Получение трансгенных линий сахарной свеклы, экспрессирующих гены устойчивости к насекомым-вредителям сry1C и сry2A
Существенным ограничением урожайности сахарной свеклы является пагубное воздействие на нее насекомых-вредителей. Одной из перспективных стратегий обеспечения устойчивости растений выступает интеграция в растительный геном генов сry-белков Bacillus thuringiensis. Целью настоящей работы была генетичес...
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Інститут клітинної біології та генетичної інженерії НАН України
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| Cite this: | Получение трансгенных линий сахарной свеклы, экспрессирующих гены устойчивости к насекомым-вредителям сry1C и сry2A / Д.И. Литвин, В.В. Сивура, В.В. Курило, В.Д. Оленева, А.И. Емец, Я.Б. Блюм // Цитология и генетика. — 2014. — Т. 48, № 2. — С. 3-11. — Бібліогр.: 45 назв. — рос. |
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irk-123456789-1266202017-11-30T03:01:04Z Получение трансгенных линий сахарной свеклы, экспрессирующих гены устойчивости к насекомым-вредителям сry1C и сry2A Литвин, Д.И. Сивура, В.В. Курило, В.В. Оленева, В.Д. Емец, А.И. Блюм, Я.Б. Оригинальные работы Существенным ограничением урожайности сахарной свеклы является пагубное воздействие на нее насекомых-вредителей. Одной из перспективных стратегий обеспечения устойчивости растений выступает интеграция в растительный геном генов сry-белков Bacillus thuringiensis. Целью настоящей работы была генетическая трансформация сахарной свеклы линии ММ1/2 векторными конструкциями pRD400-cry1C и pRD400-cry2A, несущими гены cry1C и cry2A соответственно. В результате оптимизации протокола трансформации и прямой регенерации из листовых дисков с использованием 1 мг/л бензиламинопурина, а также 0,25 мг/л бензиламинопурина и 0,1 мг/л индолил-масляной кислоты получены трансгенные линии сахарной свеклы, несущие гены cry1C и cry2A. С помощью ПЦР-анализа подтверждена интеграция cry2A и cry1C в их геном, а с помощью метода ПЦР с обратной транс-крипцией установлено наличие экспрессии указанных генов в этих линиях. Істотним обмеженням врожайності цукрових буряків є згубний вплив на цю культуру комах-шкідників. Однією з перспективних стратегій забезпечення стійкості рослин є інтеграція в рослинний геном генів сry-білків Bacillus thuringiensis. Метою даної роботи була генетична трансформація цукрового буряку лінії ММ1/2 векторними конструкціями pRD400-cry1C і pRD400-cry2A, що несуть гени cry1C та cry2A відповідно. У результаті оптимізації протоколу трансформації та прямої регенерації з листових дисків із використанням 1 мг/л бензиламінопурину, а також 0,25 мг/л бензиламінопурину та 0,1 мг/л ін-доліл-масляної кислоти отримано трансгенні лінії цукрового буряку, що несуть гени cry1C і cry2A. За допомогою ПЛР-аналізу підтверджено інтеграцію cry2A і cry1C в їхній геном, а за допомогою ПЛР зі зворотною транскрипцією встановлено наявність експресії даних генів в цих лініях. The impact of insect pests significantly limits sugar beet crop yields. The integration of cry-genes of Bacillus thuringiensis into the plant genome is a promising strategy to ensure plant resistance. The aim of this work was to obtain sugar beet lines (based on the MM1/2 line) transformed with cry2A and cry1C genes. We have optimized the transformation protocol and direct plantlet regeneration protocol from leaf explants using 1 mg/L benzylaminopurine as well as 0.25 mg/L benzylaminopurine and 0.1 mg/L indole-butyric acid. Consequently, transgenic sugar beet lines transformed with vector constructs pRD400-cry1C and pRD400-cry2A have been obtained. PCR analysis revealed integration of cry2A and cry1C into the genome of transgenic lines and expression of these genes in leaf tissues was shown by reverse transcription PCR. 2014 Article Получение трансгенных линий сахарной свеклы, экспрессирующих гены устойчивости к насекомым-вредителям сry1C и сry2A / Д.И. Литвин, В.В. Сивура, В.В. Курило, В.Д. Оленева, А.И. Емец, Я.Б. Блюм // Цитология и генетика. — 2014. — Т. 48, № 2. — С. 3-11. — Бібліогр.: 45 назв. — рос. 0564-3783 DOI: 10.3103/S0095452714020078 http://dspace.nbuv.gov.ua/handle/123456789/126620 62.37:633.6:632.7 ru Цитология и генетика Інститут клітинної біології та генетичної інженерії НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
Russian |
| topic |
Оригинальные работы Оригинальные работы |
| spellingShingle |
Оригинальные работы Оригинальные работы Литвин, Д.И. Сивура, В.В. Курило, В.В. Оленева, В.Д. Емец, А.И. Блюм, Я.Б. Получение трансгенных линий сахарной свеклы, экспрессирующих гены устойчивости к насекомым-вредителям сry1C и сry2A Цитология и генетика |
| description |
Существенным ограничением урожайности сахарной свеклы является пагубное воздействие на нее насекомых-вредителей. Одной из перспективных стратегий обеспечения устойчивости растений выступает интеграция в растительный геном генов сry-белков Bacillus thuringiensis. Целью настоящей работы была генетическая трансформация сахарной свеклы линии ММ1/2 векторными конструкциями pRD400-cry1C и pRD400-cry2A, несущими гены cry1C и cry2A соответственно. В результате оптимизации протокола трансформации и прямой регенерации из листовых дисков с использованием 1 мг/л бензиламинопурина, а также 0,25 мг/л бензиламинопурина и 0,1 мг/л индолил-масляной кислоты получены трансгенные линии сахарной свеклы, несущие гены cry1C и cry2A. С помощью ПЦР-анализа подтверждена интеграция cry2A и cry1C в их геном, а с помощью метода ПЦР с обратной транс-крипцией установлено наличие экспрессии указанных генов в этих линиях. |
| format |
Article |
| author |
Литвин, Д.И. Сивура, В.В. Курило, В.В. Оленева, В.Д. Емец, А.И. Блюм, Я.Б. |
| author_facet |
Литвин, Д.И. Сивура, В.В. Курило, В.В. Оленева, В.Д. Емец, А.И. Блюм, Я.Б. |
| author_sort |
Литвин, Д.И. |
| title |
Получение трансгенных линий сахарной свеклы, экспрессирующих гены устойчивости к насекомым-вредителям сry1C и сry2A |
| title_short |
Получение трансгенных линий сахарной свеклы, экспрессирующих гены устойчивости к насекомым-вредителям сry1C и сry2A |
| title_full |
Получение трансгенных линий сахарной свеклы, экспрессирующих гены устойчивости к насекомым-вредителям сry1C и сry2A |
| title_fullStr |
Получение трансгенных линий сахарной свеклы, экспрессирующих гены устойчивости к насекомым-вредителям сry1C и сry2A |
| title_full_unstemmed |
Получение трансгенных линий сахарной свеклы, экспрессирующих гены устойчивости к насекомым-вредителям сry1C и сry2A |
| title_sort |
получение трансгенных линий сахарной свеклы, экспрессирующих гены устойчивости к насекомым-вредителям сry1c и сry2a |
| publisher |
Інститут клітинної біології та генетичної інженерії НАН України |
| publishDate |
2014 |
| topic_facet |
Оригинальные работы |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/126620 |
| citation_txt |
Получение трансгенных линий сахарной свеклы, экспрессирующих гены устойчивости к насекомым-вредителям сry1C и сry2A / Д.И. Литвин, В.В. Сивура, В.В. Курило, В.Д. Оленева, А.И. Емец, Я.Б. Блюм // Цитология и генетика. — 2014. — Т. 48, № 2. — С. 3-11. — Бібліогр.: 45 назв. — рос. |
| series |
Цитология и генетика |
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3ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 2
ОРИГИНАЛЬНЫЕ РАБОТЫ
УДК 62.37:633.6:632.7
Д.И. ЛИТВИН1, В.В. СИВУРА1, В.В. КУРИЛО1,
В.Д. ОЛЕНЕВА2, А.И. ЕМЕЦ1, Я.Б. БЛЮМ1
1 ДУ «Институт пищевой биотехнологии и геномики Национальной академии наук Украины», Киев
2 Киевский национальный университет им. Тараса Шевченко
Е-mail: ldmi@ukr.net
ПОЛУЧЕНИЕ ТРАНСГЕННЫХ ЛИНИЙ САХАРНОЙ СВЕКЛЫ, ЭКСПРЕССИРУЮЩИХ
ГЕНЫ УСТОЙЧИВОСТИ (cry1C И cry2A) К НАСЕКОМЫМ-ВРЕДИТЕЛЯМ
© Ä.È. ËÈÒÂÈÍ, Â.Â. ÑÈÂÓÐÀ, Â.Â. ÊÓÐÈËÎ,
Â.Ä. ÎËÅÍÅÂÀ, À.È. ÅÌÅÖ, ß.Á. ÁËÞÌ, 2014
Ñóùåñòâåííûì îãðàíè÷åíèåì óðîæàéíîñòè ñàõàðíîé
ñâåêëû ÿâëÿåòñÿ ïàãóáíîå âîçäåéñòâèå íà íåå íàñåêî-
ìûõ-âðåäèòåëåé. Îäíîé èç ïåðñïåêòèâíûõ ñòðàòåãèé
îáåñïå÷åíèÿ óñòîé÷èâîñòè ðàñòåíèé âûñòóïàåò èíòåã-
ðàöèÿ â ðàñòèòåëüíûé ãåíîì ãåíîâ ñry-áåëêîâ Bacil-
lus thuringiensis. Öåëüþ íàñòîÿùåé ðàáîòû áûëà ãåíå-
òè÷åñêàÿ òðàíñôîðìàöèÿ ñàõàðíîé ñâåêëû ëèíèè ÌÌ1/2
âåêòîðíûìè êîíñòðóêöèÿìè pRD400-cry1C è pRD400-
cry2A, íåñóùèìè ãåíû cry1C è cry2A ñîîòâåòñòâåííî.
