Использование 3'-нетранслируемой области вируса некротического пожелтения жилок свеклы в качестве индуктора устойчивости к ризомании
Одно из наиболее опасных вирусных заболеваний культуры сахарной свеклы, резуха (ризомания), вызывается вирусом некротического пожелтения жилок свеклы (Beet necrotic yellow vein virus, BNYVV). Ризомания регистрировалась практически во всех районах возделывания культуры в Старом и Новом Свете (McGrann et al, 2009). При поражении ризоманией сахаристость корнеплодов снижается от 8% до 50−60% (Asher… Читать ещё >
Содержание
- Список сокращений
2
Введение.
2.1 Актуальность проблемы.
2.2 Цель и задачи работы. Ю
3 Обзор литературы.
3.1 Ризомания сахарной свеклы.
3.1.1 Географическое распространение BNYVV.
3.1.2 Вредоносность и экономическое значение BNYVV.
3.1.3 Растения-хозяева BNYVV.
3.1.4 Симптомы заболевания ризоманией.
3.1.5 Способы переноса и распространения вируса.
3.1.6 Морфология частиц и организация генома.
3.1.7 Типы и изоляты BNYVV.
3.1.8 Способы предотвращения распространения ризомании.
3.1.9 Устойчивость к вектору P. betae.
3.1.10 Селекция на устойчивость к BNYVV.
3.2 Использование трансгенных растений для индукции вирусоустойчивости.
3.2.1 Трансгенная устойчивость, обусловленная экспрессий в растении гена белка оболочки вируса.
3.2.2 Трансгенные растения, экспрессирующие репликазные гены.
3.2.3 Трансгенная устойчивость, основанная на экспрессии транспортного белка.
3.2.4 Трансгенная устойчивость, обусловленная экспрессией антител к вирусам.
3.2.5 Устойчивость, обусловленная активацией системной приобретенной устойчивости (SAR) растения.
3.2.6 Вирусоустойчивость растений, обусловленная «замолканием генов».
Использование 3'-нетранслируемой области вируса некротического пожелтения жилок свеклы в качестве индуктора устойчивости к ризомании (реферат, курсовая, диплом, контрольная)
2.1 Актуальность проблемы В мировой практике рентабельность выращивания сахарной свеклы {Beta vulgaris L.) во многом зависит от эффективности борьбы с болезнями и вредителями этой культуры. Из грибковых болезней сахарной свеклы наиболее вредоносными являются черная ножка (вызываемая Phoma betae, Pythium spp., Aphanomyceas cochlioides, Rhizoctonia solani), церкоспороз (¦Cercospora beticola), альтернариозная листовая пятнистость (Alternaria alternata), ложная мучнистая poca (Peronospora farinosa), мучнистая poca (.Erysiphe betae), ризоктониозная корневая гниль (Rhizoctonia solani), пятнистость листьев {Ramularia beticola), фузариоз корней (.Fusarium spp.) (Agrios, 2005; Draycott, 2006; Harveson et al, 2009). Культура сахарной свеклы поражается и рядом вирусов, такими как вирус желтухи свеклы (Beet yellows virus, BYV), вирус курчавости верхушки (Beet curly top virus, BCTV), вирус мозаичности свеклы (Beet mosaic virus, BtMV), вирус курчавости листьев свеклы (Beet leaf curl virus, BCLV) (Smith and Hallsworth, 1990; Wisler and Duffus, 2000; Agrios, 2005; Draycott, 2006; Harveson et al., 2009).
Одно из наиболее опасных вирусных заболеваний культуры сахарной свеклы, резуха (ризомания), вызывается вирусом некротического пожелтения жилок свеклы (Beet necrotic yellow vein virus, BNYVV). Ризомания регистрировалась практически во всех районах возделывания культуры в Старом и Новом Свете (McGrann et al, 2009). При поражении ризоманией сахаристость корнеплодов снижается от 8% до 50−60% (Asher, 1993; Henry, 1996; Johanson, 1985), а снижение урожайности может достигать 30−90% (Johanson, 1985; Шпаар, 2004).
На сегодняшний день успех борьбы с ризоманией зависит от выращивания устойчивых сортов, гибридов и линий, причем устойчивость может достигаться использованием как естественных, так и созданных генно-инженерными методами источников устойчивости к BNYVV.
Кроме методов классической селекции, с помощью которых были получены все современные сорта и гибриды сахарной свеклы, устойчивые к ризомании, существуют различные подходы генетической инженерии для дальнейшего увеличения устойчивости к этой болезни. Совокупность подходов, связанных с трансгенной экспрессией фрагментов вирусного генома, получила название патоген-зависимой устойчивости (PDR).
Ранее для создания трансгенной устойчивости к BNYVV использовали экспрессию в растениях последовательности гена белка оболочки (Mannerlof et al., 1996; Andika et al, 2005), инвертированных повторов гена репликазы (Lennefors et al, 2006; Pavli et al, 2010), RTD домена (Andika et al, 2005) и одноцепочечного фрагмента антител, специфичных к белку оболочки BNYVV (Fecker et al, 1997). В этих работах наблюдали частичную или полную трансгенную устойчивость к BNYVV.
Экспрессия в растениях фрагментов З'-нетранслируемой области (3'-НТО) вирусных РНК геномов имеет определенные преимущества с точки зрения безопасности, поскольку вирусные РНК, в отличие от белков, имеют короткое время полужизни и не являются иммуногенами. З'-НТО BNYVV представляет интерес в качестве нового индуктора патоген-зависимой устойчивости к ризомании. В настоящей работе для сравнения эффективности использования З'-НТО с ранее изученными индукторами в модельном растении Nicotiana benthamiana была также экспрессирована последовательность гена белка оболочки BNYVV. Кроме того, в трансгенных растениях был экспрессирован ген bar, обеспечивающий устойчивость к фосфинотрицину — действующему веществу гербицида BASTA.
6 Выводы.
1. Сконструирован бинарный вектор pBIJBARJBNYVVsil, несущий под контролем двух независимых 35S промоторов кДНК двухцепочечных инвертированных фрагментов З'-НТО BNYVV, разделенных интроном ubil, и кДНК гена bar.
2. Сконструирован бинарный вектор pBIBARBNYVVcp, несущий под контролем двух независимых 35S промоторов вставку кДНК белка оболочки BNYVV и кДНК гена bar.
3. Методом транзиентной экспрессии конструкции «индуктора» (BNYVVsil) и «мишени» в растениях N. benthamiana дикого типа показано 30-кратное снижение экспрессии GFP, что указывает на возможность эффективной индукции сайленсинга двунитчатой формой З'-НТО генома BNYVV.
4. Получены трансгенные растения N. benthamiana, трансформированные вектором pBIBARBNYVVsil. В трансгенных линиях наблюдали экспрессию транскрипта BNYVVsil и продукта гена bar, PAT, обеспечивающего устойчивость к гербициду BASTA.
5. Получены трансгенные растения N. benthamiana, экспрессирующие транскрипт гена белка оболочки BNYVV и PAT продукт гена bar.
6. В гомозиготном поколении Т2 выделена толерантная линия BNYVVsil-126.4, обладающая частичной устойчивостью к инфекции BNYVV в условиях механической передачи вируса.
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