Особенности трансгенных растений салата с геном интерферона-α2b, полученных путем Agrobacterium rhizogenes-опосредованной трансформации
С помощью метода Agrobacterium rhizogenes-опосредованной трансформации получена культура «бородатых» корней салата Lactuca sativa, из которой регенерированы трансгенные растения с геном интерферона α2b человека. По результатам ПЦР и ОТ-ПЦР анализов полученные растения имели транскрибируемый целевой...
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Матвеева, Н.А. Шаховский, А.М. Кучук, Н.В. 2017-11-24T16:03:08Z 2017-11-24T16:03:08Z 2012 Особенности трансгенных растений салата с геном интерферона-α2b, полученных путем Agrobacterium rhizogenes-опосредованной трансформации / Н.А. Матвеева, А.М. Шаховский, Н.В. Кучук // Цитология и генетика. — 2012. — Т. 46, № 3. — С. 27-32. — Бібліогр.: 37 назв. — рос. 0564-3783 DOI: 10.3103/S0095452712030073 https://nasplib.isofts.kiev.ua/handle/123456789/126468 575.222.7:581.1 С помощью метода Agrobacterium rhizogenes-опосредованной трансформации получена культура «бородатых» корней салата Lactuca sativa, из которой регенерированы трансгенные растения с геном интерферона α2b человека. По результатам ПЦР и ОТ-ПЦР анализов полученные растения имели транскрибируемый целевой ген ifn-α2b и отличались от растений дикого типа удлиненными междоузлиями, ранним формированием цветоноса и пурпурной окраской листьев при культивировании в условиях искусственного освещения. За допомогою Agrobacterium rhizogenes-опосередкованої трансформації отримано культуру «бородатих» коренів салату Lactuca sativa, з яких регенеровано трансгенні рослини із геном інтерферону-α2b людини. За результатами ПЛР та зт-ПЛР аналізів отримані рослини мали цільовий ген ifn-α2b, що транскрибувався і відрізнялися від рослин дикого типу подовженими міжвузлями, раннім формуванням квітконосу та пурпурним забарвленням листків при культивуванні в умовах штучного освітлення. «Hairy» roots of lettuce Lactuca sativa and regenerated plants with interferon-α2b gene had been obtained via Agrobacterium rhizogenes-mediated transformation. According to the results of PCR and rt-PCR analyses the studied plants had ifn-α2b gene. The regenerated plants differed from the plants of wild type by elongated internodes, early flower-bearing stem formation and purple coloration of leaves in artificial illumination conditions. ru Інститут клітинної біології та генетичної інженерії НАН України Цитология и генетика Оригинальные работы Особенности трансгенных растений салата с геном интерферона-α2b, полученных путем Agrobacterium rhizogenes-опосредованной трансформации Особливості трансгенних рослин салату з геном інтерферону- α2b, отриманих шляхом Agrobacterium rhizogenesопосередкованої трансформації Features of lettuce transgenic plants with ifn-α2b gene regenerated after Agrobacterium rhizogenesmediated transformation Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| title |
Особенности трансгенных растений салата с геном интерферона-α2b, полученных путем Agrobacterium rhizogenes-опосредованной трансформации |
| spellingShingle |
Особенности трансгенных растений салата с геном интерферона-α2b, полученных путем Agrobacterium rhizogenes-опосредованной трансформации Матвеева, Н.А. Шаховский, А.М. Кучук, Н.В. Оригинальные работы |
| title_short |
Особенности трансгенных растений салата с геном интерферона-α2b, полученных путем Agrobacterium rhizogenes-опосредованной трансформации |
| title_full |
Особенности трансгенных растений салата с геном интерферона-α2b, полученных путем Agrobacterium rhizogenes-опосредованной трансформации |
| title_fullStr |
Особенности трансгенных растений салата с геном интерферона-α2b, полученных путем Agrobacterium rhizogenes-опосредованной трансформации |
| title_full_unstemmed |
Особенности трансгенных растений салата с геном интерферона-α2b, полученных путем Agrobacterium rhizogenes-опосредованной трансформации |
| title_sort |
особенности трансгенных растений салата с геном интерферона-α2b, полученных путем agrobacterium rhizogenes-опосредованной трансформации |
| author |
Матвеева, Н.А. Шаховский, А.М. Кучук, Н.В. |
| author_facet |
Матвеева, Н.А. Шаховский, А.М. Кучук, Н.В. |
| topic |
Оригинальные работы |
| topic_facet |
Оригинальные работы |
| publishDate |
2012 |
| language |
Russian |
| container_title |
Цитология и генетика |
| publisher |
Інститут клітинної біології та генетичної інженерії НАН України |
| format |
Article |
| title_alt |
Особливості трансгенних рослин салату з геном інтерферону- α2b, отриманих шляхом Agrobacterium rhizogenesопосередкованої трансформації Features of lettuce transgenic plants with ifn-α2b gene regenerated after Agrobacterium rhizogenesmediated transformation |
| description |
С помощью метода Agrobacterium rhizogenes-опосредованной трансформации получена культура «бородатых» корней салата Lactuca sativa, из которой регенерированы трансгенные растения с геном интерферона α2b человека. По результатам ПЦР и ОТ-ПЦР анализов полученные растения имели транскрибируемый целевой ген ifn-α2b и отличались от растений дикого типа удлиненными междоузлиями, ранним формированием цветоноса и пурпурной окраской листьев при культивировании в условиях искусственного освещения.