 ðåçóëüòàòå îïòèìèçàöèè ïðîòîêîëà òðàíñôîðìà-
öèè è ïðÿìîé ðåãåíåðàöèè èç ëèñòîâûõ äèñêîâ ñ èñ-
ïîëüçîâàíèåì 1 ìã/ë áåíçèëàìèíîïóðèíà, à òàêæå
0,25 ìã/ë áåíçèëàìèíîïóðèíà è 0,1 ìã/ë èíäîëèë-ìàñ-
ëÿíîé êèñëîòû ïîëó÷åíû òðàíñãåííûå ëèíèè ñàõàðíîé
ñâåêëû, íåñóùèå ãåíû cry1C è cry2A. Ñ ïîìîùüþ ÏÖÐ-
àíàëèçà ïîäòâåðæäåíà èíòåãðàöèÿ cry2A è cry1C â èõ
ãåíîì, à ñ ïîìîùüþ ìåòîäà ÏÖÐ ñ îáðàòíîé òðàíñ-
êðèïöèåé óñòàíîâëåíî íàëè÷èå ýêñïðåññèè óêàçàííûõ
ãåíîâ â ýòèõ ëèíèÿõ.
Êëþ÷åâûå ñëîâà: Beta vulgaris, cry-ãåíû, ãåíåòè÷åñêàÿ
òðàíñôîðìàöèÿ.
Ââåäåíèå. Ñðåäè âîçäåëûâàåìûõ êóëüòóð ñàõàð-
íàÿ ñâåêëà (Beta vulgaris L.) íàðÿäó ñ ñàõàðíûì
òðîñòíèêîì çàíèìàåò êëþ÷åâîå ìåñòî ïðè ïðî-
èçâîäñòâå ñàõàðà, ÿâëÿÿñü ñûðüåì äëÿ 70 % åãî
ìèðîâîãî ïðîèçâîäñòâà. Ýòà äâóõëåòíÿÿ êóëü-
òóðà ïðèãîäíà äëÿ âîçäåëûâàíèÿ â óìåðåííîì
êëèìàòå, è íà ñåãîäíÿøíèé äåíü îêîëî 40 ãî-
ñóäàðñòâ âîâëå÷åíû â åå êîììåð÷åñêîå âûðà-
ùèâàíèå [1]. Êðîìå òîãî, áëàãîäàðÿ âûñîêèì
ïîêàçàòåëÿì ïðèðîñòà áèîìàññû ñàõàðíàÿ ñâåê-
ëà ðàññìàòðèâàåòñÿ â êà÷åñòâå ïåðñïåêòèâíîãî
èñòî÷íèêà ñèíòåçà è íàêîïëåíèÿ íîâûõ ìåòàáî-
ëèòîâ â êîðíÿõ [2]. Áîëåå òîãî, ââèäó âûñîêîé
ýíåðãåòè÷åñêîé öåííîñòè ïðîìåæóòî÷íûõ ïðî-
äóêòîâ ïåðåðàáîòêè ñàõàðíîé ñâåêëû åå ìîæ-
íî èñïîëüçîâàòü êàê ýôôåêòèâíîå ñûðüå äëÿ
ïðîèçâîäñòâà áèîýòàíîëà [3–5]. Îäíàêî áèîëî-
ãè÷åñêèå îñîáåííîñòè ñàõàðíîé ñâåêëû, à èìåí-
íî ïåðåêðåñòíîå îïûëåíèå è äâóõëåòíèé öèêë
ðàçâèòèÿ, â ñî÷åòàíèè ñ âûñîêèì óðîâíåì ãåòå-
ðîçèãîòíîñòè äåëàþò äîëãîâðåìåííûì ïðîöåññ
ïîëó÷åíèÿ íîâûõ ñîðòîâ ñ ïîìîùüþ ìåòîäîâ
êëàññè÷åñêîé ñåëåêöèè. Ê òîìó æå çíà÷èòåëü-
íûé óðîí ýòîé êóëüòóðå íàíîñÿò íåáëàãîïðèÿò-
íûå âîçäåéñòâèÿ îêðóæàþùåé ñðåäû, ïîðàæå-
íèå ðàçëè÷íûìè áîëåçíÿìè è âðåäèòåëÿìè. Íà
ñåãîäíÿøíèé äåíü èçâåñòíî áîëåå 250 âðåäè-
òåëåé ñàõàðíîé ñâåêëû. Ñðåäè íèõ íàèáîëåå
ðàñïðîñòðàíåíû è âðåäîíîñíû ìèíèðóþùàÿ ìó-
õà, ëóãîâîé ìîòûëåê, ñâåêëîâè÷íûé äîëãîíîñèê,
ãóñåíèöû ñîâîê, ìèíèðóþùàÿ ìîëü, à òàêæå ðÿä
êëåùåé è íåìàòîä. Â îáùåìèðîâûõ ìàñøòàáàõ
ïîòåíöèàëüíûé óðîí, íàíîñèìûé íàñåêîìûìè-
âðåäèòåëÿìè, ìîæåò äîñòèãàòü 80 % óðîæàÿ [6].
 ñâÿçè ñ ýòèì, à òàêæå ââèäó çíà÷èòåëüíûõ ìà-
òåðèàëüíûõ çàòðàò, ñâÿçàííûõ ñ èñïîëüçîâàíè-
åì èíñåêòèöèäîâ, êðàéíå öåëåñîîáðàçíî ñîç-
äàíèå ñîðòîâ è ëèíèé ñàõàðíîé ñâåêëû, óñòîé-
÷èâûõ ê ïîðàæåíèþ âðåäèòåëÿìè. Îäíîé èç
4
Ä.È. Ëèòâèí, Â.Â. Ñèâóðà, Â.Â. Êóðèëî è äð.
