Бесплатный автореферат и диссертация по биологии на тему
Получение и анализ трансгенных растений, экспрессирующих белки тройного блока генов гордеивирусов
ВАК РФ 03.00.06, Вирусология

Содержание диссертации, кандидата биологических наук, Горшкова, Елена Николаевна

Список сокращений

Г. Введение

2: Обзор литературы

2.1 Общие положения ^

2.2. Альфа-подобные фнтовирусы, содержащие тройнойблок генов.

2.2.1. Семейство Tubiviridae.

2.2.1.1. Род Benyvirus

2.2.1.2. Род Pomovirus.

2.2.1.3. Род Pecluvirus. 16 * 2.2.1.4. Род Hordeivirus

2.2.2. Порядок Tymovirales 19 2.2:2:1. Семейство Potexviridae 1 ^

2.3: Методы изучения локализации транспортных белков фитовирусов

2.3.1.1. Субклеточное фракционирование белков ТБГ 22 2.3; 1.1.1.Субклеточна» локализация 6ТБГ1 21 2.3.1.1.2. Субклеточная локализация белков-ТБГ иЛБЕЗ.

2.3.1.2. Субклеточная локализация транспортных белков тобамовирусов

2.3.1.3. Субклеточная локализация транспортных белков витивирусов, порядок Tymovirales

2.3". 1.4. Субклеточная локализация транспортных белков представителей семейства Bromoviridae

2.3.1.5. Субклеточная- локализация- транспортных белков представителей семейства Comoviridae

2.3.1.6. Субклеточная локализация транспортных

Ф белков представителей, семейства Tombusviridae

2.3.2. Электронная микроскопия транспортных белков

2.3.2.1. Электронная микроскопия белков ТБГ 28 ^ 2.3.2.2. Электронна* микроскопия: транигшртных белков других вирусов растений

2.3.3. Изучение локализации транспортных белков методом лазерной конфокальной микроскопии

2.3.3Х Изучение локализации белков ТБГ методом флуоресцентной микроскопии

2.3. Комплементационный анализ транспортных белков

2.3.1. Гомологичная комплементация транспортных белков

2.3.2. Гетерологичная комплементация транспортных белков

2.3.2.1. Гетерологичная комплементация транспортных белков в положении in cis

2.3.2.2. Гетерологичная комплементация транспортных, белков в положении in tram 37 2.3.2.2.1. Гетерологичная комплементация транспортных бежов с помощью рекомбинантных конструкций

2.3.2.2.2. Гетерологичная комплементация транспортных белков вирусов в трансгенных растениях

2.3.2.2.3. Гетерологичная комплементация вирусного транспорта с использованием вирусов-помощников

2.4. Структура и свойства белков тройного блока генов.

2.4.1. Особенности первичной структуры белка ТБГ

2.4.2. Особенности первичной структуры белков ТБГ2 и 44 Ж ТБГЗ

2.4.3. Экспрессия белков ТБГ

2.4.4. Биохимические и функциональные свойства белка

2.4.5. Функции белков ТБГ2 и ТБГЗ

2.4.6. Возможная схема транспорта ТБГ-содержащих вирусов 52 3. Материалы и методы

3.1. Реактивы и материалы

3.2. Ферменты и наборы

3.3. Векторы для клонирования и бактериальные штаммы.

3.4. Среды, использовавшиеся при культивировании 55 бактериального и растительного материала

3.5. Выделение плазмидной ДНК

3.6. Трансформация бактериальных клеток плазмидной 56 ДНК

3.7. Электрофорез в агарозном геле

3.8. Извлечение фрагментов ДНК из агарозного геля.

3.9. Цитирование фрагментов ДНК

3.10. Трансформация растительных тканей

3.11. Трансформация бактериальных клеток A. tumefaciens плазмидной ДНК

3.12. Выделение тотального препарата нуклеиновых кислот из клеток A. tumefaciens

3.14. Рестрикционный гидролиз тотального препарата ДНК, выделенного из клеток A. tumefaciens

3.15. ДНК-блотинг 61 3 Л6. Гибридизация нуклеиновы кислот, иммобилизованных на найлоновом фильтре

3.17. Синтез зонда для гибридизации.

