Escherichia coli lacZ gene as a biochemical and histochemical marker in plant cells

Gene, 66 (1988) 19-29

19

Elsevier GEN 02358

EscIzericIziacoli ZucZ gene as a biochemical and histochemical marker in plant cells (Recombinant plasmid

DNA;

vector;

rapid method

for P-galactosidase

activity assay; in situ detection

of transformants;

Ti

cells; crown gall; callus; Agrobacterium tumefaciens)

tobacco

Shogo Matsumoto *, Itaru Takebe and Yasunori Machida Department

of Biology, Faculty of Science,

Received

24 October

Revised

19 December

Nagoya

University, Chikusa-ku,

Nagoya 464 (Japan)

1987 1987

Accepted

28 December

Received

by publisher

1987 28 January

1988

SUMMARY Several IacZ chimeric genes were constructed by fusing the truncated lacZ sequence of Escherichia coli to N-terminal sequences of few other genes. Promoters used to direct expression of the chimeric genes were the promoter for 35s RNA of cauliflower mosaic virus (P,,s) as well as those of the small subunit gene of ribulose bisphosphate carboxylase and the octopine synthase gene. These constructs were introduced into tobacco cells using a Ti plasmid of Agrobacterium tumefaciens, and /I-galactosidase activity in uncloned and cloned calli derived from the crown galls were examined. The results showed that the P,,,-linked ZucZ chimeric gene is expressed very efficiently. When slices of the crown gall carrying this chimeric gene were placed on plates containing indicator XGal, localized areas of the outgrowth turned deep blue, whereas no such areas were found in the crown gall having promoter-less ZucZ. Calli from galls containing this construct expressed /?-galactosidase activity at an eight-fold higher level (approx. 7000 units/mg protein) than the endogenous activity (approx. 900 units/mg protein). Some of the calli displayed over 20-fold higher activity. Actively growing mini calli expressing activity higher than 4000 units/mg protein dyed deep blue on XGal agar medium such that they were distinguishable from calli having no ZacZ. Half of the uncloned P35s -ZucZ transformant calli showed activity higher than this level. These results indicate that the ZucZgene linked to a strong promoter such as P,,, is useful as a biochemical and histochemical marker gene in plant cells.

Correspondence to: Dr. Y. Machida,

Department

of Biology,

gene of tobacco

Faculty

of Science, Nagoya

Chikusa-ku,

Nagoya

medium;

(Japan)

Tel. (052)781-5111,

* Present Menard

address: Cosmetic

University, ext. 2502.

Biochemical Co.,

464

Ltd.,

responsible

Research Ogaki,

Institute,

Gifu-ken

Nippon

503 (Japan)

Tel. (0584) 89-5659.

mosaic virus; MS, Murashige

ocs, octopine

for phytohormone

Rubisco,

boxylase;

dodecyl

SDS, sodium

Abbreviations:

0378.1119/88/$03.50

acetyltransferase

0 1988 Elsevier

mosaic virus;

gene; cp, coat

ribulose

Science Publishers

B.V. (Biomedical

Division)

in T-DNA

o-nitrophenyl-

bisphosphate

carmosaic

B-D-galactopyranoside;

that the 5’ end (e.g., ‘lacZ) or 3’ end (e.g.,

cat’) of the gene is truncated.

protein

ONPG,

sulfate; TMV, tobacco

virus; XGal, 5-bromo-4-chloro-3-indolyl

bp, base pair(s); CaMV, cauliflower

and Skoog (1962)

gene; one, gene(s)

synthesis;

b-D-galactopyranoside;

prime (‘) indicates cat, chloramphenicol

synthase

20

INTRODUCTION

MATERIALS AND METHODS

Techniques to introduce foreign DNA into plant cells have definitely become a powerful tool in the

(a) Plants, bacteria and plasmids

study of gene expression

plants having improved properties. The development of these techniques relied upon the availability of

Nicotiana tabacum cv. Bright Yellow and Xanthi nc were used in the present study. Plants were grown either in a greenhouse at 28 ‘C in the daytime of 16

marker

h and at 20 ’ C in the nighttime

genes that permit

in plants

selection

and for creating

of transformed

conditions

of 8 h, or under axenic

on MS agar medium

at 28°C with 12 h

plant cells. The j%galactosidase-coding gene (ZucZ) of E. coli has been widely used as a marker gene in

light period (2000 lux).

prokaryotes because of many features that make it suitable for this role. The gene is well characterized

