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 the S tate Key Basic Research Project(G19990160) and the Key Project of Ch ineseM inistry of Edu cation(104191)
Au thor in troduction:Zu Yuangang (1954— ), M ale, Ph. D. , P rofessor, M ajor in p lan t science. E-m ail:zygorl@ pub lic. h r. h .l cn
Received date:2005 - 07 - 17
烟草叶片愈伤组织 BECTLIN Ⅰ基因的表达
祖元刚　刘　英　郭晓瑞　孟庆焕　祁长青　姜　洋
(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　分别以烟草盆栽实生苗和组培继代苗的叶片为外植体 ,在 MS(1. 0 mg L -1 6-BA与
0. 5mg L -1 2, 4-D)培养基中诱导形成的愈伤组织为材料 ,检测与细胞程序性死亡有关的 BECT-
LIN Ⅰ基因 。 RT-PCR检测表明 ,烟草盆栽实生苗和组培继代苗的叶片及其愈伤组织均有 BECT-
LIN Ⅰ基因的表达 ,但组培继代苗叶片愈伤组织的 BECTLIN Ⅰ基因表现出持续 、稳定的表达 ,说
明培养基中的激素可能对 BECTLIN Ⅰ基因的表达产生影响 ,同时表明烟草叶片愈伤组织形成过
程中可能产生了细胞程序化死亡 ,其结果可能导致烟草叶片原初形态不同 、功能各异的细胞在脱
分化阶段形成了形态结构和功能完全相似的胚性细胞。
关键词　烟草;愈伤组织;BECTLIN Ⅰ基因;RT-PCR;细胞程序化死亡
Expression ofBECTLIN Ⅰ gene in the leaf callus ofN icotiana bentham iana
ZU Yuan-Gang　LIU Y ing　GUO X iao-Rui　MENG Q ing-Huan　Q I Chang-Q ing　 JIANG Yang
(Key Laboratory of Fo rest P lant E co lory, M inistry of Educa tion, Northea st Fo restry Unive rsity, H arb in　150040)
Abstract　W e induced the ca llus from the leaves o fN icotiana bentham iana cultured in theMS med ium
and g rew in the g reen house. Then w e used the callus to detect the expression of BECTLIN Ⅰ gene
which w as corre lated w ith Prog rammed Ce llDeath (PCD). The detection of RT-PCR show ed that the
fragment ofBECTLIN Ⅰ gene cDNA was amp lified in the leaves of the in-v itro plantlets subcu ltured in
the medium and tho se of the plotted plan t seedling, bu tBECTLIN Ⅰ gene exp ressed continua lly and
stead ily in the leave s callus of the p lantlets. This probably ind ica ted tha t aux ins in themedium had som e
effect on the exp ression ofBECTLIN Ⅰ gene. A t the same time, PCD m ight occu r in the course o f the
forma tion of the leaves callus, and in the state of ded ifferen tiation there probably appeared the emb ryo-
b lasts w ith the same configura tion and function.
Key words　N icotiana bentham iana;callus;BECTLIN Ⅰ gene;RT-PCR;prog ramm ed cell dea th
In the long period of the evolution o f the p lant
leaves, the re appeared different ce llu la r types such as
epide rm is cells, pa lisade tissue ce lls, spongy tissue
ce lls and so on, and these ce lls possessed diffe rent
configuration and function. During the course of the
leaves g row th, a ll the tissues keep rela tive stabiliza-
tion in the configu ra tion and func tion. How ever, after
the in-v itro p lantle ts w ere cu ltured in the medium ,
the p rim ary tissues of leavesw ere a ll changed, and in
the sta te of ded ifferen tiation there probably appeared
the embryob lasts w ith the same configu ra tion and
func tion, but the mechan ism w as no t clea r so far.
Sc ientists founded the theo ry of Prog rammed ce ll
death (PCD)[ 1, 2] in 2002. PCD was a gene ral phe-
nomenon, a norma l cell changed into a w ithering cell
in the course o f the PCD. Th is had g reat sim ilarity
w ith the forma tion of the embryoblasts. The auxins in
themedium m ight be a key factor fo r fu rthe r differen-
tiation. Yule Liu et a l indicted that the bectlin I gene
in N. bentham iana leaves, a ortho log of the yeast and
mamm alian PCD gene ATG /VPS30 /bectlin I, func-
tions to restrictHR PCR to infection sites
[ 3]
. Thus,
we may find a new approach to study whe ther or no t
PCD is in the forma tion of the cou rse of embryob lasts
from callus.
W e cultu red the leaves from the in-vitro p lantlets
and the p lo tted p lant seedling, and w e induced the
callus inM Sm edium supplemen ted w ith BA (1. 0mg
L -1) and 2, 4-D (0. 5 mg L -1 ). According to
the detection of BECTLIN Ⅰ gene, we found the re
ex isted the expression o f BECTLIN Ⅰ gene in the
course of cu lture o f callus, and the study m ight be a
base for the future research of the callus diffe rentia-
tion.
1　Materials and methods
1. 1　Materia ls
The leaves induced fo r callus we re obtained from
the in-v itro p lantlets in the medium and the plotted
plant seedling in g reenhouse. The sterile leaves of to-
bacco (N icotiana bentham iana)were excised and cut
in to 1 cm ×1 cm segments and cultured onMu rash ige
and Skoog (MS) medium supplemen ted w ith 30%
glucose, 0. 7% agar and combina tion w ith BA (1
mg L -1) and 2, 4-D (0. 5 mg L - 1) fo r ca llus in-
duction.
