全 文 ：第 25卷　第 4期　　　　　　　　　　　　　植　　　物　　　研　　　究 2005年　10月
Vo .l 25　No. 4　　　　　　　　　　　　BULLETIN OF BOTAN ICAL RESEARCH Oc.t , 　2005
Foundat ion item:Supported by th e S tateKey Basic ResearchP roject(G19990160) and theKey Project of Ch ineseM inistry ofEducat ion (104191)
Au thor in troduction:Zu Yuangang (1954— ), M ale, Ph. D. , P rofessor, M ajor in p lan t science.
Received date:2005 - 07 - 17
转衣藻染色质烟草细胞核的变化及衣藻核基因的表达
祖元刚　郭晓瑞　吴双秀　孟庆焕　李艳华　张衷华　张宇亮　孙佳音
(东北林业大学森林植物生态学教育部重点实验室 , 哈尔滨　150040)
摘　要　应用反复冻融法以衣藻原生质体为材料制备衣藻染色质 ,应用显微操作技术准确将衣藻
染色质转移到烟草叶片外植体中 ,进行连续培养并镜检观察。结果表明 ,经染色质转移处理的烟
草叶片外植体 、衣藻细胞核与烟草细胞核均发生形态上的变化。同时烟草叶片外植体正常发芽并
获得再生苗 ,经 RT-PCR检测发现 ,有衣藻核基因 (rbcs2)的表达。
关键词　衣藻;染色质;显微操作技术;RT-PCR
The nuclear change of tobacco transform ed w ith chromatin of
Ch lamydomonas reinhard tii and the expression of nuclear gene
ZU Yuan-G ang　GUO X iao-Rui　WU Shuang-X iu　MENG Q ing-Huan　LIYan-Hua
ZHANG Zhong-Hua　ZHANG Yu-Liang　SUN Jia-Y in
(Key Labo ra to ry of Fo restry P lant E co logy, M inistry of Educa tion, Northeast Forestry University, H arb in　150040)
Abstract　Through them ethod o f be ing frozen in the liquid nitrogen fo r a sho rt time, and then thaw ing
in the room tempe ra tu re for severa l times, the pro toplasts o fCh lamydomonas reinhardtiiwe re broken and
chromatin w ere obtained. Then w e exactly transfo rmed the chromatin to the exp lants of the tobacco leav-
es by them ic roman ipu lation, and then w e cu ltured the explants, obse rved by the fluorescentm icroscope
every day. The resu lt indicated that the ch roma tin transfo rmed explants, the nucle i ofC. reinhard tii and
tobacco we re a ll changed. A t the same time, the explants fo rmed the ca llus and then grew into gem-
mule. Through the RT-PCR de tec tion, we found the expression of the nuclear gene rbcs2.
Key words　Chlamydomonas reinhardtii;chromatin;m icroman ipu lation;RT-PCR
S tan ley Cohen’ s andHerbert Boyer’ s basic sci-
ence d iscovery o f recomb inant DNA techno logy in
1973
[ 1]
sparked a revo lution in bio logy and spurred
deve lopment of the bio technology industry. The in-
vention is far-reach ing imp lica tions for the intercon-
nected w orlds o f science, commerce, and soc iety[ 2] .
But at the same time there are som e prob lems, such
as genetic modification and false positive. In 1997,
W ilmut and his co-workers accomplished the ir fea t by
transfe rring the nuclei from various types of sheep
cells in to unfertilized sheep eggs from wh ich the natu-
ral nuc le i had been removed by m icrosu rgery, and
Do lly w as c loned
[ 3]
.
In fac t, genes ex ist in the genome, and the ge-
nome exists in the space o f the chromosome. Ifw e de-
grade the chromosome into chrom atin , and transfe r
the chrom atin into the plan t to be improved, th is w ill
probably be a new approach to get new species. In
ou r expe riment, we prepared the chromatin from the
protop last from the g reen alga Ch lamydomonas rein-
hard tii, and then in jec ted the chromatin in to the to-
bacco, the resu lt of RT-PCR ind ica ted that the alga
chromatinmay have been transfe rred in to the tobacco.
