology Llc | Soybean variety A1024301 |
US8319038B2 (en) | 2010-07-15 | 2012-11-27 | Monsanto Technology Llc | Soybean variety A1024319 |
US8399743B2 (en) | 2010-07-15 | 2013-03-19 | Monsanto Technology Llc | Soybean variety A1024305 |
US8304623B2 (en) | 2010-07-15 | 2012-11-06 | Monsanto Technology Llc | Soybean variety A1024238 |
US8618362B2 (en) | 2010-07-15 | 2013-12-31 | Monsanto Technology Llc | Soybean variety A1024337 |
US8395026B2 (en) | 2010-07-15 | 2013-03-12 | Monsanto Technology Llc | Soybean variety A1024300 |
US8314302B2 (en) | 2010-07-16 | 2012-11-20 | Monsanto Technology Llc | Soybean variety A1024184 |
US8314303B2 (en) | 2010-07-16 | 2012-11-20 | Monsanto Technology Llc | Soybean variety A1024197 |
US8367897B2 (en) | 2010-07-16 | 2013-02-05 | Monsanto Technology Llc | Soybean variety A1024230 |
US8324467B2 (en) | 2010-07-16 | 2012-12-04 | Monsanto Technology Llc | Soybean variety A1024216 |
US8383895B2 (en) | 2010-07-16 | 2013-02-26 | Monsanto Technology Llc | Soybean variety A1024198 |
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US8324466B2 (en) | 2010-07-16 | 2012-12-04 | Monsanto Technology Llc | Soybean variety A1024181 |
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US8309813B2 (en) | 2010-07-16 | 2012-11-13 | Monsanto Technology Llc | Soybean variety A1024212 |
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US8455723B2 (en) | 2010-07-19 | 2013-06-04 | Monsanto Technology Llc | Soybean variety A1023586 |
US8404942B2 (en) | 2010-07-26 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1023673 |
US8404941B2 (en) | 2010-07-26 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1023667 |
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US8785737B2 (en) | 2011-07-29 | 2014-07-22 | Monsanto Technology Llc | Soybean variety A1026091 |
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2012
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2013
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2014
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2016
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2017
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2018
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2019
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2020
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2022
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