E. coli HBlOl Roulland-Dussoix

genetically

pGJ28 and R64 drdll was a helper strain used to transfer the intermediate vector containing chimeric genes to Agrobacterium (Van Haute et al., 1983). A. tumefaciens C58ClCm harboring octopine Ti plasmid pTiB6S3tra” (Petit et al., 1978) was used as a host strain for the introduction of manipulated ZacZ genes. The following plasmids were used: pMC1396/1403 (Casadaban et al., 1980) containing the P-galactosidase-coding sequence from which the first eight codons were removed (‘ZacZ); pCaMVCAT (Fromm et al., 1985) containing the promoter of 35s RNA from CaMV and the cat gene; pToK6 (provided by Dr. Ohno at Tokyo University of Agriculture and Technology) containing the cDNA of the cp gene of TMV; pMH2 (HerreraEstrella et al., 1984) carrying the promoter of the small subunit gene of Rubisco of pea and the cat gene; pKC7 (Rao and Rogers, 1979), pBR325 and puc12.

and

its product

biochemically;

many

mutants are available for the genes involved in the regulation of ZucZ; a simple spectrophotometric method is available for assaying /I-galactosidase activity; and finally in situ detection of its expression is possible using agar medium containing XGal, where colonies expressing the gene turn blue allowing visual identification. In contrast to the ZacZ expression system in E. coli, the possibility of using this gene for plant cells has not been systematically examined, although a low level expression of this gene in tobacco and sunflower cells was reported (Helmer et al., 1984). A disadvantage of the ZacZ gene in plant cells may be the presence of endogenous /?-galactosidase (Agrawal and Bahl, 1968; Gatt and Baker, 1970; Pierrot and Van Wielink, 1977) that may interfere with detection and assay of the expression of introduced ZacZ. Because of its features as discussed above, however, the ZacZ gene may become a useful marker gene for plants, if this disadvantage can be overcome by using a strong promoter and a suitable N-terminal region of a plant gene, or by finding conditions favorable for the assay of the introduced gene. In the present study, we examined the feasibility of using ZacZ as a marker gene in tobacco cells. To this end, we constructed various ZacZ chimeric genes and examined their in vivo and in vitro expression in transformed tobacco cells under various conditions of culture. The results showed that ZacZ was expressed at levels by far exceeding those of endogenous enzyme when directed by the promoter of 35 S RNA (P& for cauliflower mosaic virus (CaMV). The P 35s -ZacZ-transformed tissues were clearly distinguishable from untransformed tissues by incubating mini calli or crown gall slices on agar media containing XGal.

was described by Boyer and (1969). E. coli GJ23 harboring

(b) Bacterial transformation

and conjugation

E. coZi transformation was performed essentially according to the procedure described by Mandel and Higa (1970). Intermediate vector plasmids carrying the chimeric genes were transferred from E. coli to A. tumefaciens, and agrobacterial cells carrying the chimeric ZacZ in the T-DNA region of the Ti plasmid were isolated essentially as described by Van Haute et al. (1983). The structure of the chimeric gene introduced in the T-DNA was examined by Southern-blot hybridization. (c) Plant transformation Stems of 45- to 80-day-old axenic plants of N. tabacum cv. Bright Yellow were inoculated with A. tumefaciens harboring Ti plasmid containing the chi-

21

merit genes. Tumors formed on the axenic tobacco 11-17 days after inoculation were excised, cut into small pieces (approx. 2-3 mm2) and maintained at

28 ‘C in the dark on hormone-free MS agar medium containing 250 pg/ml of carbenicillin and 100 pg/ml of vancomycin.

(A)

b

tms2

$

-:mr

tmsl

e----l-H

3

tmJ

7 0

19

d

c

-+

+

-_)

11 24 !I

13

I

I

i

E

;

Hi

22

1

EcoRI BamHI

2

El3 El

(B)

pT’lacZ

619

a>P35s-

cc>

b)

lacy

I

1acZ’

El3

BSaSa

hi Pv E

p35s-

cp’-‘lacZ cat’-‘lacZ

POCS -

e>no

IacZ’

cat ‘-‘IacZ

c>Pssd)

I

SmHa

E

Hi

E

<

lKmrln3’ lacy

ocs’-‘lacZ

promoter

-

cat’-‘lacZ Pv Hi E

Fig. 1. Construction

of pT’ZucZ

octopine

pTiB6-806

(Gartinkel

mapped

(Garlinkel

Ti plasmid

which were previously 25-bp borders

of T-DNA.