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 2
íàèáîëåå ýôôåêòèâíûõ ñòðàòåãèé áîðüáû ñ
íàñåêîìûìè-âðåäèòåëÿìè ÿâëÿåòñÿ èñïîëüçî-
âàíèå òîêñè÷åñêèõ ñâîéñòâ ñry-áåëêîâ ãðàì-
ïîëîæèòåëüíîé ïàòîãåííîé áàêòåðèè Bacillus
thuringiensis. Cry-áåëêè (èëè Bt-áåëêè) ðàçëè÷-
íûõ ñåìåéñòâ ïðîÿâëÿþò âûñîêîñïåöèôè÷åñ-
êóþ òîêñè÷íîñòü ê ïðåäñòàâèòåëÿì îïðåäåëåí-
íûõ îòðÿäîâ íàñåêîìûõ. Òàê, íàïðèìåð, ãåíû
cry1 è cry9 êîäèðóþò áåëêè, òîêñè÷íûå äëÿ
÷åøóåêðûëûõ (Lepidoptera), cry2 – äëÿ ÷åøóå-
êðûëûõ è äâóêðûëûõ (Diptera), cry3 – äëÿ
æåñòêîêðûëûõ (Coleoptera), cry4 è cry11 – äëÿ
äâóêðûëûõ [7, 8]. Òàêèì îáðàçîì, ñîçäàíèå
è èñïîëüçîâàíèå ãåíåòè÷åñêè ìîäèôèöèðîâàí-
íûõ ñîðòîâ ðàñòåíèé, ýêñïðåññèðóþùèõ îïðå-
äåëåííûå òîêñè÷íûå áåëêè ýòîé ãðóïïû, ïðèç-
âàíî ïîâûñèòü óðîæàéíîñòü ñåëüñêîõîçÿéñò-
âåííûõ êóëüòóð, à òàêæå óìåíüøèòü íåãàòèâ-
íûå ýêîëîãè÷åñêèå ïîñëåäñòâèÿ, ñâÿçàííûå ñ
èñïîëüçîâàíèåì èíñåêòèöèäîâ [9–12]. Ìåõà-
íèçì öèòîòîêñè÷åñêîãî äåéñòâèÿ ñry-áåëêîâ
ðåàëèçóåòñÿ â êèøå÷íèêå íàñåêîìûõ ïîñëå ïðî-
òåîëèçà ïðîòîòîêñèíîâ è îáðàçîâàíèÿ àêòèâ-
íûõ -ýíäîòîêñèíîâ. Äåéñòâèå -ýíäîòîêñèíà
ïîñëå ñâÿçûâàíèÿ ñî ñïåöèôè÷åñêèì ìåìáðàí-
íûì ðåöåïòîðîì ïðèâîäèò ê ïåðôîðèðîâàíèþ
êëåòîê ýïèòåëèÿ êèøå÷íèêà è ãèáåëè íàñåêî-
ìîãî. Ñëåäóåò òàêæå îòìåòèòü íåòîêñè÷íîñòü
cry-áåëêîâ ïî îòíîøåíèþ ê êëåòêàì ÷åëîâåêà
è äðóãèõ ïîçâîíî÷íûõ, à òàêæå ê êëåòêàì ðàñ-
òåíèé [13].
Øèðîêîìó âíåäðåíèþ ãåííî-èíæåíåðíûõ
ïîäõîäîâ ê ñîçäàíèþ ñîðòîâ è ëèíèé ñàõàðíîé
ñâåêëû, îáëàäàþùèõ óñòîé÷èâîñòüþ ê âðåäè-
òåëÿì, ïðåïÿòñòâóåò òðóäîåìêîñòü ýòîé êóëü-
òóðû â óñëîâèÿõ in vitro. Íåñìîòðÿ íà ìíîãî-
÷èñëåííûå äàííûå, äåìîíñòðèðóþùèå ìîðôî-
ãåíåòè÷åñêóþ àêòèâíîñòü èçîëèðîâàííûõ òêà-
íåé è îðãàíîâ ñàõàðíîé ñâåêëû, åå ðåãåíåðà-
öèîííûé ïîòåíöèàë â çíà÷èòåëüíîé ñòåïåíè
çàâèñèò îò ãåíîòèïà è ñîðòà èñõîäíîãî ðàñ-
òåíèÿ. Òàê, íàèëó÷øèå ïîêàçàòåëè ðåãåíåðà-
öèè äåìîíñòðèðóþò òåòðàïëîèäíûå ôîðìû ñà-
õàðíîé ñâåêëû, òðèïëîèäíûå æå è äèïëîèäíûå
õàðàêòåðèçóþòñÿ ìåíüøèìè è íàèìåíüøèìè
ðåçóëüòàòàìè ñîîòâåòñòâåííî. Íà ðåãåíåðàöèîí-
íóþ ñïîñîáíîñòü êóëüòóðû âëèÿþò êàê òèï è
âîçðàñò èñõîäíîé òêàíè, òàê è óñëîâèÿ êóëü-
òèâèðîâàíèÿ in vitro, âêëþ÷àÿ íàëè÷èå è ñîîò-
íîøåíèå òåõ èëè èíûõ ðåãóëÿòîðîâ ðîñòà [14–
18]. Òàêèì îáðàçîì, áèîòåõíîëîãè÷åñêîå óñî-
âåðøåíñòâîâàíèå B. vulgaris òðåáóåò äàëüíåéøåé
ðàáîòû ïî îïòèìèçàöèè ìåòîäîâ åå ìèêðîêëî-
íàëüíîãî ðàçìíîæåíèÿ è ýôôåêòèâíîé ðåãåíå-
ðàöèè ðàñòåíèé â êóëüòóðå in vitro.
Ðàçðàáîòêà ìåòîäè÷åñêèõ ïîäõîäîâ ãåíå-
òè÷åñêîé òðàíñôîðìàöèè ñàõàðíîé ñâåêëû âå-
äåòñÿ â íåñêîëüêèõ íàïðàâëåíèÿõ. Íàèáîëåå
òðàäèöèîííûìè è ðàñïðîñòðàíåííûìè ÿâëÿ-
þòñÿ ìåòîäû, îñíîâàííûå íà àãðîáàêòåðèàëüíîé
òðàíñôîðìàöèè ñ èñïîëüçîâàíèåì Agrobacterium
tumefaciens è A. rhizogenes [19–21]. Îäíàêî íå-
ñìîòðÿ íà âåñüìà âûñîêóþ ÷óâñòâèòåëüíîñòü
ê èíôåêöèè Agrobacterium, ïîëó÷åíèå òðàíñãåí-
íûõ ðåãåíåðàíòîâ ñàõàðíîé ñâåêëû â áîëüøîé
ñòåïåíè çàâèñèò îò ãåíîòèïà ðàñòåíèÿ è øòàììà
áàêòåðèè [19]. Íà ñåãîäíÿøíèé äåíü ðàçðàáîòà-
íû îïòèìàëüíûå ïðîòîêîëû àãðîáàêòåðèàëüíîé
òðàíñôîðìàöèè B. vulgaris ñ ó÷åòîì ñîðòîâûõ
îñîáåííîñòåé ýòîé êóëüòóðû [22–26]. Äðóãèì
ñîâðåìåííûì ïîäõîäîì ïðè ïîëó÷åíèè òðàíñãåí-
íûõ ëèíèé ñàõàðíîé ñâåêëû ÿâëÿåòñÿ èñïîëü-
çîâàíèå áèîëèñòè÷åñêîé áîìáàðäèðîâêè êëåòîê
èëè òêàíåé ìèêðî÷àñòèöàìè ñ èñïîëüçîâàíè-
åì êîòîðîãî óäàëîñü äîñòè÷ü ïîëîæèòåëüíûõ
ðåçóëüòàòîâ ïðè òðàíñôîðìàöèè ñóñïåíçèîííîé
êóëüòóðû, ýìáðèîãåííîãî êàëëóñà, ïîëó÷åííîãî
èç ãèïîêîòèëÿ, à òàêæå òðàíñôîðìàöèè õëîðî-
ïëàñòîâ [21, 27, 28]. Â òî æå âðåìÿ íåñìîòðÿ
íà çíà÷èòåëüíóþ àêòóàëüíîñòü ïîëó÷åíèÿ ëè-
íèé ñàõàðíîé ñâåêëû ñ óñòîé÷èâîñòüþ ê âðå-
äèòåëÿì, êîòîðàÿ îáóñëîâëåíà ïðîäóêòàìè cry-
ãåíîâ, ìèðîâîé îïûò â ýòîé îáëàñòè íåâåëèê.