За допомогою Agrobacterium rhizogenes-опосередкованої трансформації отримано культуру «бородатих» коренів салату Lactuca sativa, з яких регенеровано трансгенні рослини із геном інтерферону-α2b людини. За результатами ПЛР та зт-ПЛР аналізів отримані рослини мали цільовий ген ifn-α2b, що транскрибувався і відрізнялися від рослин дикого типу подовженими міжвузлями, раннім формуванням квітконосу та пурпурним забарвленням листків при культивуванні в умовах штучного освітлення.
«Hairy» roots of lettuce Lactuca sativa and regenerated plants with interferon-α2b gene had been obtained via Agrobacterium rhizogenes-mediated transformation. According to the results of PCR and rt-PCR analyses the studied plants had ifn-α2b gene. The regenerated plants differed from the plants of wild type by elongated internodes, early flower-bearing stem formation and purple coloration of leaves in artificial illumination conditions.
|
| issn |
0564-3783 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/126468 |
| citation_txt |
Особенности трансгенных растений салата с геном интерферона-α2b, полученных путем Agrobacterium rhizogenes-опосредованной трансформации / Н.А. Матвеева, А.М. Шаховский, Н.В. Кучук // Цитология и генетика. — 2012. — Т. 46, № 3. — С. 27-32. — Бібліогр.: 37 назв. — рос. |
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27ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 3
© Í.À. ÌÀÒÂÅÅÂÀ, À.Ì. ØÀÕÎÂÑÊÈÉ,
Í.Â. ÊÓ×ÓÊ, 2012
УДК 575.222.7:581.1
Н.А. МАТВЕЕВА, А.М. ШАХОВСКИЙ, Н.В. КУЧУК
Институт клеточной биологии и генетической инженерии
НАН Украины, Киев
E-mail: joyna56@gmail.com
ОСОБЕННОСТИ ТРАНСГЕННЫХ
РАСТЕНИЙ САЛАТА С ГЕНОМ
ИНТЕРФЕРОНА- 2b, ПОЛУЧЕННЫХ
ПУТЕМ AGROBACTERIUM
RHIZOGENES-ОПОСРЕДОВАННОЙ
ТРАНСФОРМАЦИИ
Ñ ïîìîùüþ ìåòîäà Agrobacterium rhizogenes-îïîñ-
ðåäîâàííîé òðàíñôîðìàöèè ïîëó÷åíà êóëüòóðà «áî-
ðîäàòûõ» êîðíåé ñàëàòà Lactuca sativa, èç êîòî-
ðîé ðåãåíåðèðîâàíû òðàíñãåííûå ðàñòåíèÿ ñ ãåíîì
èíòåðôåðîíà- 2b ÷åëîâåêà. Ïî ðåçóëüòàòàì ÏÖÐ
è ÎÒ-ÏÖÐ àíàëèçîâ ïîëó÷åííûå ðàñòåíèÿ èìåëè
òðàíñêðèáèðóåìûé öåëåâîé ãåí ifn- 2b è îòëè÷à-
ëèñü îò ðàñòåíèé äèêîãî òèïà óäëèíåííûìè ìåæäî-
óçëèÿìè, ðàííèì ôîðìèðîâàíèåì öâåòîíîñà è ïóð-
ïóðíîé îêðàñêîé ëèñòüåâ ïðè êóëüòèâèðîâàíèè â
óñëîâèÿõ èñêóññòâåííîãî îñâåùåíèÿ.
Ââåäåíèå. Íà ïðîòÿæåíèè ïîñëåäíèõ 20–
30 ëåò àêòèâíî ðàçâèâàåòñÿ îäèí èç ìåòîäîâ
áèîòåõíîëîãè÷åñêèõ èññëåäîâàíèé – ãåíåòè-
÷åñêàÿ èíæåíåðèÿ, ïðè èñïîëüçîâàíèè êîòî-
ðîé ïîëó÷åíû íîâûå ðàñòåíèÿ ñ ìîäèôèöè-
ðîâàííûì ãåíîìîì. Ðàçðàáîòàíû ñïîñîáû
ââåäåíèÿ â ðàñòèòåëüíûå êëåòêè íîâûõ ãåíîâ,
â òîì ÷èñëå òàêèõ, êîòîðûå îïðåäåëÿþò óñòîé-
÷èâîñòü ðàñòåíèé ê ïàòîãåíàì, ãåðáèöèäàì è
äð. Èñòîðè÷åñêè ïåðâûì ìåòîäîì ãåíåòè÷åñ-
êîé òðàíñôîðìàöèè ÿâëÿåòñÿ òàê íàçûâàåìàÿ
àãðîáàêòåðèàëüíàÿ òðàíñôîðìàöèÿ. Èäåÿ ýòîãî
ìåòîäà ñîñòîèò â èñïîëüçîâàíèè ïðèðîäíîé
ñïîñîáíîñòè ïî÷âåííûõ áàêòåðèé Agrobacterium
tumefaciens è A. rhizogenes ñåìåéñòâà Rhizobia-
ceae èíôèöèðîâàòü äâóäîëüíûå ðàñòåíèÿ.