3.18. Выделение препарата тотальной РНК из растительных тканей

3.19.Электрофорез РНК в денатурирующем геле

3.20.Иммунодетекция белков

3.21. Субклеточное фракционирование растительных тканей

3.22. Разделение клеточных мембран в градиенте плотности сахарозы

3.23. Экстракция белков ш препарата мембран.

3.24. Выделение РНК из фракций протяженного градиента сахарозы

3.25. Флотация мембранных препаратов

4. Результаты

4.1. Получение трансгенных растений, экспрессирующих белок: ТБГЗ ПЛВМ и белок ТБГЗ ГОТОМ, слитого с GFP.

4.2. Иммунодетекция белков 18К и GFP-18K в трансгенных и зараженных растениях.

4.3. Эпифлуоресцентная микроскопия трансгенных растений, экспрессирующих ген. белка GFP- 18К.

4.4. Функциональная активность белков 18К и GFP-18K, экспрессируемых в трансгенных растениях

4.5. Субклеточное фракционирование растительных тканей, экспрессирующих белки 18К и GFP-18К

4.6. Исследование мембранной природы белков 18К и GFP

4.7. Локализация белков 18К и GFP-18K в эвдоплазматическом ретикулюме.

5. Обсуждение результатов

6. Выводы

Введение Диссертация по биологии, на тему "Получение и анализ трансгенных растений, экспрессирующих белки тройного блока генов гордеивирусов"

Предметом молекулярной фитовирусологии является исследование репликации и экспрессии вирусного генома, а так- же взаимодействия вирусного патогена с растением — хозяином; Одной из ключевых проблем является изучение механизмов транспорта вирусов в зараженном растении. Основную роль. в вирусном, транспорте играют вирус-кодируемые транспортные белки, которые обеспечивают перенос вирусного генома внутри клетки, от сайтов репликации к плазмодесмам, межклеточный транспорт, а так же распространение вирусного генома по всему растению (Atabekov and Dorokhov, 1984; Carrington et al, 1996).

Транспортные белки фитовирусов делятся на несколько классов, одним из которых является группа белков, кодируемых тройным блоком 1-е нов (ТБГ) (Moruzov etui., 1987; 1989). В зависимости or группы вирусов ТБГ может находиться в различных участках вирусного генома, но взаимное расположение генов ТБГ1, ТБГ2 и ТБГЗ и особенности кодируемых ими белков всегда остаются неизменными. Мутации в любом из генов ТБГ приводят к потере вирусом транспортных функций (Petty and Jackson, 1990; Beck et al, 1991; Gilmer et al, 1992; Herzog et al., 1998). Среди белков ТБГ наиболее хорошо изучен 6ТБГ1. Для него показана РНК-связывающая, НТФ-азная и хеликазная активности (Bleykasten et al.,\996; Kalinina et al., 1996; 2001; 2002; Donald et al., 1997; Wung et al., 1999; Liou et al., 2000), кроме того, хорошо изучено его распределение к клетке (Niesbach-Klosgen et al., 1990; Donald et al., 1993; Rouleau et al., 1994; Morozov et al, 1999; Erhardt et al, 1999; 2000; Lawrence and Jackson, 2001). В отличие от 6ТБГ1, биохимические свойства белков ТБГ2 и ТБГЗ изучены слабее. Это обусловлено высокой гидрофобностью этих белков. Локализация 6ТБГ2 в клетках зараженных растений была определена достаточно давно (Niesbach-Klosgen et al, 1990; Donald et al, 1993), однако попытки детектировать биохимическими методами 6ТБГЗ в клетках зараженных растений до сих пор не были успешными (Niesbach-Klosgen et al, 1990; Donald et al, 199J; Krishnamurthy et at., 2003). Тем не менее в настоящий момент появилось достаточно много сведений о локализации этого белка при экспрессии его с помощью различных векторных систем (Solovyev et al, 2000; Cowan et al, 2002; Krishnamutrfhy et al, 2003).