Ñðåäè íåìíîãèõ ðàáîò îïèñàíî ïîëó÷åíèå ëè-
íèé ñàõàðíîé ñâåêëû, òðàíñôîðìèðîâàííûõ ãå-
íîì cry1Ab [29], à òàêæå ëèíèé ðàñòåíèé, ýêñ-
ïðåññèðóþùèõ ãåíû cry1Ab è cry1C, êîòîðûå
îáëàäàþò óñòîé÷èâîñòüþ ê ÷åøóåêðûëûì [30].
Ïîýòîìó öåëüþ íàñòîÿùåãî èññëåäîâàíèÿ ñòàëà
îïòèìèçàöèÿ ïðîòîêîëîâ ðåãåíåðàöèè ñàõàðíîé
ñâåêëû è ïîëó÷åíèå òðàíñãåííûõ ëèíèé ýòîé
êóëüòóðû, ýêñïðåññèðóþùèõ ãåíû óñòîé÷èâîñ-
òè cry2A è cry1C.
Ìàòåðèàëû è ìåòîäû. Ðàñòèòåëüíûé ìàòå-
ðèàë. Èñõîäíûì ìàòåðèàëîì â ýêñïåðèìåíòàõ ïî
îïòèìèçàöèè ïðîòîêîëîâ ðåãåíåðàöèè è àãðî-
áàêòåðèàëüíîé òðàíñôîðìàöèè ñëóæèëà ðîäè-
òåëüñêàÿ ñåëåêöèîííàÿ ëèíèÿ ÌÌ1/2 (ñåëåê-
öèîííûé îïûëèòåëü ïðè ãåòåðîçèñíîé ñåëåê-
öèè) ñàõàðíîé ñâåêëû (Beta vulgaris L.), ëþáåçíî
5
Ïîëó÷åíèå òðàíñãåííûõ ëèíèé ñàõàðíîé ñâåêëû, ýêñïðåññèðóþùèõ ãåíû óñòîé÷èâîñòè
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 2 5
ïðåäîñòàâëåííàÿ Èíñòèòóòîì áèîýíåðãåòè÷åñêèõ
êóëüòóð è ñàõàðíîé ñâåêëû ÍÀÀÍ Óêðàèíû.
Òðàíñôîðìàöèÿ è ðåãåíåðàöèÿ in vitro. Äëÿ
êóëüòèâèðîâàíèÿ è ðåãåíåðàöèè ðàñòåíèé â êà-
÷åñòâå áàçîâîé èñïîëüçîâàëè àãàðèçîâàííóþ
ñðåäó MS [32], äëÿ ðåãåíåðàöèè ïîáåãîâ – ìî-
äèôèêàöèþ ýòîé ñðåäû, ñîäåðæàùóþ 1 ìã/ë
áåíçèëàìèíîïóðèíà (ÁÀÏ). Ðåãåíåðàöèþ èíäó-
öèðîâàëè, ïîìåùàÿ ýêñïëàíòû (ëèñòîâûå äèñ-
êè äèàìåòðîì 10–15 ìì) íà ðåãåíåðàöèîííóþ
ñðåäó è êóëüòèâèðóÿ ïðè ðàññåÿííîì îñâåùå-
íèè ñ 16-÷àñîâûì ñâåòîâûì èíòåðâàëîì. Êîðíå-
îáðàçîâàíèå ðåãåíåðèðîâàâøèõ ïîáåãîâ èíäó-
öèðîâàëè ïîñëåäóþùèì èõ ïåðåíåñåíèåì íà ñðå-
äó MS, ñîäåðæàùóþ 0,5 ìã/ë -íàôòèëóêñóñ-
íîé êèñëîòû (ÍÓÊ).
Äëÿ àãðîáàêòåðèàëüíîé òðàíñôîðìàöèè èñ-
ïîëüçîâàëè øòàìì Agrobacterium tumefaciens
LB 4440, à òàêæå âåêòîðíûå êîíñòðóêöèè
pRD400-cry1C è pRD400-cry2A, ñîäåðæàùèå
ãåíû cry1C è cry2A ñîîòâåòñòâåííî, ëþáåçíî
ïðåäîñòàâëåííûå ïðîô. È. Àëòîñààðîì (Óíè-
âåðñèòåò Îòòàâû, Êàíàäà) (ðèñ. 1).
Àãðîáàêòåðèàëüíóþ òðàíñôîðìàöèþ ïðîâî-
äèëè ñîãëàñíî ìåòîäèêå Norouzi et al. [24] ñ
íåêîòîðûìè ìîäèôèêàöèÿìè.  êà÷åñòâå ýêñ-
ïëàíòîâ èñïîëüçîâàëè ëèñòîâûå äèñêè äèàìåò-
ðîì 10 ìì. Øòàììû A. tumefaciens LB 4404,
ñîäåðæàùèå áèíàðíûå êîíñòðóêöèè, âûðàùè-
âàëè â æèäêîé ñðåäå LB, ñîäåðæàùåé 50 ìã/ë
êàíàìèöèíà. Êóëüòóðó àãðîáàêòåðèé, äîñòèã-
øóþ îïòè÷åñêîé ïëîòíîñòè (OD600) – 0,5, îñàæ-
äàëè öåíòðèôóãèðîâàíèåì è ðåñóñïåíäèðîâàëè
â ñðåäå MS, ñîäåðæàùåé 50 ìÌ àöåòîñåðèí-
ãîíà («Sigma-Aldrich», ÑØÀ), ñ ïîñëåäóþùåé
èíêóáàöèåé â òå÷åíèå 5 ÷. Ïåðåä òðàíñôîðìà-
öèåé äëÿ óñïåøíîãî èíôèöèðîâàíèÿ íà ýêñ-
ïëàíòàõ äåëàëè íàäðåçû è èíêóáèðîâàëè ñ ñóñ-
ïåíçèåé àãðîáàêòåðèé â òå÷åíèå 5 ìèí, ïåðèî-
äè÷åñêè ïîìåøèâàÿ.  äàëüíåéøåì ñ ýêñïëàí-
òîâ óäàëÿëè îñòàòêè áàêòåðèàëüíîé ñóñïåíçèè
è êóëüòèâèðîâàëè 3 ñóò íà àãàðèçîâàííîé
ñðåäå MS. Çàòåì îáðàçöû îòìûâàëè äâàæäû
ïî 30 ìèí â ñòåðèëüíîé âîäå, ñîäåðæàùåé
500 ìã/ë öåôîòàêñèìà, è âûñàæèâàëè íà ðåãå-
íåðàöèîííóþ ñðåäó, ñîäåðæàùóþ ñåëåêòèâíûé
àãåíò êàíàìèöèí â êîíöåíòðàöèè 200 ìã/ë, à
òàêæå öåôîòàêñèì â êîíöåíòðàöèè 300 ìã/ë
äëÿ ýëèìèíàöèè èçëèøêà àãðîáàêòåðèé. Ñïóñ-
òÿ 2 íåäåëè ýêñïëàíòû ïåðåíîñèëè íà ñâåæóþ
ïèòàòåëüíóþ ñðåäó. Ïîñëåäóþùèå ïàññàæè ñ
äâóõíåäåëüíûì èíòåðâàëîì îñóùåñòâëÿëè íà
ñðåäû ñî ñíèæåííûìè êîíöåíòðàöèÿìè àíòè-
áèîòèêîâ (100 è 250 ìã/ë äëÿ êàíàìèöèíà è
öåôîòàêñèìà ñîîòâåòñòâåííî).
Ìîëåêóëÿðíî-áèîëîãè÷åñêèé àíàëèç. Âûäåëå-
íèå ÄÍÊ è ÏÖÐ-àíàëèç òðàíñôîðìàíòîâ ïðî-
âîäèëè ñ ïîìîùüþ íàáîðà Thermo Scientific
Phire Plant Direct PCR Kit # F-130 («Thermo
Fisher Scientific Inc.», ÑØÀ) ñîãëàñíî ðåêîìåí-
äàöèÿì ïðîèçâîäèòåëÿ. Äëÿ ýòîãî èñïîëüçîâà-
ëè ïàðû ïðàéìåðîâ 5 -GGTTGTAGGAGTTG-
CCGTTGC-3 (R) è 3 -CGCAGTTCCAGATCC-
AGGGCTA-3 (F) äëÿ ãåíà cry2A è 5 -AACC-
TGTGGGAGAATCCTTGCCTG-3 (R) è 3 -TC-
TATGGAACTATGGGAAACGCCGCTC-3 (F)
äëÿ cry1C ñ àìïëèôèêàöèåé ÏÖÐ-ïðîäóêòîâ
ðàçìåðîì 1149 è 280 ï.í. ñîîòâåòñòâåííî. Ðå-
àêöèþ ïðîâîäèëè íà àìïëèôèêàòîðå PCR Ap-
plyed Biosystem 2720 (ÑØÀ) â òå÷åíèå 40 öèêëîâ
ïðè ñëåäóþùèõ óñëîâèÿõ: äåíàòóðàöèÿ – 98 ºÑ
(5 ñ), îòæèã – 66 ºÑ äëÿ cry1C è 62 ºÑ äëÿ cry2A
(5 ñ), ñèíòåç – 72 ºÑ (20 ñ), ôèíàëüíàÿ ýëîíãà-
öèÿ – 72 ºÑ (5 ìèí).