Àãðîáàêòåðèè ñïîñîáíû ïåðåíîñèòü â ðàñòå-
íèå íå òîëüêî ñîáñòâåííóþ Ò-ÄÍÊ, íî è
Ò-ÄÍÊ áèíàðíûõ âåêòîðîâ ñ ñåëåêòèâíûìè
è öåëåâûìè ãåíàìè [1, 2], ÷òî ïîçâîëÿåò
èñïîëüçîâàòü èõ äëÿ ñîçäàíèÿ òðàíñãåííûõ
ðàñòåíèé.
Ñàëàò, øèðîêî ðàñïðîñòðàíåííàÿ çåëåíàÿ
êóëüòóðà ñåìåéñòâà Àsteraceae, ÿâëÿåòñÿ îä-
íèì èç îáúåêòîâ ãåíåòè÷åñêîé èíæåíåðèè,
÷òî ñâÿçàíî ïðåæäå âñåãî ñ äîñòàòî÷íî ïðîñ-
òûì êóëüòèâèðîâàíèåì ýòîãî ðàñòåíèÿ in
vitro è ðàçðàáîòêîé ýôôåêòèâíûõ ìåòîäèê
ðåãåíåðàöèè [3]. Òðàíñãåííûå ðàñòåíèÿ ñàëàòà
ðàçíûõ ñîðòîâ ïîëó÷åíû ñ èñïîëüçîâàíèåì
A. tumefaciens [4–8], à òàêæå ñ ïîìîùüþ ýëåê-
òðîïîðàöèè [9], ÏÝÃ-èíäóöèðîâàííîé òðàíñ-
ôîðìàöèè [10] è áîìáàðäèðîâêè [11].
Åñëè öåëüþ ïåðâûõ ýêñïåðèìåíòîâ áûëî
èçó÷åíèå ïðèíöèïèàëüíîé âîçìîæíîñòè òðàíñ-
ôîðìèðîâàíèÿ ãåíîìà ñàëàòà, òî â äàëüíåé-
øåì áûëè ïîëó÷åíû òðàíñãåííûå ðàñòåíèÿ ñ
öåëåâûìè ãåíàìè, íàëè÷èå êîòîðûõ â ðàñòå-
íèÿõ ïðåäñòàâëÿåò ïðàêòè÷åñêèé èíòåðåñ. Òàê,
ñîçäàíû ðàñòåíèÿ ñ ãåíàìè ñóáúåäèíèöû Â
õîëåðíîãî òîêñèíà [12, 13], àíòèãåíà HBsAg
âèðóñà ãåïàòèòà B [14, 15] è òóáåðêóëåçíûõ
àíòèãåíîâ [16]. Ñ ïîìîùüþ A. tumefaciens òàêæå
ïîëó÷åíû òðàíñãåííûå ðàñòåíèÿ ñàëàòà ñ ãå-
íàìè, êîòîðûå ïîâûøàþò óñòîé÷èâîñòü ðàñòå-
íèé ê ãåðáèöèäàì [17,18], áèîãåííûì [19] è
àáèîãåííûì [20] ñòðåññîâûì ôàêòîðàì. Êðî-
ìå òîãî, A. rhizogenes ìîæåò ñëóæèòü ñðåäñòâîì
äëÿ ïåðåíåñåíèÿ ÷óæåðîäíûõ ãåíîâ â ðàñòè-
òåëüíûé ãåíîì, â òîì ÷èñëå â ðàñòåíèÿ ñà-
ëàòà [21, 22]. Ðàñòåíèÿ ñàëàòà èìåþò öåëåâûå
ãåíû, ïðåäñòàâëÿþùèå ïðàêòè÷åñêèé èíòåðåñ,
28 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 3
Í.À. Ìàòâååâà, À.Ì. Øàõîâñêèé, Í.Â. Êó÷óê
íàïðèìåð ãåí èíòåðôåðîíà ÷åëîâåêà, è ìîãóò
áûòü èñïîëüçîâàíû â êà÷åñòâå «ñúåäîáíûõ» âàê-
öèí – ðàñòåíèé, ïðîäóöèðóþùèõ ñïåöèôè-
÷åñêèå áåëêè, êîòîðûå ïðèìåíÿþòñÿ äëÿ ïðî-
ôèëàêòèêè è ëå÷åíèÿ çàáîëåâàíèé [23–25].
Ïîêàçàíà âîçìîæíîñòü ýêñïðåññèè ãåíà èíòåð-
ôåðîíà â ðàñòåíèÿõ êàðòîôåëÿ [26], ðèñà [27],
ñàëàòà [28] ïîñëå òðàíñôîðìàöèè ñ ïîìîùüþ
A. tumefaciens. Â òî æå âðåìÿ äî ñèõ ïîð ïóáëè-
êàöèè, êàñàþùèåñÿ èñïîëüçîâàíèÿ A. rhizo-
genes äëÿ ïîëó÷åíèÿ ðàñòåíèé ñàëàòà ñ òàêèìè
öåëåâûìè ãåíàìè, îòñóòñòâóþò.
