ES2546255T3 - Evento de Maíz MIR162 - Google Patents

Evento de Maíz MIR162 Download PDF

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ES2546255T3
ES2546255T3 ES12158734.9T ES12158734T ES2546255T3 ES 2546255 T3 ES2546255 T3 ES 2546255T3 ES 12158734 T ES12158734 T ES 12158734T ES 2546255 T3 ES2546255 T3 ES 2546255T3
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vip3aa20
mir162
vip3aa19
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Nykoll Long
Derrick Pulliam
Jeff Bottoms
Moez Meghji
Hope Hart
Qiudeng Que
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Syngenta Participations AG
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

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Abstract

Una semilla transgénica del evento de maíz MIR162 tratada con un insecticida, donde dicha semilla de maíz transgénica comprende la molécula de ácido nucleico constituida por la SEC ID NO: 49 y su complemento.

Description

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transparente (Marsh Bio Products). La PCR se Ilevó a cabo en el instrumento ABI Prism 7700 con los siguientes parámetros de amplificación: 2 min a 50oC y 10 min a 95oC, seguido por 35 ciclos de 15 s a 95oC y 1 min a 60oC.
Los resultados del análisis TAQMAN demostraron que el evento MIR162 presentó una copia del gen vip3Aa20 y una copia del gen pmi.
Los cebadores y las sondas que se usaron en las reacciones de PCR TAQMAN se muestran en la Tabla 1.
Tabla 1. Cebadores usados en el ensayo TAQMAN. Nombre del cebador Secuencia del cebador Secuencia No:
Vip3Aa-hacia adelante
5'CACCTTCAGCAACCCGAACTA3' SEQ ID NO: 4
Vip3Aa –inversa
5'GCTTAGCCTCCACGATCATCTT3' SEQ ID NO: 5
Vip3Aa –sonda
5'GTCCTCGTCGCTGCCCTTCACCT3' SEQ ID NO: 6
(etiqueta 5' = FAM, etiqueta 3' = TAMRA)
PMI-hacia adelante 5'CCGGGTGAATCAGCGTTT3' SEQ ID NO: 7 PMI-inversa 5'GCCGTGGCCTTTGACAGT3' SEQ ID NO: 8 PMI-sonda 5'TGCCGCCAACGAATCACCGG3' SEQ ID NO: 9
(etiqueta 5' = FAM, etiqueta 3' = TAMRA)
ZmADH-267 hacia adelante 5'GAACGTGTGTTGGGTTTGCAT3' SEQ ID NO: 10 ZmADH-337 inversa 5'TCCAGCAATCCTTGCACCTT3' SEQ ID NO: 11 ZmADH-316 sonda 5'TGCAGCCTAACCATGCGCAGGGTA3' SEQ ID NO: 12
(etiqueta 5' = TET, etiqueta 3' = TAMRA)
Ejemplo 3. Detección de MIR162 por Southern Blot
El ADN genómico que se utiliza para el análisis southern se aisló del tejido de hoja extraído de 10 plantas que representaba la generación BC4 del MIR162 con el uso esencialmente del método de Thomas et al. (Theor. Appl. Genet. 86:173-180, 1993). Todas las plantas usadas para el aislamiento de ADN se analizaron individualmente con PCR TAQMAN (como se describe en el Ejemplo 2) para confirmar la presencia de una sola copia del gen vip3Aa20 y del gen pmi. Para los controles segregantes negativos, se aisló ADN de un tejido de hoja extraído de segregantes negativos de la generación BC4. Estas plantas segregantes negativas se analizaron individualmente con el uso de PCR TAQMAN para confirmar la ausencia de los genes vip3Aa20 y pmi, pero fueron, tal como se esperaba, positivas para el gen adhl de maíz endógeno.