ÐÍÊ äëÿ ÏÖÐ ñ îáðàòíîé òðàíñêðèïöèåé
âûäåëÿëè ïðè ïîìîùè ðåàãåíòà TRIzol Reagent
(«Life Technologies Co.», ÑØÀ). Ïðîöåäóðó îá-
ðàòíîé òðàíñêðèïöèè ïðîâîäèëè ñ èñïîëüçî-
âàíèåì íàáîðà Thermo Scientific RevertAid Re-
verse Transcriptase («Thermo Fisher Scientific Inc.»,
ÑØÀ) ñîãëàñíî ïðîòîêîëó è ðåêîìåíäàöèÿì
ïðîèçâîäèòåëÿ. Ñèíòåç êÄÍÊ îñóùåñòâëÿëè
íà ìàòðèöå òîòàëüíîé ÐÍÊ (0,5 ìêã) â òå÷åíèå
1 ÷ ïðè 42 ºÑ â 20 ìêë ðåàêöèîííîé ñìåñè,
ñîäåðæàùåé 20 ïìîëü ãåíîñïåöèôè÷åñêèõ ïðàé-
Ðèñ. 1. Ñõåìû ãåíåòè÷åñêèõ êîíñòðóêöèé íà îñíîâå
áèíàðíîãî âåêòîðà pRD-400: d35S – óñèëåííûé â äâà
ðàçà 35S ïðîìîòîð âèðóñà ìîçàèêè öâåòíîé êàïóñòû,
AMV – óñèëèòåëü òðàíñëÿöèè âèðóñà ìîçàèêè ëþ-
öåðíû, Nos-Ter – òåðìèíàòîð NOS
6
Ä.È. Ëèòâèí, Â.Â. Ñèâóðà, Â.Â. Êóðèëî è äð.
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 2
ìåðîâ (5 -GGTTGTAGGAGTTGCCGTTGC-3 è
5 -AACCTGTGGGAGAATCCTTGCCTG-3 äëÿ
cry2À è cry1Ñ ñîîòâåòñòâåííî) è 200 åä. îáðàò-
íîé òðàíñêðèïòàçû. Ïî îêîí÷àíèè ðåàêöèè îá-
ðàçöû èíêóáèðîâàëè 10 ìèí ïðè 70 ºÑ äëÿ
èíàêòèâèðîâàíèÿ òðàíñêðèïòàçû. Äëÿ ýëåêòðî-
ôîðåòè÷åñêîãî ðàçäåëåíèÿ ïðîäóêòîâ ÏÖÐ èñ-
ïîëüçîâàëè 2%-íûé àãàðîçíûé ãåëü íà îñíîâå
1× òðèñ-áîðàòíîãî áóôåðà.
Ðåçóëüòàòû èññëåäîâàíèé è èõ îáñóæäåíèå.
Ïîäáîð óñëîâèé äëÿ ðåãåíåðàöèè è ãåíåòè÷åñêàÿ
òðàíñôîðìàöèÿ. Îäíîé èç ãëàâíûõ ïðåäïîñû-
ëîê ïîëó÷åíèÿ òðàíñãåííûõ êóëüòóð â óñëîâè-
ÿõ in vitro ÿâëÿåòñÿ ýôôåêòèâíàÿ è âîñïðîèçâî-
äèìàÿ ïðîöåäóðà ðåãåíåðàöèè òðàíñôîðìàíòîâ
èç ýêñïëàíòîâ èñõîäíîé òêàíè. Ââèäó ñëîæíîñ-
òåé, ñâÿçàííûõ ñ îïòèìèçàöèåé ðåãåíåðàöèè ñà-
õàðíîé ñâåêëû, êîòîðûå ìîãóò áûòü îáóñëîâ-
ëåíû ñîðòîâûìè è ãåíîòèïè÷åñêèìè îñîáåí-
íîñòÿìè, ïðåäâàðèòåëüíî ïðîâåäåíà ðàáîòà ïî
ïîäáîðó óñëîâèé ðåãåíåðàöèè ðàñòåíèé èç ëèñ-
òîâûõ äèñêîâ ëèíèè ÌÌ1/2 B. vulgaris. Èñ-
ïîëüçîâàíèå êóëüòóðàëüíîé ñðåäû, ñîäåðæàùåé
ÁÀÏ â êîíöåíòðàöèè 1 ìã/ë, îáåñïå÷èëî îïòè-
ìàëüíûå ïîêàçàòåëè ðåãåíåðàöèè ðàñòåíèé óêà-
çàííîé ñåëåêöèîííîé ëèíèè. Ðåãåíåðàöèÿ ðàñ-
òåíèé (ïðîöåíòíîå ñîîòíîøåíèå êîëè÷åñòâà
ïîëó÷åííûõ ðåãåíåðàíòîâ ê îáùåìó ÷èñëó âû-
ñàæåííûõ ýêñïëàíòîâ ÷åðåç 10–14 ñóò ïîñëå
âûñàäêè) íà ýòîé ñðåäå ñîñòàâëÿëà ïðèìåðíî
60 %. Ñðàâíèòåëüíûå èññëåäîâàíèÿ ïîêàçàëè,
÷òî ó ñàõàðíîé ñâåêëû ëèñòîâûå äèñêè ÿâëÿ-
þòñÿ íàèáîëåå ïðèåìëåìûìè ýêñïëàíòàìè äëÿ
èíäóêöèè ïðÿìîé ðåãåíåðàöèè ðàñòåíèé. Îïûò
äðóãèõ ëàáîðàòîðèé äåìîíñòðèðóåò ýôôåêòèâ-
íîå ïðèìåíåíèå öèòîêèíèíîâ êàê èíäóêòîðîâ
ðåãåíåðàöèè ñàõàðíîé ñâåêëû in vitro, â ÷àñò-
íîñòè ÁÀÏ [14, 32–34]. Èñïîëüçîâàíèå ôèòî-
ãîðìîíà â êîíöåíòðàöèÿõ íå íèæå 1 ìã/ë, êàê
ïðàâèëî, ýôôåêòèâíî äëÿ ïðÿìîé ðåãåíåðàöèè
èç ýêñïëàíòîâ ëèñòüåâ, ÷åðåøêîâ, ñòåáëåé, à
òàêæå êàëëóñà. Àíàëîãè÷íûå äàííûå ïîëó÷åíû
íàìè ïðè ðàáîòå ñ ëèíèåé ÌÌ1/2, ãäå äëÿ
èíäóêöèè ïðÿìîé ðåãåíåðàöèè ïîáåãîâ èç ëèñ-
òîâûõ ýêñïëàíòîâ â ïèòàòåëüíûå ñðåäû äîáàâ-
ëÿëè 1 ìã/ë ÁÀÏ. Íåîáõîäèìî îòìåòèòü, ÷òî
âî ìíîãèõ ñëó÷àÿõ ðåçóëüòàòèâíûì òàêæå ÿâëÿ-
åòñÿ êîìáèíèðîâàííîå èñïîëüçîâàíèå öèòîêè-
íèíîâ è àóêñèíîâ, ïðè ýòîì çíà÷èòåëüíî óìåíü-
øàþòñÿ ýôôåêòèâíûå ðàáî÷èå êîíöåíòðàöèè
èñïîëüçóåìûõ ôèòîãîðìîíîâ. Òàê, â îïóáëèêî-
âàííûõ ðàíåå ðàáîòàõ ïðîäåìîíñòðèðîâàíî äîñ-
òàòî÷íî ýôôåêòèâíîå ïðèìåíåíèå êîìáèíà-
öèè ÁÀÏ è ÍÓÊ [35], à òàêæå ÁÀÏ, ÈÓÊ è
2,3,5-òðèéîäáåíçîéíîé êèñëîòû [28].