intermediate

(B) Physical

vector

containing

chimeric

‘IucZ genes. (A) Physical

et al., 1981). tms, tmr and tml are genetic loci responsible et al., 1981: Barker and genetic

et al., 1983). Arrows

structures

of the critical

fragment

containing

right-to-left

truncation

is shown on the right (N) end (ZucZ’). The DNA fragments Details of the construction

‘ZucZ gene in pT’ZucZ. Km’, kanamycin-resistance 90-l 126 (Bevan et al., 1983)). (C) Structure the orientation

of promoter.

box, cut’, vertically

Striped

the DNA fragment pCaMVCAT,

procedure.

of The

oriented

the BumHI

The arrow indicates

signal of the nopaline

and control regions of chimeric the N-terminal

between

of

the positions

et al., 1980). It is shown

were inserted

elsewhere.

n3’, 3’ poly(A)

P3ss- cat’-‘ZucZ, the EcoRI-PruII

To construct

containing

gene from pKC7; regions indicate

ofcut gene (cut’) was isolated from pCaMVCAT TMV coordinate

will be published

and its construction

(Casadaban

indicated

of phytohormones,

site and

the orientation

synthase

of

gene (positions

‘IucZ genes. Black heavy arrows represent

regions of coding sequences

used: horizontally

striped

cp', hatched box, ocs’ . E, EcoRI; B, BumHI; Sa, SuZI; Sm, SmuI; Hi, HindIII; Ha, HueIII; Pv, PvuII;

striped box,

R, RsuI; Sau, Sau3A.

of pT’lacZ

of the N-terminal

or hatched

from pMC1396/1403

map of T-DNA

by a, b, c and d indicate

part of pT’ZacZ

EcoRI-Sal1

the Hind111 site of pBR325.

‘ZucZ and ZucY wasderived

marked

and genetic for synthesis

the N-terminal

5795 to the BumHI

and then the fiuI-EcoRI

fragment

region of the coat protein

site in pToK6) fragment

and SmaI sites in pT’ZucZ. A DNA fragment

containing

the 35s promoter

and the N-terminal

and inserted between the EcoRI and SmuI sites in pT’ZacZ. To construct gene (cp’) in TMV cDNA

was first joined to the Suu3A-EcoRI

containing

containing

the 35s promoter

the promoter

fragment

and the cp’ sequence

for the Rubisco

small subunit

region

P,,,-cp’-‘ IucZ,

(from the RruI site at position containing

the 35s promoter

was inserted

between

of

from

the EcoRI

gene (P,,) and the cut’ sequence

was isolated by digesting pMH2 with EcoRI + PvuII, and was then inserted into the cloning sites of pT’ZucZ to create the P,,-cut’-‘ZacZ. The EcoRI fragment also inserted by introducing containing

was isolated

sequencing

containing

the promoter

(PO,,) and the N-terminal

region of the octopine

synthase

gene (ocs’) was

the PO,,-ocs’-‘IucZ. We also constructed a promoter-less cat’-‘ZucZ region of the cut gene into the intermediate vector. To do this, the HindIII-PvuII fragment from pMH2

cloning site of pT’Zuc2

only the N-terminal

only the N-terminal

EcoRI fragment nucleotide

24 of T-DNA

into the EcoRI

to construct

region of cut was first inserted from the resulting

that these sequences

between

DNA and reinserted

were joined in frame.

the Hind111 and the PvuII sites of pBR322,

between

then the PvuII and

the SmaI and EcoRI sites of pT’ZucZ. We confirmed

by

22

RESULTS ANDDISCUSSION

(a) Construction

of chimeric

formed with the chimeric gene and 01zcgenes. Other areas of the slices remained colorless or were only genes and plant cell

transformation Fig. 1 shows the structures of chimeric 1acZ genes we constructed. The chimeric genes consist of the truncated IacZ coding sequence (‘ZacZ) and the N-terminal region of the coding DNA (or cDNA) sequence

of either cat gene, TMV cp gene, or ocs

gene, joined in frame to the ‘IacZ sequence. We used the 5’ sequences from various sources, because the 5’-untranslated

faintly blue, as were the slices of tumors bacteria

and N-terminal

regions

may affect

the efficiency of mRNA translation in plant cells. To express these chimeric genes in tobacco cells, they were joined to the promoters of the CaMV 3.5s RNA, the Rubisco small subunit gene and the ocs gene. These chimeric genes were inserted into T-DNA of pTiB6S3 carried by A. tumefaciens C58ClCm as describedin MATERIALS AND METHODS, section b. The resulting Ti plasmids retained all of the oylc genes for synthesis of phytohormones as well as the border sequences (Fig. l), so that transformed plant cells can be selected by phytohormone-independent growth.