Öåëüþ íàñòîÿùåé ðàáîòû áûëî èñïîëü-
çîâàíèå A. rhizogenes äëÿ ïîëó÷åíèÿ êóëüòóðû
«áîðîäàòûõ» êîðíåé L. sativa ñîðòà Ðóáèíî-
âîå êðóæåâî è ðåãåíåðàöèÿ èç íèõ òðàíñãåííûõ
ðàñòåíèé ñàëàòà ñ ãåíîì èíòåðôåðîíà- 2b
(ifn- 2b) ÷åëîâåêà, êîòîðûå ìîãóò ñòàòü «ñúå-
äîáíûìè» âàêöèíàìè, à òàêæå èçó÷åíèå îñî-
áåííîñòåé ïîëó÷åííûõ òðàíñãåííûõ ðàñòåíèé.
Ìàòåðèàëû è ìåòîäû. Èñõîäíûì ìàòåðèà-
ëîì ñëóæèëè ñåìåíà ñàëàòà ñîðòà Ðóáèíîâîå
êðóæåâî, êîòîðûå ñòåðèëèçîâàëè ïî ìåòîäèêå,
îïèñàííîé íàìè ðàíåå [16]. Ñåìåíà ïðîðà-
ùèâàëè â ÷àøêàõ Ïåòðè íà àãàðèçîâàííîé
ñðåäå ÌÑ [29]. Äëÿ òðàíñôîðìàöèè èñïîëü-
çîâàëè ñåìÿäîëè 7–10-äíåâíûõ ïðîðîñòêîâ.
Òðàíñôîðìàöèþ îñóùåñòâëÿëè ïî ìåòîäèêå
[16] ñ ïîìîùüþ A. rhizogenes À4 ñ âåêòîðîì
ðÑÂ161, êîòîðûé ñîäåðæàë öåëåâîé ãåí ifn- 2b
ïîä êîíòðîëåì êîðíåñïåöèôè÷åñêîãî ïðî-
ìîòîðà Mll ñàõàðíîé ñâåêëû è ñåëåêòèâíûé
ãåí íåîìèöèíôîñôîòðàíñôåðàçû II (nptII) ïîä
êîíòðîëåì ïðîìîòîðà ãåíà íîïàëèíñèíòåòàçû
[30]. Íàëè÷èå ãåíà nptII äàâàëî âîçìîæíîñòü
îñóùåñòâëÿòü îòáîð òðàíñãåííûõ êîðíåé è
ðàñòåíèé ïî ïðèçíàêó óñòîé÷èâîñòè ê êàíà-
ìèöèíó. Ïîñëå òðàíñôîðìàöèè äëÿ ïîëó÷å-
íèÿ «áîðîäàòûõ» êîðíåé ýêñïëàíòû êóëüòè-
âèðîâàëè íà àãàðèçîâàííîé ñðåäå ÌÑ ñ 25 ìã/ë
êàíàìèöèíà è 600 ìã/ë öåôîòàêñèìà. Äëÿ
ðåãåíåðàöèè ðàñòåíèé êîðíè ïåðåíîñèëè íà
òó æå ñðåäó ñ äîáàâëåíèåì 5 ìã/ë êèíåòèíà è
0,5 ìã/ë -íàôòèëóêñóñíîé êèñëîòû (ÍÓÊ).
Ðàñòèòåëüíóþ ÄÍÊ è ÐÍÊ âûäåëÿëè ïî
ìåòîäèêàì [31, 32]. Ïðèñóòñòâèå è òðàíñêðèï-
öèþ ïåðåíåñåííûõ ãåíîâ îïðåäåëÿëè ñ ïîìî-
ùüþ ÏÖÐ è ÎÒ-ÏÖÐ íà àìïëèôèêàòîðå Mas-
tercycler personal 5332 («Eppendorf», Ãåðìàíèÿ).
Äëÿ ïðîâåäåíèÿ ÏÖÐ ðåàêöèîííóþ ñìåñü ãî-
òîâèëè èç îäíîêðàòíîãî ÏÖÐ-áóôåðà ñ ñóëü-
ôàòîì àììîíèÿ, 0,25 ìêÌ ïðàéìåðîâ, 200 ìêÌ
äåçîêñèíóêëåîçèäòðèôîñôàòîâ, 0,5 åä. Taq-ïî-
ëèìåðàçû, 10–50 íã ÄÍÊ (îáùèé îáúåì ðåàê-
öèîííîé ñìåñè 20 ìêë). Äëÿ àìïëèôèêàöèè
èñïîëüçîâàëè ïðàéìåðû, ñïåöèôè÷íûå ê ãåíàì
rolB, nptII è ifn- 2b (ñîîòâåòñòâåííî 5'-5'atg-
gatcccaaattgctattccttccacga-3' è 5'-ttaggcttctttctt-
caggtttactgcagc-3', 780 ï.í., 5'-cctgaatgaactccag-
gacgaggca-3' è 5'-gctctagatccagagtcccgctcagaag-3',
622 ï.í., 5'-ttgatgctcctggcacag-3' è 5'-ttctgctctga-
caacctc-3', 396 ï.í.). Óñëîâèÿ àìïëèôèêàöèè:
ïåðâè÷íàÿ äåíàòóðàöèÿ – 94 °Ñ, ìèí, 30 öèê-
ëîâ àìïëèôèêàöèè (94 °Ñ, 30 ñ – 62 °Ñ, 30 ñ –
72 °Ñ, 30 ñ. äëÿ ãåíîâ nptII è ifn- 2b è
94 °Ñ, 30 ñ – 56 °Ñ, 30 ñ – 72 °Ñ, 45 ñ äëÿ
rolB), çàêëþ÷èòåëüíûé ñèíòåç – 72 °Ñ, 3 ìèí.
