RU2019128647A - Оптимальные локусы кукурузы - Google Patents

Оптимальные локусы кукурузы Download PDF

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RU2019128647A
RU2019128647A RU2019128647A RU2019128647A RU2019128647A RU 2019128647 A RU2019128647 A RU 2019128647A RU 2019128647 A RU2019128647 A RU 2019128647A RU 2019128647 A RU2019128647 A RU 2019128647A RU 2019128647 A RU2019128647 A RU 2019128647A
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nucleic acid
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Лакшми САСТРИ-ДЕНТ
Цзэхуэй ЦАО
Шридхаран СРИРАМ
Стивен Р. Уэбб
Дебра Л. КАМПЕР
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ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи
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Claims (33)

1. Способ получения трансгенной клетки растения кукурузы, содержащей представляющую интерес ДНК, причем способ включает:
a. выбор негенного геномного локуса–мишени кукурузы, обладающего по меньшей мере 99% идентичностью последовательности с молекулой нуклеиновой кислоты, выбранной из группы, состоящей из SEQ ID NO: 1, SEQ ID NO: 100, SEQ ID NO: 203, SEQ ID NO: 295, SEQ ID NO: 384, SEQ ID NO: 424, SEQ ID NO: 451, SEQ ID NO: 547, SEQ ID NO: 671, SEQ ID NO: 687, SEQ ID NO: 781, SEQ ID NO: 843, SEQ ID NO: 875, SEQ ID NO: 967, SEQ ID NO: 1107, SEQ ID NO: 1147, SEQ ID NO: 1190, SEQ ID NO: 1252, SEQ ID NO: 1300 b SEQ ID NO: 1371;
b. выбор сайт–специфической нуклеазы, которая специфически связывает и расщепляет указанный негенный геномный локус–мишень кукурузы, где указанная сайт–специфическая нуклеаза выбрана из группы, состоящей из нуклеазы с цинковыми пальцами, нуклеазы CRISPR, нуклеазы TALEN;
c. введение указанной сайт–специфической нуклеазы в клетку растения кукурузы;
d. введение представляющей интерес ДНК в клетку растения, где указанная представляющая интерес ДНК содержит трансген;
e. встраивание представляющей интерес ДНК в указанные негенные геномные локусы–мишени кукурузы; и
f. выбор трансгенных клеток растения, содержащих представляющую интерес ДНК, вставленную в указанный негенный геномный локус–мишень кукурузы.
2. Способ получения трансгенной клетки растения по п.1, где указанная представляющая интерес ДНК содержит ген устойчивости к насекомым или ген толерантности к гербицидам.
3. Способ получения трансгенной клетки растения по п.1, где указанная представляющая интерес ДНК кодирует пептид.
4. Способ получения трансгенной клетки растения по п.1, где указанная представляющая интерес ДНК содержит ген устойчивости к насекомым, ген толерантности к гербицидам, ген эффективности утилизации азота, ген эффективности утилизации воды, ген пищевой ценности, ген ДНК–связывающего продукта или ген селективного маркера.
5. Способ получения трансгенной клетки растения по п.1, где указанная представляющая интерес ДНК содержит две или более кассет для экспрессии гена.
6. Способ получения трансгенной клетки растения по п.1, где указанная сайт–специфическая нуклеаза является нуклеазой с цинковыми пальцами.
7. Способ получения трансгенной клетки растения по п.1, где указанная представляющая интерес ДНК встраивается в указанный негенный локус способом встраивания по типу направляемой гомологией репарации.
8. Способ получения трансгенной клетки растения по п.1, где указанная представляющая интерес ДНК встраивается в указанный негенный локус способом встраивания по типу негомологичного связывания концов.
9. Способ получения трансгенной клетки растения по п.1, где две или более из указанных представляющих интерес ДНК встраиваются в два или более из указанных негенных геномных локуса–мишени кукурузы.
10. Способ получения трансгенной клетки растения по п.9, где два или более из указанных негенных геномных локуса–мишени кукурузы располагаются на одной хромосоме.
11. Клетка растения кукурузы, содержащая рекомбинантную молекулы нуклеиновой кислоты, где указанная рекомбинантная молекула нуклеиновой кислоты содержит:
негенную молекулу нуклеиновой кислоты, имеющую длину в диапазоне от примерно 1 т.п.о. до примерно 8,4 т.п.о., где
a. уровень метилирования указанной негеной молекулы нуклеиновой кислоты составляет 1% или менее;
b. негенная молекула нуклеиновой кислоты обладает менее чем 40% идентичностью последовательности с любыми другими молекулами нуклеиновой кислоты, содержащиеся в геноме кукурузы;
c. негенная молекула нуклеиновой кислоты расположена в пределах области из 40 т.п.о. известной или спрогнозированной экспрессивной кодирующей молекулы нуклеиновой кислоты кукурузы; и
d. негенная молекула нуклеиновой кислоты проявляет частоту рекомбинации в пределах генома кукурузы более чем 0,00041 cM/м.п.о., дополнительно где указанная негенная молекула нуклеиновой кислоты содержит последовательность нуклеиновой кислоты, выбранную из группы, состоящей из SEQ ID NO: 1, SEQ ID NO: 100, SEQ ID NO: 203, SEQ ID NO: 295, SEQ ID NO: 384, SEQ ID NO: 424, SEQ ID NO: 451, SEQ ID NO: 547, SEQ ID NO: 671, SEQ ID NO: 687, SEQ ID NO: 781, SEQ ID NO: 843, SEQ ID NO: 875, SEQ ID NO: 967, SEQ ID NO: 1107, SEQ ID NO: 1147, SEQ ID NO: 1190, SEQ ID NO: 1252, SEQ ID NO: 1300 и SEQ ID NO: 1371, и
и представляющую интерес ДНК, содержащую ген устойчивости к насекомым или ген толерантности к гербицидам, где представляющая интерес ДНК вставлена в указанную негенную молекулу нуклеиновой кислоты, где клетка растения кукурузы имеет улучшенную экспрессию трансгена относительно указанного гена устойчивости к насекомым или гена толерантности к гербицидам.
12. Растение кукурузы, имеющее устойчивость к насекомым или толерантность к гербицидам, где указанное растение содержит клетку растения кукурузы по п.11, где указанная клетка растения содержит две или более указанных негенных молекул нуклеиновой кислоты, причем каждая имеет представляющую интерес ДНК, вставленную в соответствующую негенную нуклеиновую кислоту.
13. Растение по п.12, где указанные рекомбинантные молекулы нуклеиновой кислоты расположены на одной и той же хромосоме.
14. Растение по п.12, где каждая из указанных представляющих интерес ДНК встроена в пределах 1,5 т.п.о., 1,25 т.п.о., 1,0 т.п.о., 0,75 т.п.о., 0,5 т.п.о., или 0,25 т.п.о. участка–мишени цинковых пальцев, специфического для указанной негенной молекулы нуклеиновой кислоты.
15. Растение по п.12, где каждая из указанных представляющих интерес ДНК встроена между парой участков–мишеней цинковых пальцев, специфических для указанной негенной молекулы нуклеиновой кислоты.
16. Растение по п.12, где по меньшей мере одна из указанных представляющих интерес ДНК содержит аналитический домен.
17. Растение по п.12, где по меньшей мере одна из указанных представляющих интерес ДНК не кодирует пептид.
18. Растение по п.12, где по меньшей мере одна из указанных представляющих интерес ДНК кодирует пептид.
19. Растение по п.12, где каждая из указанных представляющих интерес ДНК содержит кассету для экспрессии гена, содержащую ген устойчивости к насекомым, ген толерантности к гербицидам, ген эффективности утилизации азота, ген эффективности утилизации воды, ген пищевой ценности, ген ДНК–связывающего продукта или ген селективного маркера.
20. Клетка растения кукурузы по п. 12, где каждая их указанных представляющих интерес ДНК содержит кассету экспрессии гена, содержащую ген устойчивости к насекомым или ген устойчивости к гербицидам.
21. Растение кукурузы, имеющее устойчивость к насекомым или толерантность к гербицидам, где указанное растение кукурузы содержит клетку растения кукурузы по п. 11, где растение кукурузы имеет улучшенную экспрессию трансгена относительно гена устойчивости к насекомым или гена толерантности к гербицидам.
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WO2015066636A3 (en) 2015-06-25
MX2016005875A (es) 2016-10-13
CL2016001065A1 (es) 2016-10-14
RU2016122067A3 (ru) 2018-07-05
US20190024100A1 (en) 2019-01-24
RU2016122067A (ru) 2017-12-11
AU2018201717A1 (en) 2018-04-05
BR102014027442A2 (pt) 2016-09-20

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