carrying

promoter-less

incited by

cat’-‘IacZ or PO,,_

-ocs’-‘ZacZ (Fig. 2). The extract of P,,,-cat’-‘lacZtransformed cells was shown to contain p-galactosidase activity higher than that of POCS-ocd-‘IacZ transformed cells (see the next section). Fig. 2 also showed that the intensity of blue color and the size of colored regions have a parallel relationship to the efficiency of the expression

of the chimeric genes. It

is, therefore, likely that the strong blue color observed in the P35s- cat’-‘ZacZ slices is due to the efficient expression of the P35s -cat’-‘IacZ gene introduced into crown-gall cells. When only Agrobacterium carrying P,,,-cat’-‘IacZ in T-DNA was inoculated on the same agar medium containing XGal and antibiotics, bacteria did not grow and blue color was not observed (not shown), indicating that the observed blue color was not due to P-galactosidase expressed in Agrobacterium. (c) jLGalactosidase calli

activity of uncloned transformed

Crown-gall tumors should contain non-transformed as well as transformed cells. We tested first if the chimeric ZacZ genes inserted in T-DNA can be

Since the expression level of genes inserted into chromosomal DNA is expected to vary from one clone to another, we independently inoculated two to five plants with each A. tumefaciens strain (designated A through S in Table I). The tumors formed were cut into several pieces, which were cultured on hormone-free MS medium. The calli thus obtained, which are probably mixtures of transformed and

used to locate transformed tissues in crown gall. Galls incited by A. tumefaciens carrying P35S-cat’‘lacZ, POCS-ocd-‘IacZ, or promoter-less cat’-‘lacZ in its T-DNA were sliced and placed on MS agar medium containing XGal. As shown in Fig. 2 (see Plate I), slices of the tumor incited by Agrobacterium carrying PW cat’-‘2acZ showed a localized area which dyed deep blue. The dark blue area was located in the region of slices which represented an outgrowth from normal tissues (swelled regions in Fig. 2). This area should contain transformed cells in which IacZ and the one gene are expressed. When the slices were transferred to hormone-free MS agar medium without XGal, callus in fact developed from the stained region (not shown), indicating that the region which dyed blue contained the cells trans-

non-transformed cells, were surveyed for /Lgalactosidase activity using the rapid method by which a large number of samples can be processed. As shown in Fig. 3, the values obtained by the rapid method were roughly proportional to activity in soluble extracts determined more accurately by the conventional method. Table I summarizes the results obtained. Activities of the PSSs-cat’-’ ZacZ calli (first column) assayed by the rapid method were on the average eightfold higher than those of negative control calli, i.e., calli transformed with Ti plasmids containing promoter-less chimeric ‘ZacZ gene (6th column) or no 1acZ (5th column). Some of the P,,,-cat’-‘ZacZ calli (B-4 and D-4) expressed 20-fold higher P-galactosidase activity than the control calli. Note that the

(b) Histochemical in crown gall

detection of transformed regions

23

P35s -cat’-‘IacZ

Pots -ocs’-‘IacZ

(Fig. 2)

a

-

b

,

(Fig. 4)

Plate I. (Fig. 2) Histochemical

identification

plants (A, B, C) of N. tabacum cv. Xanthi The tumors

were excised and sterilized

of transformed

as described

into OS- to l-mm slices and these were placed 12 h, they were transferred and vancomycin

transformed

stem in the representative

(Fig. 4) In situ detection

at 250 pg/ml

(Takebe

four layers of gauze. The filtrate thus obtained

contained

derive from a single cell. The filtrate containing 3MM) placed

cat’-‘ZacZ transformant

in I were further

analysed

at 28°C for

for 12 h. The agar medium

to kill Agrobacterium. The regions

dissociated

of crown

single cells and clusters developed

200 ng/ml of XGal,

essentially

contained

gall and non-

as described

into tine masses by pipetting, consisting

by Horsch

and filtered through

of two to ten cells which are presumed

and Jones,

1980) was spread

after 40 to 45 days were transferred and were incubated

over the surface

to of

onto filter paper

at 28°C in the dark. They were

I, derived from P35s- cat’-‘IacZ transformant B-4 ofTable I; II, derived R-l of Table I; III, derived from pTiB6S3-induced transformant Q-5 of Table I. Calli

at 5 h (a), 15 h (b), and 40 h (c) after transferring.