Ïðåïàðàòû ñóììàðíîé ÐÍÊ, ïðåäâàðèòåëü-
íî îáðàáîòàííûå ÄÍÊàçîé I, ñâîáîäíîé îò
ÐÍÊàçû, èñïîëüçîâàëè â êà÷åñòâå ìàòðèöû
Ðèñ. 1. Îáðàçîâàíèå «áîðîäàòûõ» êîðíåé (à) è ðå-
ãåíåðàöèÿ ðàñòåíèé (á) ïîñëå òðàíñôîðìàöèè ñà-
ëàòà ñ ïîìîùüþ A. rhizogenes À4
Âëèÿíèå ñîñòàâà ñðåäû íà ðîñò òðàíñãåííûõ êîðíåé
ñàëàòà è ðåãåíåðàöèþ ðàñòåíèé
¹
ñðåäû
Êîíöåíòðàöèÿ
ðåãóëÿòîðîâ
ðîñòà, ìã/ë
Ðîñò
Êèíå-
òèí
ÍÓÊ êîðíåé
êàëëóñ-
íîé
òêàíè
ïîáå-
ãîâ
1
2
3
4
0,5
0,5
5
0
0,05
5
0,5
0
+
–
–
+
–
+
+
–
–
–
+
–
29ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 3
Îñîáåííîñòè òðàíñãåííûõ ðàñòåíèé ñàëàòà ñ ãåíîì èíòåðôåðîíà
äëÿ ñèíòåçà ïåðâîé öåïè êÄÍÊ. Ñèíòåç îñó-
ùåñòâëÿëè ñ ïîìîùüþ íàáîðà ðåàêòèâîâ «Fer-
mentas» (Ëèòâà) ïî èíñòðóêöèè ôèðìû-èçãî-
òîâèòåëÿ. Äëÿ êàæäîé ïðîáû ÐÍÊ ïðîâîäèëè
äâå ðåàêöèè – â ïðèñóòñòâèè è â îòñóòñòâèå
îáðàòíîé òðàíñêðèïòàçû.
Ðåçóëüòàòû èññëåäîâàíèé è èõ îáñóæäåíèå.
Ðîñò êîðíåé íà áåçãîðìîíàëüíîé ñðåäå ñ
ñåëåêòèâíûì àíòèáèîòèêîì íà÷èíàëñÿ óæå ÷å-
ðåç 10–14 ñóò. ×àñòîòà îáðàçîâàíèÿ êîðíåé
ñîñòàâëÿëà 43,4 %. Íà ëèñòüÿõ êîíòðîëü-
íûõ ðàñòåíèé ïðè òåõ æå óñëîâèÿõ êóëüòè-
âèðîâàíèÿ êîðíè íå îáðàçîâûâàëèñü. Ïîëó-
÷åííûå êîðíè îòäåëÿëè îò ýêñïëàíòîâ è
ïåðåíîñèëè íà ñðåäó òîãî æå ñîñòàâà. Îíè èìå-
ëè õàðàêòåðíûå äëÿ «áîðîäàòûõ» êîðíåé ïðèç-
íàêè (ðèñ. 1, à): ãîðìîíîíåçàâèñèìûé ðîñò,
îòñóòñòâèå ïîçèòèâíîãî ãåîòðîïèçìà, ñèëüíîå
âåòâëåíèå, ÷òî îáóñëîâëåíî ïåðåíîñîì â ãåíîì
TL ôðàãìåíòà Ò-ÄÍÊ ðRi-ïëàçìèäû. ÏÖÐ-àíà-
ëèç ïîëó÷åííûõ ëèíèé ïîêàçàë ïðèñóòñòâèå êàê
ñåëåêòèâíîãî, òàê è öåëåâîãî ãåíîâ (äàííûå íå
ïðèâåäåíû).