Agrobacterium-îïîñðåäîâàííóþ òðàíñôîðìà-
öèþ ñàõàðíîé ñâåêëû îñóùåñòâëÿëè ñîãëàñíî
ìåòîäó [23] ñ íåêîòîðûìè âíåñåííûìè ìîäè-
ôèêàöèÿìè, êîòîðûå êàñàëèñü ðàçìåðà è òèïà
Ðèñ. 2. Ýòàïû òðàíñôîðìàöèè è ðåãåíåðàöèè òðàíñ-
ãåííûõ ïîáåãîâ ñàõàðíîé ñâåêëû: à – îáùàÿ ñõåìà
ïîëó÷åíèÿ òðàíñãåííûõ ëèíèé ñàõàðíîé ñâåêëû, òðàíñ-
ôîðìèðîâàííûõ ãåíàìè cry2A è cry1C; á – ðåãå-
íåðàöèÿ ïîáåãà â çîíå öåíòðàëüíîé æèëêè ëèñòîâî-
ãî ýêñïëàíòà, â – 4-íåäåëüíûå òðàíñôîðìèðîâàííûå
ïîáåãè ïåðåä ïåðåñàäêîé íà ñðåäó äëÿ óêîðåíåíèÿ
7
Ïîëó÷åíèå òðàíñãåííûõ ëèíèé ñàõàðíîé ñâåêëû, ýêñïðåññèðóþùèõ ãåíû óñòîé÷èâîñòè
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 2
ýêñïëàíòîâ, êîíöåíòðàöèé ñåëåêòèâíûõ àãåí-
òîâ, à òàêæå äëèòåëüíîñòè êóëüòèâèðîâàíèÿ ìà-
òåðèàëà íà ñåëåêòèâíûõ ñðåäàõ (ðèñ. 2, à). ×å-
ðåç 2 íåäåëè ïîñëå ïåðåñàäêè ýêñïëàíòîâ íà
ðåãåíåðàöèîííóþ ñðåäó, ñîäåðæàùóþ 200 ìã/ë
êàíàìèöèíà è 300 ìã/ë öåôîòàêñèìà, íàáëþäà-
ëè ôîðìèðîâàíèå ðåãåíåðèðóþùèõ ïîáåãîâ íà
èõ ïîâåðõíîñòè.
 áîëüøèíñòâå ñëó÷àåâ îáðàçîâàíèå ðåãåíå-
ðàíòîâ ïðîèñõîäèëî â çîíå ðàíåâûõ ïîâåðõíîñ-
òåé öåíòðàëüíûõ ñîñóäîâ (ðèñ. 2, á), à ÷åðåç 4–
6 íåäåëü ïîñëå òðàíñôîðìàöèè óäàâàëîñü ïîëó-
÷èòü íîðìàëüíî ñôîðìèðîâàííûå ïîáåãè, ãî-
òîâûå ê äàëüíåéøåìó óêîðåíåíèþ (ðèñ. 2, â).
Ýôôåêòèâíîñòü ãåíåòè÷åñêîé òðàíñôîðìàöèè
ãåíàìè cry1C è cry2A îòëè÷àëàñü. Äëÿ óñòàíîâ-
ëåíèÿ ýòîãî ïîêàçàòåëÿ îïðåäåëÿëè ïðîöåíòíîå
ñîîòíîøåíèå êîëè÷åñòâà îòîáðàííûõ ïîñëå òðàíñ-
ôîðìàöèè íà ñåëåêòèâíîé ñðåäå ðåãåíåðàíòîâ ê
îáùåìó ÷èñëó âûñàæåííûõ ýêñïëàíòîâ. Èçó÷å-
íèþ è ñðàâíåíèþ ïîäëåæàëè ñëåäóþùèå ïî-
êàçàòåëè: îáùåå êîëè÷åñòâî ýêñïëàíòîâ; êîëè-
÷åñòâî ýêñïëàíòîâ, ó êîòîðûõ íàáëþäàëè ïðèç-
íàêè ðåãåíåðàöèè ïðè âûðàùèâàíèè èõ â òå÷å-
íèå 4 íåäåëü íà ñåëåêòèâíîé ñðåäå ñ 200 ìã/ë
êàíàìèöèíà; êîëè÷åñòâî ýêñïëàíòîâ, íà êîòî-
ðûõ ïðîèñõîäèëà ðåãåíåðàöèÿ ðàñòåíèé íà ñðå-
äå ñî 100 ìã/ë êàíàìèöèíà ÷åðåç 6 íåäåëü ïîñëå
òðàíñôîðìàöèè. Â ðåçóëüòàòå òðàíñôîðìàöèè
îáåèìè ãåíåòè÷åñêèìè êîíñòðóêöèÿìè áûë ïðî-
äåìîíñòðèðîâàí äîñòàòî÷íî âûñîêèé âûõîä
òðàíñãåííûõ ðàñòåíèé ñ íåáîëüøèì ïðåâîñõîä-
ñòâîì ïîêàçàòåëÿ ýôôåêòèâíîñòè òðàíñôîðìà-
öèè â ñëó÷àå èñïîëüçîâàíèÿ ãåíà cry1C (ðèñ. 3).
Ïîäáîð óñëîâèé äëÿ óêîðåíåíèÿ òðàíñôîðìàí-
òîâ. Çàêëþ÷èòåëüíûì ýòàïîì ÿâëÿëñÿ ïîä-
áîð îïòèìàëüíûõ óñëîâèé äëÿ êîðíåîáðàçîâà-
íèÿ ðàñòåíèé ñàõàðíîé ñâåêëû. Êàê è â ðà-
áîòå Norouzi [24], îïòèìèçàöèþ ñðåäû äëÿ óêî-
ðåíåíèÿ ïðîâîäèëè ñ ïîìîùüþ ïîäáîðà ýôôåê-
òèâíîé êîíöåíòðàöèè ÍÓÊ.  ñëó÷àå ðàáîòû ñ
ëèíèåé ÌÌ1/2 íàèáîëåå ïîäõîäÿùèì îêàçàëîñü
èñïîëüçîâàíèå ÍÓÊ â êîíöåíòðàöèè 0,5 ìã/ë.
Ïðè ýòîì ýôôåêòèâíîñòü êîðíåîáðàçîâàíèÿ ÷å-
ðåç 3–4 íåäåëè ïîñëå âûñàäêè ïîáåãîâ íà óïî-
ìÿíóòóþ ñðåäó ñîñòàâëÿëà 80 % (ðèñ. 4, à).
Ïðèìå÷àòåëüíî, ÷òî â ðàáîòå Bekheet et al.
[35] èñïîëüçîâàíèå ÍÓÊ ïðèâîäèëî ê äîâîëü-
íî ïîñðåäñòâåííûì ðåçóëüòàòàì, â òî âðåìÿ êàê
ýôôåêòèâíîé äëÿ êîðíåîáðàçîâàíèÿ îêàçàëàñü
ñðåäà, ñîäåðæàùàÿ èíäîëèëìàñëÿíóþ êèñëîòó.