from promoter-less numbered

containing

on different

40 pg/ml of XGal. After incubation

50 to 500 such plating units (Horsch

on MS agar medium

were induced

by c and s, respectively.

the inner filter of the feeder plate. Mini calli (1 to 2 mm diameter) photographed

containing

activity on XGal agar medium. Cell clones were obtained

and Jones (1980). Briefly, friable calli were placed in liquid MS medium,

(Whatman

tumors

PO,,-ocs’-‘IacZ and promoter-less cat’-‘1acZ. 1984) 20 days after Agrobacterium infection. They were cut

200 pg/ml of XGal and incubated

and 100 fig/ml, respectively,

slices are indicated

of p-galactosidase

and Nagata,

containing

slices. Crown-gall

P,,,-cat’-‘lacZ,

first on MS agar medium

to the same medium

carbenicillin

region in crown-gall

nc by A. tumefaciens carrying

as described

in RESULTS

AND DISCUSSION,

sections

d to f.

24

crown-gall

tissues

from

three

out of five plants

(plants A, B and D) yielded calli expressing higher

activity.

TABLE

I

As expected,

the

in activity was

observed even among the calli derived from the same

varied

tumor (plant D). Half (eleven out of 22) of the calli

activity

callus of Nicotiana tabacum cv. Bright Yellow

p-Galactosidase

activity

of transformed

p-Galactosidase

activity

in transformants

P 35s -cat’-‘lacZ A 420

among calli and a 3%fold variation

ten-fold

(tobacco)

carrying

Ps,,-cp’-‘1acZ

P -cat’-‘lacZ

A 420

AL

a: P OCS -ocs’-‘lacZ A 420

Wild-type

T-DNA

Promoter-less

A 420

cat’-‘la&T

A 420

A

R ‘0.117

0.101 (729) (R-l)

0.105 1;;;;

(10500)

I!!::

(959)

1;;

(2140)

0.091

S

0.111 L G 0 B

0.091 (659)

0.063

0.139

0.049

r

_0.102 (723)

0.200

r0.210 0.186 0.762 2.665 (14800)

(B-4)

1.665 _ C

I

J

E 0.410 [

0.110

Summary

of the results b

n = 22

n=

n=9

n=7

n=

x = 0.775

x = 0.493

x = 0.139

x = 0.163

x = 0.097

s = 0.949

s = 0.487

s = 0.053

s = 0.023

s = 0.023

a Absorbance represent produced. A-E

at 420 nm/5 mg fresh weight of callus was measured

units/mg

protein

determined

Tissues bracketed

were transformed

PO,,-ocd-‘IacZ,

by using soluble extracts

and marked

A-Q

with P ,,,-cat’-‘lacZ,

plants P and Q with wild-type

AND DISCUSSION, b n, number

18

of samples;

sections

by the rapid method

F-J

T-DNA,

c to e, are referred

n=2 x = 0.096

(see legend to Fig. 3). Numbers

(see legend to Fig. 3); one unit corresponds

were derived from different plants

12

parts of the same crown-gall

with P 35s-cp’-‘lacZ, plants

K-M

x, mean value of absorbance

tumor

with P,,-cat’-‘lacZ,

of each plant. Plants plants

and plants R and S with cat’-‘ZacZ. Calli, which were described

to as B-4, C-l, D-2, D-4, Q-5 and R-l (as marked at A,,,;

s, standard

deviation

of the mean.

in parentheses

to 1 nmol of o-nitrophenol

in the Table).

N and 0 with in RESULTS

25

examined showed activity higher than absorbance at 420 nm of 0.4, corresponding to approx. 4000 units of fl-galactosidase (see Fig. 3). As described in the following section, this level of expression is high enough to distinguish 1uc.Ztransformants from nontransformed tissues on XGal agar medium.