2. Обзор литературы

2Л. Общие положения транспорта вирусов растений

Распространение фитовирусов по тканям растения-хозяина происходит по принципиально отличной стратегии; по сравнению с вирусами животных или бактериофагами. Из первично-инфицированной клетки, пользуясь-специальными механизмами и не разрушая клетки, вирус транспортирует свой геном в соседние клетки растений (Atabekov and Taliansky, 1990; Carrington et al, 1996; Lazarowitz, 1999; Oparka and Roberts, 2001). Такое распространение фитовирусов называют ближним или межклеточным траснпортом (Carrington et al, Т996; Ding, 1998; Lazarowitz and Beachy 1999; Oparka and Roberts, 2001). При попадании в проводящие пучки вирус способен перемещаться по флоэме пассивно с током жидкости. Таким образом, вирус перемещается но стеблю на достаточно далекие дистанции к другим листьям и органам. Такой процесс именуется дальним транспортом (Atabekov and Taliansky, 1990; Carrington et al, 1996; Lazarowitz, 1999; Oparka and Roberts, 2001). Попав в неинфицированный лист, для дальнейшего распространения фитовирусы снова пользуются механизмами ближнего транспорта. Следует отметить, что существует несколько групп фитовирусов, неспособных к ближнему транспорту в мезофилле листовой пластины, и весь их жизненный цикл проходит во флоэме. Такие вирусы называют флоэмноограниченными (Leisner and Turgeon, 1993; Lucas and Wolf, 1999; Santa Cruz 1999).

Механизм попадания фитовирусов в первичную клетку изучен недостаточно хорошо. Считается, что такое проникновение вируса в клетку происходит через механические повреждения в клеточной стенке (Carrington et al, 1996). Известно, что вирусы, передающиеся насекомыми и патогенными грибами, попадают в первую очередь во флоэму (Tamada and Kusume 1991; Richards and Tamada 1992; Robinson et al, 1997). Некоторые вирусы способны передаваться через семена от поколения к поколению (Jackson et al.,1989; Edwards, 1995; Wang et al., 1997).

После попадания вириона в первично инфицированную клетку, начинаются процессы, связанные с выражением и репликацией вирусного i-енома. Среди синтезируемых вирусснецифических белков имеется один или несколько белковых продуктов, функциями которого является обеспечение процессов ближнего и дальнего транспорта. Такие белки получили название транспортных белков. В частности, в функции ТБ входит внутриклеточных транспорт вирусного генома от места репликации к плазмодесмам. Местом репликации вирусного генома может быть ядро для ДНК-содержащих фитовирусов или цитоплазма для РНК-содержащих вирусов. У некоторых вирусов транспортной функцией в дополнение к ТБ может обладать белок оболочки. Иногда наличие БО необходимо для обеспечения только дальнего транспорта.

2.2. Альфа-подобные фитовирусы, содержащие тройной блок генов

Геном вирусов, имеющих тройной блок генов, представляет собой однонитевую РНК положительной полярности. Все вирусы, имеющие в составе генома ТБГ, относятся к супергруппе альфа-подобных вирусов и имеют два характерных признака. Во-первых, их геномные РНК всегда несут кэп-структуру па 5'конце и поли-А-тракт и/или тРНК подобную структуру на З'конце. Во-вторых, они обладают характерным набором инвариантных доменов репликационных белков, а именно, домен, характерный для РНК-зависимой РНК-полимеразы, относящейся к супергруппе 3, хеликазный домен, характерный для суперсемейства 1, и метил-гуанилил-трансферазный домен (Goldbach, 1987; Koonin and Dolja, 1993).

В состав супергруппы альфа-подобных вирусов входят два семейства, геном представителей которых содержит ТБГ. Это семейства Tubiviridae и Potexviridae.

Заключение Диссертация по теме "Вирусология", Горшкова, Елена Николаевна

б. Выводы

1. Получены трансгенные растения N. benthamiana, экспрессирующие белки 18К и GFP-18K ПЛВМ.

2. Комплементационный анализ показал функциональную активность белков 18К и GFP-18K, экспрессируемых в трансгенных растениях.

3. Белки 18К и GFP-18K детектированы в трансгенных и зараженных растениях. Выявлена склонность белков к образованию высокомолекулярных агрегатов.

4. Субклеточное фракционирование тканей трансгенных растений, экспрессирующих белки 18К и GFP-18K и результаты экстракции этих белков показали их принадлежность к классу интегральных мембранных бежов.

5. Анализ распределения бежа 18К и GFP-18K относительно белков-маркеров в протяженном градиенте плотности сахарозы показал, что эти белки ассоциированы со структурами ЭР.

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