Ïîñêîëüêó ïðè êóëüòèâèðîâàíèè òðàíñãåí-
íûõ êîðíåé íà ñðåäå áåç ðåãóëÿòîðîâ ðîñòà
ðåãåíåðàöèÿ ïîáåãîâ íå íàáëþäàëàñü, äëÿ ïî-
ëó÷åíèÿ òðàíñôîðìèðîâàííûõ ðàñòåíèé ê ñðå-
äå ÌÑ äîáàâëÿëè êèíåòèí è ÍÓÊ â êîíöåí-
òðàöèÿõ ñîîòâåòñòâåííî 0,5–5,0 è 0,05–5 ìã/ë
(òàáëèöà). Íèçêèå êîíöåíòðàöèè êèíåòèíà è
ÍÓÊ íå ïðèâîäèëè ê ðîñòó êàëëóñà èëè ïî-
áåãîâ (ñðåäà ¹ 1), ïîâûøåíèå êîíöåíòðàöèè
ÍÓÊ äî 5 ìã/ë òîðìîçèëî ðîñò êîðíåé è
èíèöèèðîâàëî ðîñò êàëëóñíîé òêàíè. Íà ïè-
òàòåëüíîé ñðåäå, ñîäåðæàùåé 5 ìã/ë êèíåòèíà
è 0,5 ìã/ë ÍÓÊ (ñðåäà ¹ 3), ïðè îòñóòñòâèè
ðîñòà êîðíåé íà÷èíàëñÿ ðîñò êàëëóñà, à ÷å-
ðåç 20–30 ñóò – ðåãåíåðàöèÿ ïîáåãîâ (ðèñ. 1,
á). Îáðàçîâàâøèåñÿ ðàñòåíèÿ ïåðåñàæèâàëè â
÷àøêè Ïåòðè íà áåçãîðìîíàëüíóþ ñðåäó ÌÑ
ñ àíòèáèîòèêàìè äëÿ óêîðåíåíèÿ.
Äëÿ ïîäòâåðæäåíèÿ ïðèñóòñòâèÿ ãåíîâ rolB,
íåîìèöèíôîñôîòðàíñôåðàçû II è èíòåðôåðî-
íà â ïîëó÷åííûõ ðàñòåíèÿõ èñïîëüçîâàëè ìåòîä
ÏÖÐ. Ïðîàíàëèçèðîâàíî 8 ëèíèé êîðíåé, â
êîòîðûõ äåòåêòèðîâàëè ãåí rolB (ðèñ. 2, à), à
òàêæå öåëåâîé (ðèñ. 2, á) è ñåëåêòèâíûé (ðèñ. 2,
â) ãåíû. Àíàëèç ÎÒ-ÏÖÐ ïîêàçàë, ÷òî âî âñåõ ëè-
íèÿõ îáíàðóæåíà òðàíñêðèïöèÿ êàê ñåëåêòèâ-
íîãî (ðèñ. 2, ã), òàê è öåëåâîãî (ðèñ. 2, ä) ãåíîâ.
Ðèñ. 2. ÏÖÐ-àíàëèç òîòàëüíîé ÄÍÊ (à–â) è ÎÒ-ÏÖÐ (ã, ä): rolB (à), ifn- 2b (á, ä) è nptII (â, ã); à: 1 – ÄÍÊ
êîíòðîëüíîãî ðàñòåíèÿ; 2–4 – ÄÍÊ òðàíñãåííûõ ðàñòåíèé; á, â: 1–3 – ÄÍÊ òðàíñãåííûõ ðàñòåíèé; 4 –
ÄÍÊ êîíòðîëüíîãî ðàñòåíèÿ; 5 – ïëàçìèäíàÿ ÄÍÊ; ã, ä: 1–8 – ÄÍÊ òðàíñãåííûõ ðàñòåíèé; íå÷åòíûå
òðåêè – ñèíòåç îáðàòíûõ òðàíñêðèïòîâ â ïðèñóòñòâèè ðåâåðòàçû, ÷åòíûå – ÎÒ-ÏÖÐ áåç ðåâåðòàçû; Ì –
ìàðêåð (ðàçìåð ôðàãìåíòîâ, ï.í.)
30 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 3
Í.À. Ìàòâååâà, À.Ì. Øàõîâñêèé, Í.Â. Êó÷óê
Ðàñòåíèÿ, ïîëó÷àåìûå ïîñëå òðàíñôîðìà-
öèè A. rhizogenes, êàê ïðàâèëî, èìåþò ñïåöè-
ôè÷åñêèé ôåíîòèï. Ó íèõ ìîãóò áûòü ñìîð-
ùåííûå ëèñòüÿ, óêîðî÷åííûå ìåæäîóçëèÿ, ðàç-
âåòâëåííûå ñòåáëè, íàðóøåíî àïèêàëüíîå äî-
ìèíèðîâàíèå, ñèëüíî ðàçâèòàÿ ïëàãèîòðîïíàÿ
êîðíåâàÿ ñèñòåìà. Îäíàêî ìîãóò áûòü ïîëó-
÷åíû è ðàñòåíèÿ ñ íåáîëüøèìè íàðóøåíèÿìè
ìîðôîëîãèè ëèáî âîîáùå áåç íèõ [33–36], ïðè-
÷åì âûðàæåííîñòü Ri-ôåíîòèïà òðàíñãåííûõ
ðàñòåíèé îïðåäåëÿåòñÿ ýêñïðåññèåé rol-ãåíîâ;
«ìîë÷àíèå» ýòèõ ãåíîâ ïðèâîäèò ê îáðàçîâà-
íèþ ðàñòåíèé ñ ôåíîòèïîì äèêîãî òèïà.