Ïîäîáíûå íåñîîòâåòñòâèÿ ýôôåêòèâíîñòè ðàç-
ëè÷íûõ ôèòîãîðìîíîâ ïðè èíäóêöèè ðåãåíå-
ðàöèè è êîðíåîáðàçîâàíèÿ in vitro ëîãè÷íî
îáúÿñíèòü óæå óïîìÿíóòîé ãåíåòè÷åñêîé âàðèà-
áåëüíîñòüþ ñàõàðíîé ñâåêëû. Ââèäó ýòîãî ñëå-
Ðèñ. 3. Ïîêàçàòåëè ýôôåêòèâíîñòè ãåíåòè÷åñêîé
òðàíñôîðìàöèè ñàõàðíîé ñâåêëû êîíñòðóêöèÿìè
pRD400-cry2À (1) è pRD400-cry1Ñ(2): – ïðîöåíò
ýêñïëàíòîâ ñ ïðèçíàêàìè ðåãåíåðàöèè ïîáåãîâ
ñïóñòÿ 4 íåäåëè êóëüòèâèðîâàíèÿ íà ñåëåêòèâíîé
ñðåäå, – ïðîöåíò ýêñïëàíòîâ, íà êîòîðûõ îáðà-
çîâàëèñü ðàñòåíèÿ ñïóñòÿ 6 íåäåëü ïîñëå òðàíñôîð-
ìàöèè è ñåëåêöèè. Óñðåäíåííûé ðåçóëüòàò òðåõ ýêñ-
ïåðèìåíòîâ
Ðèñ. 4. Êîðíåîáðàçîâàíèå ó òðàíñôîðìèðîâàííûõ
ðàñòåíèé ñàõàðíîé ñâåêëû: à – îáðàçîâàíèå êîðíåé
íà ñðåäå, ñîäåðæàùåé 0,5 ìã/ë ÍÓÊ; á – ïîñëåäóþùèé
ìîðôîãåíåç êîðíåé íà áåçãîðìîíàëüíîé ñðåäå MS
8
Ä.È. Ëèòâèí, Â.Â. Ñèâóðà, Â.Â. Êóðèëî è äð.
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 2
äóåò ïðåäïîëîæèòü íåîáõîäèìîñòü àäàïòàöèè
ïðîòîêîëîâ ðåãåíåðàöèè ñàõàðíîé ñâåêëû â
êóëüòóðå in vitro ïðè òðàíñôîðìàöèè ðàçëè÷íûõ
ëèíèé è ñîðòîâ. Êðîìå òîãî, óêîðåíåíèå ðàñ-
òåíèé ïðîèñõîäèëî òàêæå ñïîíòàííî íà áåç-
ãîðìîíàëüíîé ñðåäå MS, îäíàêî ïðîöåíò ðàñ-
òåíèé, îáðàçîâàâøèõ êîðíè, áûë íèçêèì. Ïî
äîñòèæåíèè äëèíû êîðíåé îêîëî 20 ìì ðàñòå-
íèÿ ïåðåíîñèëè íà áåçãîðìîíàëüíóþ ñðåäó MS íà
2 íåäåëè äëÿ ôîðìèðîâàíèÿ è àäàïòàöèè êîð-
íåâîé ñèñòåìû ê îòêðûòîìó ãðóíòó (ðèñ. 4, á).
Ìîëåêóëÿðíî-ãåíåòè÷åñêèé àíàëèç. Ñïîñîá-
íîñòü òðàíñôîðìàíòîâ äëèòåëüíîå âðåìÿ ðàçâè-
âàòüñÿ â ïðèñóòñòâèè ñåëåêòèâíûõ àãåíòîâ íå
ÿâëÿåòñÿ îêîí÷àòåëüíûì äîêàçàòåëüñòâîì òðàíñ-
ãåííîé ïðèðîäû ïîëó÷åííûõ ðàñòåíèé. Äëÿ
óñòàíîâëåíèÿ èíòåãðàöèè ãåíîâ èíòåðåñà â ãå-
íîì ðàñòåíèé íàìè ïðîâåäåí ÏÖÐ-àíàëèç ïî-
ëó÷åííûõ òðàíñôîðìèðîâàííûõ ëèíèé ñàõàð-
íîé ñâåêëû, ðåãåíåðèðîâàâøèõ íà ñåëåêòèâíîé
ñðåäå, ñ èñïîëüçîâàíèåì ñïåöèôè÷åñêèõ ïðàé-
ìåðîâ ê ãåíàì cry1Ñ è cry2A. Â ðåçóëüòàòå óñ-
òàíîâëåíî íàëè÷èå èññëåäóåìûõ ãåíîâ ó òðàíñ-
ôîðìèðîâàííûõ ðàñòåíèé (ðèñ. 5, äàííûå ïðåä-
ñòàâëåíû äëÿ íåêîòîðûõ ëèíèé).
Êðîìå ýòîãî äëÿ ïîäòâåðæäåíèÿ íå òîëüêî
âñòðàèâàíèÿ èññëåäóåìûõ ãåíîâ â ãåíîì ðàñ-
òåíèé, íî è èõ ýôôåêòèâíîé ýêñïðåññèè, ïðî-
âåäåíî âûäåëåíèå ÐÍÊ òðàíñôîðìàíòîâ, ïîëó-
÷åíèå êÄÍÊ â ðåàêöèè îáðàòíîé òðàíñêðèïöèè
è àìïëèôèêàöèÿ ãåíîâ cry1C è cry2A. Â ðåçóëü-
òàòå áûëà ïîäòâåðæäåíà ýêñïðåññèÿ óïîìÿíó-
òûõ ãåíîâ. Ñëåäóåò òàêæå îòìåòèòü, ÷òî óðîâåíü
ýêñïðåññèè ãåíà cry1Ñ îêàçàëñÿ çíà÷èòåëüíî
âûøå, ÷åì ãåíà cry2A (ðèñ. 6).
Òàêèì îáðàçîì, íàìè ïîëó÷åíû òðàíñãåí-
íûå ëèíèè ñàõàðíîé ñâåêëû, ýêñïðåññèðóþ-
ùèå ãåíû cry2A è cry1C, ñ ïîòåíöèàëüíîé óñ-
òîé÷èâîñòüþ ê íàñåêîìûì-âðåäèòåëÿì îòðÿäîâ
Lepidoptera è Diptera. Ê íàñòîÿùåìó âðåìåíè
î÷åâèäíû çàìåòíûå óñïåõè â èñïîëüçîâàíèè
ñâîéñòâ ñry-áåëêîâ äëÿ ïîëó÷åíèÿ óñòîé÷èâûõ
ê âðåäèòåëÿì ðàñòåíèé. Ñðåäè êóëüòóð, òðàíñ-
ôîðìàöèÿ êîòîðûõ ñry-ãåíàìè óñïåøíî çàðå-
êîìåíäîâàëà ñåáÿ, ìîæíî íàçâàòü êàðòîôåëü
[36, 37], òîìàò [38–40], ðèñ [41, 42], êóêóðóçó
[43, 44], áàòàò [45]. Ðàáîò â äàííîé îáëàñòè, ïî-
ñâÿùåííûõ ñàõàðíîé ñâåêëå, íåäîñòàòî÷íî äëÿ
ýôôåêòèâíîãî îáåñïå÷åíèÿ âîçäåëûâàíèÿ êóëü-
òóðû, îòâå÷àþùåãî ñîâðåìåííûì ïîòðåáíîñòÿì
Ðèñ. 5. Ðåçóëüòàòû àíàëèçà òðàíñôîðìèðîâàííûõ
ðàñòåíèé ñàõàðíîé ñâåêëû ñ ïîìîùüþ ÏÖÐ (àìï-
ëèôèêàöèÿ ôðàãìåíòîâ ãåíîâ cry2A è cry1C): 1 –
êîíòðîëü (àìïëèôèêàöèÿ áåç ÄÍÊ-ìàòðèöû ñ ïðàé-
ìåðàìè ê ãåíó cry2A); 2 – àìïëèôèêàöèÿ ãåíà cry2A
â êîíòðîëüíîì íåòðàíñôîðìèðîâàííîì ðàñòåíèè
ëèíèè ÌÌ1/2; 3 – àìïëèôèêàöèÿ ãåíà cry2A ïëàç-
ìèäû pRD400-cry2A; 4 – àìïëèôèêàöèÿ ãåíà
cry2A â ðàñòåíèè, òðàíñôîðìèðîâàííîì ïëàçìèäîé
pRD400-cry2A; 5 – êîíòðîëü (àìïëèôèêàöèÿ áåç
ÄÍÊ-ìàòðèöû ñ ïðàéìåðàìè ê ãåíó cry1C); 6 –
àìïëèôèêàöèÿ ãåíà cry1C â êîíòðîëüíîì íåòðàíñ-
ôîðìèðîâàííîì ðàñòåíèè ëèíèè ÌÌ1/2; 7 – àìïëè-
ôèêàöèÿ ãåíà cry1C ïëàçìèäû pRD400-cry1C; 8 –
àìïëèôèêàöèÿ ãåíà cry1C â ðàñòåíèè, òðàíñôîðìè-
ðîâàííîì ïëàçìèäîé pRD400-cry1C
Ðèñ. 6. Ðåçóëüòàòû ýëåêòðîôîðåòè÷åñêîãî àíàëèçà
ïðîäóêòîâ àìïëèôèêàöèè êÄÍÊ òðàíñôîðìèðîâàí-
íûõ ðàñòåíèé ñàõàðíîé ñâåêëû: 1 – ýêñïðåññèÿ ãåíà
cry2A â êîíòðîëüíîì ðàñòåíèè; 2 – ýêñïðåññèÿ ãåíà
cry2A â òðàíñôîðìèðîâàííîé ëèíèè; 3 – ýêñïðåññèÿ
ãåíà cry1Ñ â êîíòðîëüíîì ðàñòåíèè; 4 – ýêñïðåññèÿ
ãåíà cry1Ñ â òðàíñôîðìèðîâàííîé ëèíèè
9
Ïîëó÷åíèå òðàíñãåííûõ ëèíèé ñàõàðíîé ñâåêëû, ýêñïðåññèðóþùèõ ãåíû óñòîé÷èâîñòè
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2014. Ò. 48. ¹ 2
ê åå âûðàùèâàíèþ. Ïîëó÷åííûå íàìè ëèíèè
ÿâëÿþòñÿ îñíîâîé äëÿ ñîçäàíèÿ íîâûõ, óñòîé-
÷èâûõ ê íàñåêîìûì-âðåäèòåëÿì, ñîðòîâ ñàõàð-
íîé ñâåêëû.