1　Materials and methods
1. 1　S tra ins and cu lture conditions
C. reinhardtii strain 849 w as inocu la ted in Tris-
acetate-phospha te-medium (TAP)[ 4] in E rlenmeyer
flasks and ag itated on a ro tatory shake r (150 rpm)
unde r constant illum ination w ith w hite ligh t (120
μmo l quanta m -2 s-1 PAR) at 25℃. And the
green alga in our experimentw as removed its cellw all
and f lage llum. Fo r freezing and thaw ing treatmen t,
the cu lture o f stra in 849 w as g row n to a cell density o f
6×106 ce lls mL -1 and div ided into 1. 5mL subcul-
tures. F irstly the subcu lturew as frozen in liquid nitro-
gen fo r 1m inute and thaw ed in room temperature re-
peated for 3 times, and then filtra ted by filte rmem-
b rane (3 μm).
1. 2　Tissue culture of the leaf explants
The sterile leaves o f tobacco (N ico tiana
Ben tham iana)were excised and cu t in to 1 cm ×1 cm
segments and cultured onM urashige and Skoog (MS)
medium supplemen ted w ith 30% g lucose, 0. 7% agar
and combina tion w ith BA (1 mg L -1 ) and 2, 4-D
(0. 5mg L - 1) fo r callus induction.
1. 3　The transfo rmation o f chromatin in to tobacco
A fte r the filtration, we dyed then the filtrate u-
sing the Hoechst 33258 (5 μg mL - 1)[ 5] and ob-
served under the fluorescen t m icroscope. W e found
that there w as no any entire pro toplast in the liqu id,
then w e used the m icrom anipula to r to abso rb filtrate
(2 μL) and then in jected into the leaves o f tobacco
unde r the strict sterile conditions. The cu lture w as
kep t a t 25℃ w ith the light /dark cycle of 16 h /8 h in
an environmen t chambe r (DongTuo ZPW - 40)w ith
the light intensity o f 150 μE m - 1 s- 1 provided by
coo lwhite fluorescen t lamps du ring the ligh t period.
Du ring the course of the cu lture, we go t one segment
of the leave and sliced, then dyed w ith Hoechst
33258 (5μg mL -1) and obse rved eve ry day.
1. 4　RNA isola tion and RT-PCR
Fo r the RNA iso lation o f C. reinhardtii strain
849, cells of 15 mL of indiv idua l cu ltures w ere ha r-
vested by centrifugation and to ta lRNA was isola ted by
the acid guanidine iso th iocyana te-phenol-ch lo ro form
me thod
[ 6]
using TRIzo lReagen t(Inv itrogen) fo llow ing
the supplier’ s instructions. And the RNA isolation of
the leaves o f tobacco a lso used the TRIzo l Reagent
(Invitrogen). The qua lityw as checked by agarose gel
e lec trophoreses and ethidium brom ide staining.
For reverse transcription, 1 μg o f DN ase I-trea-
ted to tal RNA was incubated in a 20 μL reaction in-
c lud ing 4 μL of a 5x Reverse Transcriptase buffer,
0. 75 μL o ligo (dT)18 primer (0. 1 mmo l L -1), 5
μL dNTP m ix ture(10 mmo l L- 1), and 10 U AMV
Reverse T ranscrip tase (Takara) for 1 h at 42℃. The
reaction w as stopped by coo ling at 2℃ fo r 2m in. The
PCR reaction was performed on the PCR m achine
(Eppendorf).
2　Results and analysis
2. 1　The changes o fC. reinhardtii
When observ ing under the fluorescen t m icro-
scope, we cou ld find that be fo re the trea tmen t of
freezing and thaw ing again and aga in, the pro toplasts
of the C. reinhardtii we re in tac t and ova te in shape
(PlateⅠ :1, 2);and afte r the trea tmen t, the pro to-
plastsw ere broken (PlateⅠ:3 , 4), then we immedi-
a te ly dyed the filtrate, and incuba ted fo r 20 m inu tes
in the darkness, and then w e obse rved unde r the fluo-
rescentm icroscope (P lateⅠ :5, 6). The resu lt indi-
cated that the protoplasts we re broken (fo r there w as
no any intact pro top last in the eyeshot) and the chro-
m atin w ere ob tained ( the chromatin we re in the sta te
of dispe rsion).