El análisis Southern se realizó con el uso de técnicas de biología molecular convencionales. (Véase Chomczynski, P. 1992. Analytical Biochemistry 201:34-139). El ADN genómico DNA (7,5 μg) se digirió con las enzimas de restricción que digieren dentro del inserto del evento MIR162, pero no dentro de la secuencia codificadora que corresponde a la sonda específica utilizada en el experimento. Esta estrategia permitió la determinación de la cantidad de copias de cada gen, que corresponde a la sonda específica utilizada para cada análisis Southern, que se incorporó en el evento MIR162.
Se realizó otra serie de digestiones de restricción en las que el inserto se digirió con enzimas de restricción que liberarían un fragmento de tamaño conocido del inserto. Esta estrategia proveyó una prueba adicional para la presencia de una sola copia de cada secuencia codificadora presente en MIR162 y permitió la detección de copias parciales del inserto que pueden estar unidas estrechamente al inserto de MIR162. Luego de una electroforesis de gel de agarosa y la transferencia alcalina a una membrana de GT Zeta-Probe® (Bio-Rad, Cat. No. 162-0195), se realizaron hibridaciones con sondas de elementos generados por PCR de longitud completa. Las sondas se etiquetaron con cebación aleatoria via 32P con el sistema MegaPrimeTM (Amersham Biosciences, Cat. No. RPN1607). La hibridación se realizó a 65°C, seguida de múltiples lavados en 2X SSC, 0,1% de SDS y luego 0,1X de SSC y 0,1% de SDS. Las membranas se sometieron luego a autorradiografía.
Se incluyeron en cada análisis Southern tres muestras de control: (1) ADN del segregante negativo (no transformado) usado para identificar cualesquiera secuencias de Zea mays endógenas que pudieran hibridarse por cruzamiento con la sonda de elemento específico; (2) se introdujo ADN de un segregante negativo en el que se introduce una cantidad de pNOV1300 digerido que es igual a un número de copias basado en el tamaño del plásmido para demostrar la sensibilidad del experimento para detectar una sola copia del gen dentro del genoma de Zea mays; y (3) el plásmido pNOV1300 digerido igual a un número de copias basado en el tamaño del plásmido, para actuar como un control positivo para la hibridación, así como también para demostrar la sensibilidad del experimento.
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SEC ID NO: 33
b00203e TCTACGACGTGAGCATCAAG SEC ID NO: 34
b00203f GTAGAAGTGCACGATCGGG b00203g CGGTGCTGGTCCAGTTG SEC ID NO: 35
Otras dos reacciones de PCR se superpusieron con la secuencia codificadora completa de vip3Aa20. El extremo 5' de vip3Aa20 se cubrió con una amplificación de PCR con los cebadores 162INSERT-F2: 5 5'ACACCAATGATGCAAATAGGC3' (SEC ID NO: 36) y VIP_R4 5'GAAGGTGTTCAGGTAGAACTCGAAG3' (SEC ID NO: 37) y con la enzima de expansión de alta fidelidad. La segunda reacción cubrió el extremo 3’ de vip3Aa20; el producto se amplificó con los cebadores VIP-F3: 5'GGTGCTGTTCGAGAAGAGGT3' (SEC ID NO: 42) y PMI_REV1: 5'CGATTTATCACTCTCAATCACAT3' (SEC ID NO: 43) y la enzima de expansión de alta fidelidad. Los amplicones generados por estas reacciones comprendieron una secuencia de nucleótidos de 2946 bp (SEC ID NO: 38) y una
10 secuencia de nucleótidos de 2577 bp (SEC ID NO: 44), respectivamente.