A0

0

1.5 0.5 1.0 Absorbance at 420nm

Fig. 3. Correlation rapid method

of fi-galactosidase

with the extractable

was divided into two portions, tosidase

activity

method,

respectively.

transferred

assayed

by the

activity. A transformed

callus

which were analyzed

by the rapid

method

Rapid method:

into a glass homogenizer

(0.06 M Na,HPO,, MgSO,,

activity

for /%galac-

and the conventional 1 g of fresh

containing

0.04 M NaH,PO,,

0.05 M 2-mercaptoethanol,

gently to dissociate

2.0

callus

was

1 ml of Z buffer

0.01 M KCl, 0.001 M

Since the coat protein is synthesized in large quantities throughout TMV propagation (Takamatsu et al., 1987), the N-terminal region of the cp gene might direct expression of the chimeric gene at a higher level than that of the prokaryotic cat gene. Average activity of the P,,s-cp’-‘lacZ calli was, however, slightly lower than that of calli having P 35S-cat’-‘ZacZ, but still higher than that of the control transformants (Table I, second column). Both P,,-cat’-‘IacZ and PO,,-ocs’-‘IacZ were expressed only at levels several fold higher than control, as was reported for a chimeric EacZ gene linked to the promoter of the nopaline synthase gene (Helmer et al., 1984). The P,, promoter is shown to turn on specifically in differentiated leaves under light (Herrera-Estrella et al., 1984; Fluhr et al., 1986). When the P,,-cat’-‘IacZ calli were grown under light, they turned green, although shoots did not differentiate under these conditions. However, the j?-galactosidase activity did not increase in the light. p-Galactosidase activity was assayed more accurately using soluble extracts for representative callus from each plant. As shown in Table I, the results contu-rned that calli transformed with the P,,,-linked chimeric genes expressed activity at levels much higher than the negative controls.

pH 7.0) and homogenized

the tissues. A 200~~1 aliquot of the homogen-

ate was used for assay. If the sample volume was smaller than

(d) Direct identification XGal agar medium

of transformed

calli on

200 ~1, Z buffer was added to make 200 ~1. Toluene (10 ~1) was added

to the 200 pl sample,

vortexed

and the mixture

for 10 s and pre-incubated

was started potassium

by adding 40 ~1 of 4 mg/ml ONPG phosphate

The mixture was centrifuged phenol

nm, we also carried each sample background subtracted

compounds

out parallel

values,

which

without

from

callus. The absorbance

of absorbance

absorbance.

value thus obtained to the fresh weight of

here was normalised

tissue

extracts

at 420 nm were carried out as described temperature

standard.

supplied

by BioRad

was determined using bovine

by Miller

was 37°C. One unit

per h. The units were normalized

protein. The protein content

by the

and measurement

of enzyme activity was defined as that which releases

reagents

to the

were prepared

et al. (1984). The reaction

(1972) except that the reaction o-nitrophenol

for The

by using 5 mg fresh weight of callus. Assay

using soluble extracts: of Helmer

presented

at 420

ONPG

0.020 to 0.100, were

since it was proportional

value to be obtained

and Since

with absorbance

reactions

the background ranged

materials,

was measured.

for each sample. The absorbance

was quantitative

method

to remove insoluble

and measured

at 28°C

was added to stop the reaction.

at 420 nm of the supernatant

plant cells contain

solution in 0.1 M

buffer (pH 7.0). After incubation

for 30 min, 100 ~1 of 1 M Na,CO, absorbance

was immediately

at 28’ C for 5 min. Reaction

1 nmol of

to those per mg

using protein serum albumin

assay as a

Calli transformed with chimeric ZacZ genes were cultured on agar medium containing XGal to test whether they can be distinguishable from non-transformed cells with endogenous p-galactosidase on the basis of the intensity of blue color. Putative single cell clones were isolated from the uncloned callus B-4 (transformed with P,,, -cat’-‘ZacZ), R-l (transformed with promoter-less cat’-‘ZucZ) and Q-5 (transformed with Ti plasmid containing no ZacZ). After 40-45 days of culture mini calli (1 to 2 mm) developed were transferred to MS agar medium containing XGal. Fig. 4 (see Plate I) shows that most of the cloned mini calli transformed with P35s-cat’-‘ZucZ became distinctly blue within 5 h and that the color grew darker with time. In contrast, mini calli transformed with promoter-less cat’-‘ZacZ or with Ti plasmid containing no ZacZ were hardly stained, although a few turned blue after prolonged incubation. Thus, P,,,-cat’-‘ZacZ calli could be dis-

26

tinguished from the negative controls after transfer to XGal medium. To examine a correlation

as early as 5 h

between the intensity

blue color and the activity of /3-galactosidase

of

in the

cloned calli, we further grew three calli (marked

no IacZ, the intensity of the band visualized by immunoblotting was much weaker (lane 9) than that of the purified enzyme alone (lane 2). The extract of tobacco

cells thus

appears

to contain

materials

1,2

and 3 in Fig. 4, Plate I) on MS agar medium without XGal and assayed /%galactosidase activity in their extracts. showed

The cloned

calli B-4-1, B-4-2 and B-4-3

3150, 8540 and 20000 units,

respectively.