Ðàáîòà âûïîëíåíà ïðè ôèíàíñîâîé ïîääåðæêå
ïðîåêòà «Ñîçäàíèå óñòîé÷èâûõ ê äîëãîíîñèêó ëè-
íèé ñàõàðíîé ñâåêëû ïóòåì èñïîëüçîâàíèÿ ñèí-
òåòè÷åñêèõ Bt ãåíîâ äëÿ ïðîèçâîäñòâà áèîýòà-
íîëà» â ðàìêàõ öåëåâîé êîìïëåêñíîé ïðîãðàììû
íàó÷íûõ èññëåäîâàíèé ÍÀÍ Óêðàèíû «Áèîìàññà
êàê òîïëèâíîå ñûðüå» («Áèîòîïëèâî»), 2010–
2012 ãã.
D.I. Lytvyn, V.V. Syvura, V.V. Kurylo,
V.D. Olenieva, A.I. Yemets, Ya.B. Blume
Institute of Food Biotechnology and Genomics,
National Academy of Sciences of Ukraine, Kyiv
Taras Shevchenko National University of Kyiv, Ukraine
Å-mail: ldmi@ukr.net
CREATION OF TRANSGENIC SUGAR BEET
LINES EXPRESSING INSECT PEST RESISTANCE
GENES cry1C AND cry2A
Impact of insect pests makes a significant limitation of
the sugar beet crop yield. Integration of cry-genes of
Bacillus thuringiensis into plant genome is one of the
promising strategies to ensure plant resistance. The aim of
this work was to obtain sugar beet lines (based on the
MM1/2 line) transformed with cry2A and cry1C genes. We
have optimized transformation protocol and direct plant
let regeneration protocol from leaf explants using 1 mg/l
benzylaminopurine as well as 0,25 mg/l benzylamino-
purine and 0,1 mg/l indole-butyric acid. Consequently,
transgenic sugar beet lines transformed with vector cons-
tructs pRD400-cry1C and pRD400-cry2A have been ob-
tained. PCR analysis revealed integration of cry2A and
cry1C into genome of transgenic lines and expression of
these genes in leaf tissues was shown by reverse transcrip-
tion PCR.
Ä.². Ëèòâèí, Â.Â. Ñèâóðà, Â.Â. Êóðèëî,
Â.Ä. Îëåíºâà, À.². Åìåöü, ß.Á. Áëþì
ÎÒÐÈÌÀÍÍß ÒÐÀÍÑÃÅÍÍÈÕ Ë²Í²É
ÖÓÊÐÎÂÎÃÎ ÁÓÐßÊÓ, ÙÎ ÅÊÑÏÐÅÑÓÞÒÜ
ÃÅÍÈ ÑÒ²ÉÊÎÑÒ² (cry1C ÒÀ cry2A)
ÄÎ ÊÎÌÀÕ-ØʲÄÍÈʲÂ
²ñòîòíèì îáìåæåííÿì âðîæàéíîñò³ öóêðîâèõ áó-
ðÿê³â º çãóáíèé âïëèâ íà öþ êóëüòóðó êîìàõ-øê³äíè-
ê³â. Îäí³ºþ ç ïåðñïåêòèâíèõ ñòðàòåã³é çàáåçïå÷åííÿ
ñò³éêîñò³ ðîñëèí º ³íòåãðàö³ÿ â ðîñëèííèé ãåíîì
ãåí³â ñry-á³ëê³â Bacillus thuringiensis. Ìåòîþ äàíî¿ ðî-
áîòè áóëà ãåíåòè÷íà òðàíñôîðìàö³ÿ öóêðîâîãî áóðÿ-
êó ë³í³¿ ÌÌ1/2 âåêòîðíèìè êîíñòðóêö³ÿìè pRD400-
cry1C ³ pRD400-cry2A, ùî íåñóòü ãåíè cry1C òà cry2A
â³äïîâ³äíî. Ó ðåçóëüòàò³ îïòèì³çàö³¿ ïðîòîêîëó òðàíñ-
ôîðìàö³¿ òà ïðÿìî¿ ðåãåíåðàö³¿ ç ëèñòîâèõ äèñê³â
³ç âèêîðèñòàííÿì 1 ìã/ë áåíçèëàì³íîïóðèíó, à òà-
êîæ 0,25 ìã/ë áåíçèëàì³íîïóðèíó òà 0,1 ìã/ë ³í-
äîë³ë-ìàñëÿíî¿ êèñëîòè îòðèìàíî òðàíñãåíí³ ë³í³¿
öóêðîâîãî áóðÿêó, ùî íåñóòü ãåíè cry1C ³ cry2A. Çà
äîïîìîãîþ ÏËÐ-àíàë³çó ï³äòâåðäæåíî ³íòåãðàö³þ
cry2A ³ cry1C â ¿õí³é ãåíîì, à çà äîïîìîãîþ ÏËÐ
ç³ çâîðîòíîþ òðàíñêðèïö³ºþ âñòàíîâëåíî íàÿâí³ñòü
åêñïðåñ³¿ äàíèõ ãåí³â â öèõ ë³í³ÿõ.
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Ïîñòóïèëà 21.03.13
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/NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.)
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/UKR <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>
/ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.)
>>
/Namespace [
(Adobe)
(Common)
(1.0)
]
/OtherNamespaces [
<<
/AsReaderSpreads false
/CropImagesToFrames true
/ErrorControl /WarnAndContinue
/FlattenerIgnoreSpreadOverrides false
/IncludeGuidesGrids false
/IncludeNonPrinting false
/IncludeSlug false
/Namespace [
(Adobe)
(InDesign)
(4.0)
]
/OmitPlacedBitmaps false
/OmitPlacedEPS false
/OmitPlacedPDF false
/SimulateOverprint /Legacy
>>
<<
/AddBleedMarks false
/AddColorBars false
/AddCropMarks false
/AddPageInfo false
/AddRegMarks false
/ConvertColors /ConvertToCMYK
/DestinationProfileName ()
/DestinationProfileSelector /DocumentCMYK
/Downsample16BitImages true
/FlattenerPreset <<
/PresetSelector /MediumResolution
>>
/FormElements false
/GenerateStructure false
/IncludeBookmarks false
/IncludeHyperlinks false
/IncludeInteractive false
/IncludeLayers false
/IncludeProfiles false
/MultimediaHandling /UseObjectSettings
/Namespace [
(Adobe)
(CreativeSuite)
(2.0)
]
/PDFXOutputIntentProfileSelector /DocumentCMYK
/PreserveEditing true
/UntaggedCMYKHandling /LeaveUntagged
/UntaggedRGBHandling /UseDocumentProfile
/UseDocumentBleed false
>>
]
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice
|