1. 2　Methods
1. 2. 1　Leafma terials obtainment and obse rvation by
fluorescen t-m icroscope
During the cou rse of the culture, we go t one seg-
ment of the leaves and sliced, then dyed w ithH oechst
33258 (5 μg mL -1) and observed every day unde r
the inverted fluorescen t m icroscope (N icon TE
2000—U).
1. 2. 2　To tal RNA iso lation
Tota l RNA was extracted by using TRIzol reagent
(Invitrogen) fo llow ing the supplier’ s instruc tions.
W e iso la ted to tal RNA from the leaf segmen ts cultured
in the medium. And the segments from the in-vitro
plan tle ts w ere cultu red for 1, 2, 4 , 6 d; the seg-
ments fo r the p lo tted plant seedlingsw ere cu ltured fo r
3, 5, 7 , 12d. The RNA qua lity w as detected by 1%
denaturealization fo rmaldehyde-aga rose e lectrophoresis
and u ltrav iole t spectropho tomete r.
1. 2. 3　RT-PCR de tection ofBECTLIN Ⅰ gene
The first BECTLIN Ⅰ cDNA from RNA tem-
plates that w as synthesized by M-MuLv reve rse tran-
scrip tase w as used directly fo r amplifica tion by PCR
w ith one pa irs o f prime rs:
upper primer　5′CAGACCCAGATTGAGCAACC 3′
low er prime r　5′GGCCAAATAGTTCATAGGTAT 3′
PCR was perfo rmed via 33 circ les inc luding de-
na ture step (94℃, 30 sec), annea ling step (52℃,
30sec), extension step (72℃, 1 m in), extension
step (72℃, 10 m in), and ending step (ho lding at
4℃).
F ig. 1　Obse rvation of nuc lea r changes
a:The nucleus of the leaves of p lotted plan t seed ling;b:The nucleu s of th e leaves of th e in-vitro p lan tlets
424 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　25卷
2　Results and analysis
2. 1　Observa tion of nuclea r changes
W e sliced the two k inds of the segments cultu red
in themedium and obse rved unde r the inverted fluo-
rescen t m icroscope (F ig. 1), we found that befo re
being cu ltured the nuc lei we re in compact and round
shape;and after be ing cu ltured in med ium , the nu-
cle iw ere irregula r. But the nuc lear change of the seg-
men ts from the in-vitro plantle tsw asmuchmore obvi-
ous than tha t from the p lo tted plant seedling s. This
indica ted there m ight be some sim ilarity betw een the
nuc lear change o f the tissue culture and that in the
proce ss o f PCD.
2. 2　Detection o f to tal RNA qua lity
The detected re sult of dena tu realization forma lde-
hyde-agarose electropho resis show ed: the to tal RNA
from the leaf segments from the in-vitro plantle ts and
the p lo tted p lant seedlings w ere almost un-deg raded
(F ig. 2:a and b). Through spectropho tometermeas-
u ring, the ratio of to tal RNA was 1. 7 ～ 1. 8, and the
concentration w as 500 ～ 700 μg mL- 1.
F ig. 2　The qua lity detection of to ta l RNA
a:The total RNA of leaf segm en ts from th e plotted p lant seed lings
b:Th e totalRNA of leaf segm ents from the in-vitro p lant lets
2. 3　Detection o f RT-PCR product
E thidium brom ide-stained ge l represents
RT-PCR product from 3 samp les (F ig. 3). Lane 1 is
the RT-PCR product o f the leaves from the w ild N.
ben tham iana plan ted in the g reen house, the band
w as not sing le and steady, this could show that the
expre ssion of BECTLIN Ⅰ gene w as restricted by
time;Lane 4 , 5 , 7 and 8 w ere the RT-PCR prod-
uc ts o f the leaf segments cultured in the medium fo r
3, 5, 7, 12 d respectively, and the leaf segments
we re from the leaves of the plotted plan t seed lings,
this show ed that the BECTLIN Ⅰ gene sing ly ex-
pressed;Lane 2, 3, 6 , and 9 w ere the RT-PCR
products o f leaf segments cultu red in the medium fo r
1, 2, 4 , 6 d respec tively, and the leaf segmentsw ere
from the leaves o f the in-vitro plan tle ts, from the
bandsw e could see the exp ression o f theBECTLIN Ⅰ
genes w ere all sing le, and the result indica ted the
ho rmones in the medium had some effect on the ex-
pression ofBECTLIN Ⅰ gene. The results further in-
dicated tha tBECTLIN Ⅰ gene continuously expressed
during the course o f the leaf in-vitro cu lture.
F ig. 3　The detection of RT-PCR product
Lan eM isDNA m arker;Lane 1～ 9 are the p roducts of the PT-PCR
3　Dicussion
It w as reported thatBECTLIN Ⅰ gene expressed
much stab ly, and the cellsm ight be rea ligned and re-
constructed during the process o f leaf g row th and de-
velopmen t
[ 4]
. In our research that the BECTLIN Ⅰ
gene expressed continuously under the influence of
ho rmones, th is m igh t cause the au tophagy. Xues in-
ferred tha t the autophagym ight ac tivate the Caspase of
apoptosis
[ 5] , and it indica ted tha t apop tosis had
something to do w ith the autophagy. Ce lls in the same
tissue could die by the means of apoptosis or au toph-
agy
[ 6]
. BECTLIN Ⅰ gene expressed stably in the
course of tissue culture, this p layed a p ro tec tive ro le
in the early period o f PCD
[ 7] , but in the late pe riod,
4254期 祖元刚等:烟草叶片愈伤组织 BECTLIN Ⅰ基因的表达
it cou ld promote ce lls death
[ 8]
. W e cou ld included
that the expression o fBECTLIN Ⅰ gene m ight have
much sc ientific significance in the further study o f
callus fo rmation.
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