Two clones from another P,,s-cat’-‘ZacZcallus D-2, also exhibited an intense blue color on an XGal plate and contained

12900 and 9000 units of p-galacto-

sidase. These results indicate that transformed cells expressing p-galactosidase at levels higher than 4000 units can be identified on XGal medium by the intensity of their blue color. The results in Table I also show that half of the P,,,-cat’-‘1acZ transformants should be distinguishable on a XGal plate even as uncloned calli. Fig. 5. Detection

(e) Detection of /?-galactosidase

protein and chime-

Tissue extracts

ric genes introduced

acetone,

Cloned B-4-3 and uncloned C-l tissues, which contained 20 000 and 3 100 units of /?-galactosidase, respectively, were used to demonstrate the protein specified by P,,,- cat’-‘ZucZ. Proteins in the extracts of these calli were separated on SDS-polyacrylamide gel and visualized by Western blotting with an antibody against j?-galactosidase of E. coli. As

glycerol,

containing

formed were dissolved

gation, the supernatants troblotted

and 25 PM bromphenol

at 100°C for 10 min. After centrifuwere electrophoresed

1970). The resolved

to nitrocellulose

membranes

membrane

were washed

on 10% polyacryproteins

were elec-

at 70 V for 4 h. The

in 150 mM NaCl in 50 mM Tris . HCl

(pH 7.5) (saline) for 10 min, and in saline containing milk (Yukijirushi

with saline containing

0.1 y0 Triton X-100 and 0.5% skim milk. were incubated

in the same rinse solution

againstE.

c&/3-galactosidase

University).

in the same solution rabbit

IgG conjugated

containing

at room temrabbit

were then washed

containing

antibody

by Dr. Tsuboi at Nagoya in the same

for 60 min, and subsequently

immersed

l/400 diluted goat IgG against

with horseradish

for 2 h. The membranes

peroxidase

(purchased

were washed

in saline

0.1 y0 Triton X-100 for 25 min and then in saline for

35 min. Protein bands were visualized in 24 ml of saline containing reagent

overnight

containing

(provided

The membranes

solution without antibody

from Cappel)

3% skim

Milk Corp.) for 2 h. They were then rinsed twice

The rinsed membranes perature

(BioRad)

by dipping the membranes

12 mg of horseradish

and 12 ~1 of H,O,.

peroxidase

Lanes 1 and 2,4 ng and 40

ng of purified E. coli /I-galactosidase

(Sigma),

3 and 4, 100 pg and 500 pg protein

of the callus extract

pTiB6S3 transformant

I), respectively;

(Q-5 ofTable

respectively;

6, 100 pg and

500 pg protein

P ,,,-cat’-‘lad

callus (B-4-3 in Fig. 4), respectively;

8, 100 pg and 500 pg protein P,,,-cat’-‘ZacZ of purified extract markers.

with

in 40 pl of

7 mM Tris . HCl (pH 7.5), 1 y0 SDS, 15%

by heating

gel (Laemmli,

analysis.

above were treated

0.175 M 2-mercaptoethanol

blue, followed lamide

by Western-blot

as described

and the precipitates

a solution

shown in Fig. 5, these calli contained a protein recognized by the antibody (lanes 5 and 7), whereas no such protein was detectable in the extracts of calli transformed with the wild-type Ti plasmid (lanes 3 and 4). The size of this protein (approx. 116 kDa) was to be expected from that of the chimeric gene. By comparing the intensity of the protein band of B-4-3 (lane 5) with that of purified /3-galactosidase as a marker (lane 9), it was estimated that B-4-3 cells produce the ‘ZacZ product at a level of approx. 0.1% of total extractable protein. These results show that the chimeric gene introduced is efficiently expressed in the plant cells under the direction of P,,,. Although the calli definitely contained the protein recognized by the antibody, it was difficult to quantitate; when a five-fold larger amount of protein (500 pg) was used for the immunoblotting, no band was observed (lanes 6 and 8). When purified P-galactosidase was mixed with the extract of a callus having

of 1ucZ product prepared

of the extract of the extract

callus (C-l ofTable

E. coli ,5’-galactosidase

from a pTiB6S3

callus

The numbers

represent

lanes from a

lanes 5 and

from

a cloned

lanes 7 and

from an uncloned

I), respectively;

lane 9,40 ng

plus 100 pg of protein (Q-5 of Table I). Lane molecular

sizes in kDa.

of the 10, M,

21

which interfere with the transfer of j?-galactosidase protein to nitrocellulose membrane, as was previously suggested (Bevan et al., 1985). We also examined the copy number and the structure of the introduced P,,,-cat’-‘IacZ chimeric gene in B-4- 1 and B-4-3 cell clones (expressing 3 150 units and 20 000 units, respectively) by Southern blotting. The results showed that both B-4-l and B-4-3 cells contained one copy of the chimeric gene per diploid genome (not shown), indicating that the level of expression of the introduced gene is influenced by other factors than copy number. No DNA rearrangement was observed in the region of the chimeric gene and the one gene of each clone.