Los datos de la secuencia de consenso revelaron dos cambios de nucleótidos en la secuencia codificadora de vip3Aa en MIR162 (denominada vip3Aa20) en comparación con la secuencia condificadora de vip3Aa en pNOV1300 (denominada vip3Aa19), que se utilizó para transformar el MIR162. El primer cambio de nucleótidos, una mutación 15 de G a T, se produjo en la posición 387 de la secuencia codificadora vip3Aa19 (SEC ID NO: 3). Esta mutación resultó en que la metionina en la posición 129 de la Vip3Aa19 cambió a isoleucina en la Vip3Aa20 (M129I). El segundo cambio de nucleótidos se produjo en la posición 1683 de la secuencia codificadora, una mutación de G a C, pero no resultó en un cambio de aminoácidos. Por lo tanto, la secuencia codificadora vip3Aa20 y la proteína Vip3Aa20 son únicas para el evento MIR162 y se pueden usar para identificar cualquier planta que comprenda al
20 genotipo transgénico MIR162. El MIR162 de la secuencia codificadora de pmi fue idéntico a aquel del plásmido de transformación pNOV1300. Una alineación de las proteínas insecticidas de Vip3Aa20 y Vip3Aa19 se muestra en la Tabla 3.
Tabla 3. Comparación de las secuencias de aminoácidos Vip3Aa20 y Vip3Aa19 25 Nombre Alineación de Secuencias
Vip3Aa20 (1) MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDE Vip3Aa19 (1) MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDE
Vip3Aa20 (51) ILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL 30 Vip3Aa19 (51) ILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL
Vip3Aa20 (101) NDVNNKLDAINTMLRVYLPKITSMLSDVimagen17KQNYALSLQIEYLSKQLQEIS Vip3Aa19 (101) NDVNNKLDAINTMLRVYLPKITSMLSDVMKQNYALSLQIEYLSKQLQEIS
35 Vip3Aa20 (151) DKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDG Vip3Aa19 (151) DKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDG
Vip3Aa20 (201) SPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALK Vip3Aa19 (201) SPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALK
40 Vip3Aa20 (251) TASELITKENVKTSGSEVGNVYNFLIVLTALQAQAFLTLTTCRKLLGLAD Vip3Aa19 (251) TASELITKENVKTSGSEVGNVYNFLIVLTALQAQAFLTLTTCRKLLGLAD
Vip3Aa20 (301) IDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVE 45 Vip3Aa19 (301) IDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVE
Vip3Aa20 (351) AKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLL Vip3Aa19 (351) AKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLL
50 Vip3Aa20 (401) CPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEI Vip3Aa19 (401) CPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEI
Vip3Aa20 (451) DLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENS Vip3Aa19 (451) DLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENS
55 Vip3Aa20 (501) RLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLE Vip3Aa19 (501) RLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLE
Vip3Aa20 (551) PWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYT 60 Vip3Aa19 (551) PWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYT
Vip3Aa20 (601) VKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILK
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el siguiente programa: un ciclo a 94°C durante cinco minutos, 35 ciclos de 94°C durante 40 segundos, 60°C durante 40 segundos, y 72°C durante 90 segundos, y un ciclo a 72°C durante siete minutos.
Diez μl de los productos de la PCR se analizaron sobre un 1% de gel de agarosa que contenía bromuro de etidio. La banda adecuada se extrajo del gel de agarosa y se purificó con un conjunto de extracción de gel Qiagen Qiaquick Gel Extraction Kit de acuerdo con las instrucciones del fabricante. El ADN extraído se clonó en un vector de clonación TOPO-XL de Invitrogen de acuerdo con las instrucciones del fabricante. Este clon se transformó en E. coli, y el ADN del plásmido se extrajo de las células al otro día cultivado con un conjunto Qiagen Miniprep de acuerdo con las instrucciones del fabricante. Este plásmido se utilizó para el secuenciamiento del análisis final.