Âñå ïðîàíàëèçèðîâàííûå ëèíèè ñàëàòà èìå-
ëè òðàíñêðèáèðóåìûé ãåí rol (ðèñ. 2, à, ã),
îäíàêî èõ ôåíîòèï íå èìåë îñîáåííîñòåé,
õàðàêòåðíûõ äëÿ A. rhizogenes-òðàíñôîðìèðî-
âàííûõ ðàñòåíèé. Ïîëó÷åííûå íàìè òðàíñ-
ãåííûå ðàñòåíèÿ óæå íà íà÷àëüíîì ýòàïå
ðåãåíåðàöèè ïðè îäèíàêîâûõ óñëîâèÿõ êóëü-
òèâèðîâàíèÿ (ñîñòàâ ñðåäû, òåìïåðàòóðà, îñ-
âåùåíèå) îòëè÷àëèñü êàê îò êîíòðîëüíûõ,
òàê è îò ðàñòåíèé òîãî æå ñîðòà, òðàíñôîð-
ìèðîâàííûõ íàìè ðàíåå ñ ïîìîùüþ A. tume-
faciens [16]. Äëÿ íèõ õàðàêòåðíûì áûëî áû-
ñòðîå âûòÿãèâàíèå ñòåáëÿ â âûñîòó, óäëèíå-
íèå ìåæäîóçëèé è ðàííåå ôîðìèðîâàíèå öâåòî-
íîñà (ðèñ. 3, à), â òî âðåìÿ êàê êîíòðîëüíûå
è ðàíåå ïîëó÷åííûå A. tumefaciens-òðàíñ-
ôîðìèðîâàííûå ðàñòåíèÿ [16] èìåëè ðîçåòî÷-
íóþ ôîðìó (ðèñ. 3, á). Àíàëîãè÷íûé èçìåíåí-
íûé ôåíîòèï ìû ðàíåå íàáëþäàëè ó ðàñòåíèé
öèêîðèÿ, òðàíñôîðìèðîâàííûõ A. tumefaciens
ñ âåêòîðîì ðÑÂ124 (ãåí èíòåðôåðîíà- 2b ÷åëî-
âåêà) [37].
Åùå îäíà îñîáåííîñòü ÷àñòè ðåãåíåðèðî-
âàííûõ ðàñòåíèé ñàëàòà çàêëþ÷àëàñü â ñïåöè-
ôè÷åñêîé îêðàñêå ëèñòüåâ.  ïðèðîäíûõ óñëî-
âèÿõ ïðè åñòåñòâåííîì ñîëíå÷íîì îñâåùåíèè
ëèñòüÿ ðàñòåíèé ýòîãî ñîðòà èìåþò ïóðïóð-
íóþ îêðàñêó, ÷òî è äàëî íàçâàíèå ñîðòó –
Ðóáèíîâîå êðóæåâî. Ïðè êóëüòèâèðîâàíèè â
òåðìàëüíûõ êîìíàòàõ èëè òåïëèöå (èñêóññò-
âåííîå îñâåùåíèå) îíè áûëè çåëåíûìè (ðèñ. 3,
á, ã). Îêðàñêà ëèñòüåâ íåêîòîðûõ À. rhizo-
genes-òðàíñôîðìèðîâàííûõ ëèíèé ïðè êóëüòè-
âèðîâàíèè in vitro íàïîìèíàëà îêðàñêó ëèñ-
òüåâ â åñòåñòâåííûõ óñëîâèÿõ (ðèñ. 3, â).
Îáíàðóæåííûå îñîáåííîñòè, â ÷àñòíîñòè
ðàííåå ôîðìèðîâàíèå öâåòîíîñà ó ïîëó÷åí-
íûõ òðàíñãåííûõ ðàñòåíèé, ïðåäñòàâëÿþò èí-
òåðåñ, à äëÿ âûÿâëåíèÿ ïðè÷èí òðåáóþòñÿ äî-
ïîëíèòåëüíûå èññëåäîâàíèÿ.
Òàêèì îáðàçîì, áàêòåðèè A. rhizogenes ñ
âåêòîðîì, íåñóùèì ãåí èíòåðôåðîíà ÷åëîâåêà,
ìîãóò áûòü ýôôåêòèâíî èñïîëüçîâàíû â êà-
÷åñòâå èíñòðóìåíòà äëÿ òðàíñôîðìàöèè ðàñòå-
íèé ñàëàòà. ×àñòîòà òðàíñôîðìàöèè ðàñòåíèé
ñîðòà Ðóáèíîâîå êðóæåâî ñîñòàâëÿëà 43 %.
