Изучение стабильности экспрессии чужеродных генов у трансгенных растений табака (Nicotiana tabacum L.)
Для изучения наследования и стабильности экспрессии чужеродных генов были выделены модельные линии трансгенных растений табака: 1) трансгенные растения с одиночными и множественными вставками Т-ДНК- 2) трансформанты, имеющие в геноме Т-ДНК инсерцию с дупликацией гена uidA и одной копией гена nptll- 3) трансгенное растение табака Nu 21 с мозаичным характером проявления экспрессии маркерного гена… Читать ещё >
Содержание
- СПИСОК СОКРАЩЕНИЙ
- ГЛАВА 1. ОБЗОР ЛИТЕРАТУРЫ
- 1. 1. Получение трансгенных растений: общие сведения
- 1. 2. Наследование чужеродных генов и вариабельность их экспрессии у трансгенных растений
- 1. 3. Инактивация чужеродных генов в растительном геноме
- 1. 4. Молекулярно-генетические механизмы инактивации чужеродной ДНК в ядерном геноме трансгенных растений
- 1. 4. 1. Структура и местоположение экзогенной ДНК в растительном геноме
- 1. 4. 2. Замолкание экспрессии трансгена и модификация ДНК
- 1. 4. 3. Мутации растительных генов, влияющих на процесс инактивации экспрессии чужеродной ДНК
- 1. 5. Примеры инактивации экспрессии множественных копий собственных растительных генов
- 1. 6. Инактивация экспрессии множественных копий генов у грибов-аскомицетов
- 1. 7. Использование феномена инактивации экспрессии множественных копий генов для целенаправленного выключения экспрессии растительных генов
- 1. 8. Стратегия предотвращения инактивации целевых генов в трансформированных растениях
- ГЛАВА 2. МАТЕРИАЛЫ И МЕТОДЫ
- 2. 1. Получение транс генных растений табака
- 2. 2. Типы генетических конструкций
- 2. 3. Гибридологический анализ трансгенных растений табака
- 2. 4. Определение активности неомицинфосфотрансферазы II in situ
- 2. 5. Флуориметрическое определение активности p-глюкуронидазы в листьях трансгенных растений табака
- 2. 6. Выделение геномной ДНК из листьев табака
- 2. 7. Саузерн блот гибридизация геномной ДНК трансгенных растений табака
- 2. 8. Выделение РНК из растительной ткани и точечный Нозерн блотгинг
- 2. 9. Подтверждение наличия дупликаций в области инсерции Т-ДНК с помощью метода ПЦР
- 2. 10. Проверка исходных трансформантов на присутствие в растительном геноме векторных последовательностей с помощью метода ПЦР
- 2. 11. Электрофоретический анализ ДНК
- ГЛАВА 3. РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ
- 3. 1. Стабильность экспрессии маркерного гена nptll у гибридов от скрещивания трансгенных растений табака с одной инсерцией Т-ДНК
- 3. 2. Анализ стабильности наследования гена nptll у трансгенных растений табака со множественными инсерциями Т-ДНК
- 3. 3. Моделирование влияния дупликации в структуре Т-ДНК на стабильность экспрессии маркерных генов nptll и uidA в трансгенных растениях табака
3.3.1 Сравнительный анализ стабильности экспрессии гена nptll в первом и втором поколениях от самоопыления трансгенных растений табака (с одной копией гена uidA и с дупликациями данного гена в составе Т-области).
3.3.2 Анализ активности ферментов (З-глюкуронидазы и NPTII в двух группах трансгенных растений табака (с одной копией гена uidA и прямой дупликацией данного гена).
3.3.3 Анализ стабильности экспрессии гена nptll у гибридов Fj от различных типов скрещиваний.
3.3.4 Встраивание векторных последовательностей в растительный геном исходных трансгенных растений табака.
3.4 Мозаичный характер проявления экспрессии гена nptll в трансгенных растениях табака модельной линии Nu 21.
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