Se diseñó un nuevo cebador dentro de la secuencia nueva conocida previamente para utilizarse con un cebador en el inserto de ADN heterólogo para amplificar el 1 kb completo de la secuencia flanqueante fuera del ADN genómico. El cebador de la secuencia flanqueante 162DWConf3: 5'CCTGTGTTGTTGGAACAGACTTCTGTC3' (SEC ID NO: 53) y el cebador del ADN del inserto FE0900: 5'GGCTCCTTCAACGTTGCGGTTCTGTC3' (SEC ID NO: 54) se utilizaron para amplificar una molécula de ácido nucleico que comprende la secuencia flanqueante 5' para la confirmación. La secuencia del amplicón resultante se encuentra establecida en la SEC ID NO: 55. Este amplicón 5' comprende la secuencia de unión de 5' establecida en la SEC ID NO: 45. Diez μl del producto de la PCR (amplicón) se analizaron sobre un 1% de gel de agarosa que contenía bromuro de etidio. La banda adecuada se extrajo del gel de agarosa y se purificó con un conjunto de extracción de gel Qiagen Qiaquick Gel Extraction de acuerdo con las instrucciones del fabricante. El ADN extraído se clonó en un vector de clonación TOPO-XL de Invitrogen de acuerdo con las instrucciones del fabricante. Este clon se transformó en ADN del plásmido de E. coli. El ADN del plásmido se extrajo de las células al otro día en un medio con un conjunto Qiagen Miniprep de acuerdo con las instrucciones del fabricante. Se secuenciaron completamente tres plásmidos con los cebadores que se muestran en la Tabla 4. Las secuencias de los plásmidos se alinearon para generar la escuencia flanqueante 5’ confirmada. Con el uso de este método, se determinó aproximadamente 1 kb de la secuencia flanqueante 5' (SEC ID NO: 46).
Tabla 4. Secuencias de Cebadores
Nombre del cebador Secuencia (5'3') Secuencia No. b00201h TTCACGGGAGACTTTATCTG SEC ID NO: 60 b00605a CCGATTCATTAATGCAG SEC ID NO: 61 b00701b ACGTAAAACGGCTTGTC SEC ID NO: 62 b00702b GTTTAAACTGAAGGCGG SEC ID NO: 63 b00704h AATAATATCACTCTGTACATCC SEC ID NO: 64
SEC ID NO: 65
b01106f GTTGTAAAACGACGG SEC ID NO: 66
b01709f TAGGCACCCCAGGCTTTA SEC ID NO: 67
b03504a AATTGAATTTAGCGGCCG SEC ID NO: 68
b05102f GGTCCCTACAACATAAATAG
b05102g TTCGTCCCTACTATCAACGC SEC ID NO: 69 SEC ID NO: 70
b05102h CTTTAGGCATCAGCGGGT SEC ID NO: 71
b05103a AGCATCTGCGTAAGCACA SEC ID NO: 72
b05103b CTGATGACACCAATGATGC SEC ID NO: 73
b05103c GATCAGATTGTCGTTTCCC SEC ID NO: 74
b05103d GCATCATTGGTGTCATCAG SEC ID NO: 75
b05103e TGTGCTTACGCAGATGCT SEC ID NO: 76
b05103f ACCCGCTGATGCCTAAAG
b05103g GCGTTGATAGTAGGGACGAA SEC ID NO: 77 SEC ID NO: 78
b05103h CTATTTATGTTGTAGGGACC SEC ID NO: 79
b05210a CTAGACTGGAAAGCGGAG SEC ID NO: 80
b05210b CCACTTTCATCCCTAGTTG
La secuencia flanqueante 3' del evento MIR162 se generó con el uso del conjunto Clonetech GenomeWalkerTM Universal (Clonetech Laboratories, Inc.) de acuerdo con las instrucciones del fabricante.
En primer lugar, se construyeron conjuntos de fragmentos de ADN genómico ligados a adaptadores, no clonados, conocidos como “colecciones” de GenomeWalker. Cada colección se construyó mediante la digestión del ADN genómico de MIR162 con una enzima de restricción (DraI, EcoRV, PvuII, StuI, y XmnI) de la siguiente manera: Por ejemplo, 25 μl de ADN genómico de MIR162 (0,1 μg/μl), 8 μl de la enzima de restricción (10 unidades/μl), 10 μl del tampón de la enzima de restricción (10X), y 57 μl de H2O destilada se mezclaron en un tubo y se incubaron a 37°C durante la noche.
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