Òðàíñôîðìàöèÿ ïðèâåëà ê èçìåíåíèþ ôåíî-
òèïà ðàñòåíèé, ðåãåíåðèðîâàííûõ èç êîðíåé;
îíè îòëè÷àëèñü îò ðàñòåíèé äèêîãî òèïà óäëè-
íåííûìè ìåæäîóçëèÿìè, ðàííèì ôîðìèðîâà-
íèåì öâåòîíîñà, à íåêîòîðûå ëèíèè èìåëè
ïóðïóðíóþ îêðàñêó ëèñòüåâ ïðè êóëüòèâèðî-
Ðèñ. 3. Îñîáåííîñòè ðàñòåíèé ñàëàòà, ïîëó÷åííûõ
ïîñëå òðàíñôîðìàöèè ñ ïîìîùüþ A. rhizogenes: à –
òðàíñãåííîå ðàñòåíèå, ôîðìèðîâàíèå öâåòîíîñà;
á – êîíòðîëüíîå ðàñòåíèå, ðîçåòî÷íàÿ ôîðìà; â, ã –
äâà òèïà îêðàñêè ëèñòüåâ òðàíñãåííûõ ðàñòåíèé
31ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 3
Îñîáåííîñòè òðàíñãåííûõ ðàñòåíèé ñàëàòà ñ ãåíîì èíòåðôåðîíà
âàíèè â óñëîâèÿõ èñêóññòâåííîãî îñâåùåíèÿ,
÷òî, âåðîÿòíî, ÿâëÿåòñÿ ñëåäñòâèåì ãåíåòè-
÷åñêîé òðàíñôîðìàöèè. A. rhizogenes ìîæåò
áûòü èñïîëüçîâàíà äëÿ ïåðåíåñåíèÿ â ðàñòè-
òåëüíûé ãåíîì ãåíîâ, ïðåäñòàâëÿþùèõ ïðàêòè-
÷åñêèé èíòåðåñ, ïîñêîëüêó òðàíñãåííûå êîðíè
è ðåãåíåðèðîâàííûå èç íèõ ðàñòåíèÿ ñàëàòà ïî
ðåçóëüòàòàì ÏÖÐ è ÎÒ-ÏÖÐ èìåëè öåëåâîé
ãåí èíòåðôåðîíà, à ïîëó÷åííûå ðàñòåíèÿ, èìå-
þùèå ãåí ifn- 2b, ïîñëå äîïîëíèòåëüíûõ àíà-
ëèçîâ íà áèîëîãè÷åñêóþ àêòèâíîñòü èíòåð-
ôåðîíà «ðàñòèòåëüíîãî» ïðîèñõîæäåíèÿ ìîãóò
áûòü «ñúåäîáíûìè» âàêöèíàìè.
N.A. Matvieieva, À.Ì. Shahovsky, Ì.V. Kuchuk
FEATURES OF LETTUCE TRANSGENIC
PLANTS WITH ifn- 2b GENE REGENERATED
AFTER AGROBACTERIUM RHIZOGENES-
MEDIATED TRANSFORMATION
«Hairy» roots of lettuce Lactuca sativa and regener-
ated plants with interferon- 2b gene had been obtained
via Agrobacterium rhizogenes-mediated transformation.
According to the results of PCR and rt-PCR analyses
the studied plants had ifn- 2b gene. The regenerated
plants differed from the plants of wild type by elon-
gated internodes, early flower-bearing stem formation
and purple coloration of leaves in artificial illumina-
tion conditions.
Í.À. Ìàò⺺âà, À.Ì. Øàõîâñüêèé, Ì.Â.Êó÷óê
ÎÑÎÁËÈÂÎÑÒ² ÒÐÀÍÑÃÅÍÍÈÕ ÐÎÑËÈÍ
ÑÀËÀÒÓ Ç ÃÅÍÎÌ ²ÍÒÅÐÔÅÐÎÍÓ- 2b,
ÎÒÐÈÌÀÍÈÕ ØËßÕÎÌ
AGROBACTERIUM RHIZOGENES-
ÎÏÎÑÅÐÅÄÊÎÂÀÍί ÒÐÀÍÑÔÎÐÌÀÖ²¯
Çà äîïîìîãîþ Agrobacterium rhizogenes-îïîñåðåä-
êîâàíî¿ òðàíñôîðìàö³¿ îòðèìàíî êóëüòóðó «áîðî-
äàòèõ» êîðåí³â ñàëàòó Lactuca sativa, ç ÿêèõ ðåãåíå-
ðîâàíî òðàíñãåíí³ ðîñëèíè ³ç ãåíîì ³íòåðôåðî-
íó- 2b ëþäèíè. Çà ðåçóëüòàòàìè ÏËÐ òà çò-ÏËÐ
àíàë³ç³â îòðèìàí³ ðîñëèíè ìàëè ö³ëüîâèé ãåí ifn-
2b, ùî òðàíñêðèáóâàâñÿ ³ â³äð³çíÿëèñÿ â³ä ðîñ-
ëèí äèêîãî òèïó ïîäîâæåíèìè ì³æâóçëÿìè, ðàíí³ì
ôîðìóâàííÿì êâ³òêîíîñó òà ïóðïóðíèì çàáàðâëåí-
íÿì ëèñòê³â ïðè êóëüòèâóâàíí³ â óìîâàõ øòó÷íîãî
îñâ³òëåííÿ.
ÑÏÈÑÎÊ ËÈÒÅÐÀÒÓÐÛ
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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
|