Структурно-функциональные особенности запасных и защитных белков растений и их использование в генетических исследованиях
Впервые разработана методология разделения и очистки различных запасных и защитных растительных белков: мономерных и полимерных проламинов (сем. Роасеае), 2Э альбуминов (сем. Аз1егасеае) и глобулинов (сем. СисигЬкасеае), РЯ-белков (сем. 8о1апасеае) и антимикробных пептидов (сем. Роасеае). Впервые изучены структурные особенности глиадинов гексаплоидной пшеницы ТгШсит аезймит, кодируемых одним… Читать ещё >
Содержание
- ВВЕДЕНИЕ 7 ОБЗОР ЛИТЕРАТУРЫ
- ГЛАВА I. ЗАПАСНЫЕ БЕЛКИ
- 1. Классификация белков растений
- 2. 0. бщая характеристика запасных белков
- 3. 2. S альбумины
- 4. Глобулины
- 4. 1. 11S глобулины
- 4. 2. 7S глобулины
- 4. 3. Запасные глобулины злаков
- 5. Проламины
- 5. 1. Общая характеристика
- 5. 2. Проламины пшеницы
- 5. 2. 1. Глиадины
- 5. 2. 2. Глютенины
- 5. 2. 3. Клейковина
- 5. 2. 3. Роль дисульфидных связей в формировании структуры полимеров глютенина
- 5. 2. 4. Генетические исследования роли высоко- и низкомолекулярных субъединиц глютенина в вязкоэластичных свойствах клейковины
- 5. 3. Суперсемейство проламинов Triticeae
- 5. 4. Суперсемейство проламинов других видов растений
- 1. Классификация белков растений
- 1. Общая характеристика
- 2. Основные семейства антимикробных белков
- 3. Экспрессия и локализация антимикробных белков
- 3. 1. Экспрессия АМБ в листьях
- 3. 2. Экспрессия АМБ в корнях
- 3. 3. Экспрессия АМБ в цветках
- 3. 4. Экспрессия в семенах
- 4. Роль антимикробных белков в устойчивости растений
- 4. 1. Суперэкспрессия генов АМБ
- 4. 2. Подавление экспрессии генов АМБ
- 4. 3. Трансгенные или мутантные растения с нарушениями сигнальных путей, участвующих в регуляции экспрессии генов АМБ
- 5. Структурная характеристика и антимикробные свойства основных семейств антимикробных белков растений
- 5. 1. Белки семейства PR
- 5. 2. Р-1,3- Глюканазы (семейство PR-2) и хитиназы (семейства PR-3, PR-8, PR-11)
- 5. 2. 1. Белки семейства PR
- 5. 2. 2. Белки семейства PR
- 5. 2. 3. Белки семейства PR
- 5. 2. 4. Белки семейства PR
- 5. 3. Белки семейства PR
- 5. 4. Белки семейства PR-5 (тауматиноподобные белки)
- 5. 5. Ингибиторы протеиназ (белки семейства PR-6)
- 5. 6. Рибосомо-инактивирующие белки (RIPS)
- 6. Антимикробные пептиды
- 6. 1. Гевеиноподобные антимикробные пептиды
- 6. 2. Липидпереносящие белки (ЛПБ)
- 6. 3. Тионины
- 6. 4. Растительные дефензины
- 6. 5. Другие антимикробные пептиды
- 6. 5. 1. Антимикробный пептид Mirabilis jalapa (Mj-AMPs) (Ноттиноподобные пептиды)
- 6. 5. 2. Антимикробный пептид Macadamia integrifolia (MIAMP1)
- 6. 5. 3. Пептид кукурузы (МВР-1)
- 6. 5. 4. Антимикробные пептиды Impatiens balsamina (Ib-AMPs)
- 6. 5. 5. Антимикробные свойства
- 6. 5. 6. Снакины
- 6. 5. 7. Глицин-гистидин-богатые пептиды
Выделение спирторастворимых полимерных глютелинов Выделение ш-глиадинов и D-субъединиц глютенина Выделение HMW субъединиц глютенина 17 и 18 и D субъединиц из изогенной линии пшеницы Triticum aestivum L88−31 Выделение полимеров глютенина
Экстракция белков из семян представителей Cucurbitaceae Экстракция кукурбитина
Обращенно-фазовая жидкостная хроматография кислоторастворимых альбуминов
Выделение кислоторастворимых белков из листьев табака Выделение кислоторастворимых белков из листьев томата Разделение кислоторастворимых белков листьев томата путем обращенно-фазовой жидкостной хроматографии Выделение белка Р15 листьев томата с использованием препаративного электрофореза Очистка белка Р
Экстракция белков листьев трансгенных растений Выделение антимикробных пептидов из зерновок пшеницы Triticum kiharae Dorofet Migusch. Выделение дефензинов из зерновок пшеницы Triticum kiharae Dorofet Migusch.