2. 2　The change o f tobacco leaf explan ts
During the cou rse of the tobacco explan ts cu l-
ture, we found that after cu lturing the leaves for 7
days, the ca llus grew in to gemmu le(P lateⅠ :13),
we w ashed it c lean to avoid being contam ina ted by the
ch roma tin, and then w e sliced the gemmule by hand
and dyed w ith Hoechst 33258 (5 μg mL -1), also
then incubated in the darkness fo r 20 m inu tes, afte r
then w e observed unde r the fluorescent m icroscope,
we surprising ly found tha t on the nuc leus o f the tobac-
co, there w ere some sm all, brigh t spo ts, we deduced
tha t theyw e re the nuc le i of theC. reinhard tii. (Pla te
4334期 祖元刚等:转衣藻染色质烟草细胞核的变化及衣藻核基因的表达
P lateⅠ 　The p ictures of protop lasts ofCh lam ydom ona s reinha rdt ii(1:Under the natu ral ligh t;2:Under the fluorescen ce);The p ictu res of bro-
ken p rotop lasts (3:Under the natu ral ligh t;4:Under th e fluorescence);The pictu res of th e chrom atin(5:Under th e natural light;6:Under the
fluorescen ce);7 ～ 12:The s lices of the leaves of tobacco cu ltu red for another 7 days after the gemmu le b eing sub cu ltured in theMS m ed ium. (7,
9, 11:Under the natu ral light;8, 10, 12:Under the f luorescence);13～ 15:13. Ob servation of the gemmu le using the stereoscope;14. The
photo pictu re of th e gemmu le transferred to newM Sm ed ium for sub cu ltu re;15. The photo p icture of the sma ll seed lings after being subcu ltu red for
7days.
434 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　25卷
Ⅰ :7 ～ 12). W e subcultu red the gemmu le fo r anoth-
er 7 day s(P lateⅠ :14, 15), and sliced the leaf by
hand and dyed w ith Hoechst 33258 (5 μg mL -1),
then incuba ted in the darkness for 20 m inu te s, after
the observa tion w e a lso found tha t on the nucleus o f
the tobacco, there we re some sma ll, bright spo ts.
2. 3　Detection o f RT-PCR product
W e ran the 1% agarose gel to de tect the RT-
PCR produc.t W e could see in the gel that the posi-
tive contro l injected w ith chromatin had w eak signal
(F ig. 1:Lane 6 and 7). This result indicated that
perhaps the chromatin had already integrated into the
leaves o f tobacco.
F ig. 1　The de tection of RT-PCR produc t
From left to righ t, Lane 1w asDNA m ark er, Lane 2 and 3 w ere RT-
PCR p roduct ofCh lamydomonas reinhard ti iw ith p rim er rbcs2;Lane
4 and 5 were the RT-PCR produ ct of tobacco leaves (negative con-
trol) w ith prim er rbcs2;Lane 6 and 7 w ere the RT-PCR produ ct of
tobacco leaves(posi tive control) w ith prim er rbcs2.
3　Discussion
During thew ho le experimen t, we ope ra ted unde r
the sterile cond ition , so the exp lants grew w ell and no
m icroorganism s. A nd befo re slicing the leaf exp lants,
we had w ashed them c lean severa l times to insure that
there w as no any chrom atin on the surface.
A fte r be ing in jec ted o f the chromatin into the leaf
explants, the explants g rew w ell as tha t o f the con-
tro.l About 7 days la ter, the ca llus induction began
and the gemmule grew in to seedlings, and the g row th
speed w as the same as that o f the contro .l
Running the product o fRT-PCR in 1% agar gel,
there w as band in the ge.l The weak band gave us
some confidence in one hand, in the o ther hand w e
must do many hard wo rk to exp lo re the re search.
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4354期 祖元刚等:转衣藻染色质烟草细胞核的变化及衣藻核基因的表达