Бесплатный автореферат и диссертация по биологии на тему

Идентификация методом инсерционного мутагенеза генов, участвующих в контроле морфогенеза Arabidopsis thaliana

ВАК РФ 03.00.15, Генетика

Содержание диссертации, кандидата биологических наук, Томилов, Алексей Александрович

1 Введение

Заключение Диссертация по теме "Генетика", Томилов, Алексей Александрович

5. Выводы.

1. Показано, что обработка семян A. thaliana ультразвуком в присутствии окиси алюминия увеличивает эффективность последующей агробактериальной трансформации прорастающих семян. Это приводит к увеличению эффективности процесса индукции инсерционных мутаций у A. thaliana не менее, чем в 3 раза.

2. Для плазмид серии рВ1 разработана эффективная система селекции трансгенных растений, позволяющая в Т2 поколении отбирать растения, геном которых содержит инсерцию Т - ДНК.

3. При использовании двух векторов (pLD3 и pPCVRN4) создана коллекция инсерционных мутантов, включающая в себя 2175 линий растений. Из этой коллекции отобрано и с помощью генетического и молекулярно - генетического анализа охарактеризовано 83 линии растений, имеющих различные морфологические нарушения.

4. Исследование мутантного фенотипа линии 76, геном клеток которой содержит одну инсерцию Т - ДНК, показало, что в его основе лежит условно летальная гормон - зависимая рецессивная мутация.

5. Проведенный косегрегационный анализ наследования маркеров инсерции и мутантного фенотипа показал их тесное сцепление. Это свидетельствует в пользу того, что в основе фенотипически

164 регистрируемой мутации у растений линии 76 лежит одиночная инсерция Т - области.

6. При использовании метода «TAIL» - ПЦР амплифицирован фрагмент растительной ДНК генома мутантной линии 76, примыкающий к левому бордеру и определена его нуклеотидная последовательность после клонирования амплифицированного фрагмента в бактериальную плазмиду.

7. Использование компьютерного анализа позволило установить, что инсерция в геноме линии 76 расположена в 32 экзоне гена YUP8H12R.44, находящегося в первой хромосоме. Функции этого гена ранее были неизвестны, однако, судя по нуклеотидной последовательности, он является представителем семейства генов, кодирующих оксидоскуаленциклазы.

Выражаю глубокую благодарность:

Своим научным руководителям д.б.н. проф. Тарасову В.А. и к.б.н. Огарковой О.А за постоянную помощь в решении экспериментальных и организационных проблем, обсуждении результатов работы, и неоценимую помощь в работе в целом; к.б.н. Ежовой Т.А. и к.б.н. Солдатовой О.П. (каф. генетики и селекции Биологического факультета МГУ им. М.В. Ломоносова) за помощь в выращивании трансформантов Т1 поколения, сборе семян, сегрегационном анализе некоторых трансформантов, проведении эмбрионального теста; к.б.н. Кочиевой А.З. (лаб. генетики растений ИОГен им. Н.И.Вавилова РАН) за консультации по методам извлечения окружений Т-ДНК инсерций и транспозонов, помощь в подборе условий проведения "ТА1Ь"-ПЦР и подборе праймеров к Т-области векторов системы рВ1 для метода "ТА1Г"-ПЦР; к.б.н. Хадеевой Н.В. (лаб. генетики растений ИОГен им. Н.И.Вавилова РАН) за консультации по ведению асептических культур растений, подбору гормонов и их концентраций, гистохимическим анализам; к.б.н. Сидоруку К.В. (лаб. белковой инженерии ГосНИИ "Генетика") за консультации и конструктивные беседы по проведению секвенирования фрагментов ПЦР и работы в целом; к.б.н. Шайхаеву Г.О. (ИОГен им. Н.И.Вавилова РАН) за консультации по вопросу подбора режимов ПЦР, подбор и проверку реактивов для ПЦР; д.б.н., проф. Пухальскому В.А. и д.б.н. Гапоненко А.К. за рецензию данной работы и высказанные ценные замечания; всем сотрудникам Института общей генетики им. Н.И.Вавилова РАН за доброжелательное отношение и помощь в работе.

Библиография Диссертация по биологии, кандидата биологических наук, Томилов, Алексей Александрович, Москва

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