Выделение кислоторастворимых белков из семян Taraxacum officinale Разделение белков семян Taraxacum officinale путем гель-фильтрации Обращенно-фазовая жидкостная хроматография белков семян Taraxacum officinale
Обнаружение интерфероноподобных белков в листьях томатов методом иммуноблоттинга с антителами к-интерферону человека
Методы структурного анализа белков
Восстановление и алкилирование белков и пептидов Масс-спектрометрия
Ограниченный протеолиз глиадина у-46 химотрипсином Ферментативный гидролиз дисульфидсодержащих фрагментов глиадина у
Идентификация дисульфидсодержащих фрагментов
Селективное выделение триптических цистеинсодержащих пептидов
Расщепление ш-глиадинов трипсином
Расщепление белков и пептидов бромцианом
Расщепление пептидов эндопротеиназой Glu-C
Гидролиз белка в геле
Обращенно-фазовая жидкостная хроматография триптических Пептидов
Иммобилизация на хитине Электрофоретические методы исследования белков
Одномерный электрофорез белков Двумерное разделение белков в полиакриламидном геле Аналитические методы
Установление аминокислотной последовательности Определение аминокислотного состава УФ-спектрофотометрия и определение концентрации белка Круговой дихроизм (КД)
Определение размеров полимеров глютенина методом светорассеяния Выделение вирусов и их компонентов и заражение растений
Выращивание растений
Инокуляция растений препаратами вирусов ВТМ и ВОМ-1 Получение препарата вируса ВТМ Получение препарата вируса ВОМ-1 Определение относительного содержания ВТМ в ткани зараженных растений по тесту инфекционности Выделение белка оболочки ВТМ Определение активности ингибиторов ферментов
Определение антимикробной активности
Определение антигрибной активности Определение антибактериальной активности Методы исследования хлоропластов
Выделение хлоропластов
Выделение мембранной и растворимой фракции хлоропластов Определение РНК-полимеразной активности
Электронно-микроскопическое исследование структуры хлоропластов Экспрессия рекомбинантного пептида WAMP-1a в клетках Е. coli
Конструирование экспрессионного вектора Получение и очистка гибридного белка
Расщепление гибридного белка и выделение целевого пептида Установление структуры предшественника пептида WAMP
Выделение тотальной РНК и синтез первой цепи кДНК Процедура ПЦР
Определение нуклеотидной последовательности кДНК методом 3'- и 5'-RACE
Определение изменения уровня экспрессии гена WAMP-1 а под действием биотических и абиотических стрессовых факторов методом ОТ-ПЦР
Статистические методы
РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ
Глава I. ЗАПАСНЫЕ БЕЛКИ
1. Выделение глиадинов
2. Исследование структурных особенностей глиадинов пшеницы и родственных видов
2.1. Определение N-концевых аминокислотных последовательностей проламинов различных глиадинкодирующих локусов пшеницы
2.2. Выделение и характеристика глиадинов семян Ае. squarrosa
2.3. Выделение и характеристика глиадинов семян Ае. longissima
3. Дисульфидное картирование глиадинов
3.1. Определение частичной первичной структуры глиадина у
3.2. Определение дисульфидных связей в глиадине у
4. Дисульфидное картирование альбумина семян подсолнечника: консервативные и вариабельные дисульфидные связи в суперсемействе проламинов злаков
5. Выделение и структурный анализ глютенинов
5.1. Выделение D-субъединиц глютенина
5.2. Выделение и характеристика высокомолекулярных (HMW) субъединиц глютенина 17 и 18, а также D субъединиц глютенина из изогенной линии пшеницы L88
5.3. Характеристика высокомолекулярных полимеров глютенина методами электрофореза в агарозном геле и светорассеяния
6. Исследование полиморфизма белков семян огурца
6.1. Исследование полиморфизма альбуминов
6.2. Исследование полиморфизма глобулинов
7. Исследование полиморфизма белков семян тыквы.
7.1. Элекгрофоретический анализ кукурбитина
7.2. Элекгрофоретический анализ основных альбуминов
7.3. Высокоэффективная жидкостная хроматография белков семян тыквенных культур
8. Генетический контроль компонентного состава кукурбитина
8.1. Гибридологический анализ компонентного состава кукурбитина в гибридной комбинации Белые 13 х Грибовские
8.2. Гибридологический анализ компонентного состава кукурбитина в комбинации скрещивания Якорь х Белые
8.3. Гибридологический анализ компонентного состава кукурбитина в комбинации скрещивания Миндальная 35 х Сотэ
8.4. Полиморфизм кукурбитина у сортов тыквы
8.4.1. Идентификация аллелей кукурбитинкодирующих локусов
8.4.2. Межвидовой полиморфизм кукурбитина
8.4.3. Межсортовой и внутрисортовой полиморфизм кукурбитина
8.4.4. Использование электрофореза кукурбитина в качестве экспресс-метода определения сортовой чистоты
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- Я благодарна всем сотрудникам лаборатории генетики растений за критические замечания, дружескую помощь и доброжелательную атмосферу в лаборатории.