Malhotra, 196 1). On the other hand, the pH optimum of plant endogenous fl-galactosidase was not in the neutral range, but appeared to be in an acidic range (Fig. 6A), in agreement with previous reports (Agrawal and Bahl, 1968; Gatt and Baker, 1970; Pier-rot and Van Wielink, 1977). To examine heat stability of P-galactosidase, samples were assayed for activity after heating at 50°C. As shown in Fig. 6B, the enzyme of B-4-3 cells exhibited a stability close to that of E. coli /?-galactosidase being much more stable than the plant endogenous enzyme. These results indicate that the high level of P-galactosidase activity detected in the B-4-3 cells is due to the introduced chimeric gene.

(f) pH dependence and heat stability of /Sgalactosidase activity in transformed cells

(g) Use of the Zuc.2 chimeric genes Several features should be pointed out in visualization and maintaining the transformed cells that are efficiently expressing the P,,,-cat’-‘ZacZ gene. First, it is crucial to use actively growing mini calli for

Fig. 6A shows that the enzyme activity in the B-4-3 cells has a pH optimum of 6.9, the optimum of purified P-galactosidase of E. coli (Wallenfels and

(A)

(B) 10

100 -

-

Q

0

0 2

t

V 2-l + .-

h

0

‘;”

0

> .c

0

5;

ki

.-r c 3

aJ .-> z

50

z I

6.6

1

I

6.8

n

,

7.0

7.2

Time (min)

PH Fig. 6. Physical from tobacco

/3-galactosidase

of the /?-galactosidase

and E. coli cells. Soluble

pTiB6S3-induced at indicated

parameters

extracts

activities

-0-,

P,,,-cat’-‘1uc.Z;

from tobacco

-O-, pTiB6S3

Reactions

-x -, purified

cells. (A) pH dependence

of /I-galactosidase

activities

were carried

tumor callus (B-4-3 in Fig. 4) and from a out at 28°C for 60 min using 100 mM phosphate buffer

E. coli /I-galactosidase;

and E. coli cells. Plant extracts were then carried

callus. Values are means

tobacco

from a P ,,,-cat’-‘ZucZ

were prepared

callus (Q-5 of Table I), respectively.

pH values.

30 min and then quickly chilled on ice. Reactions /I-galactosidase;

activity in transformed

-O-,

pTiB6S3

and purified /I-galactosidase out for 60 mm at 28°C. -O-,

f S.D. of four sets of experimental

data.

callus.

were heated P ,,,-cat’-‘1acZ;

(B) Heat

stability

at 50°C for lo,20

of and

- x -, purified E. coli

28

identification

of transformants

When P35s- cat’-‘lad

by their blue color.

calli were transferred

to XGal

containing agar medium after extensive growth on MS agar medium, they hardly dyed blue so that it was difficult to distinguish them from control calli.

Research

(62107003)

and Grant-in-Aid

for Special

Research on Priority Areas (62622001) from the Ministry of Education, Science and Culture, Japan and a grant to Y.M. from the Naito Foundation.

Second, it is necessary to isolate single cell clones at an early stage of selection of transformants. When an uncloned

callus which initially showed a high level of

expression

was cultured for six months without phy-

tohormones, ly. In contrast,

the enzyme activity decreased

marked-

cloned lines such as B-4-1, B-4-2 and

B-4-3, however, maintained

the initial levels of p-gal-

actosidase activity for a year. These observations indicate that during culture the uncloned calli become predominant by faster growing cells containing no or low activity of /I-galactosidase. Third, it is recommended to transfer calli from XGal medium to regular MS medium within 12 h if they are to be cultured further. Calli did not grow on the medium containing 200 pg/ml of XGal. When calli were maintained for 24 h on XGal plate, they lost the ability to grow on MS medium. It may be difficult to use ZacZ for fine analysis of the DNA region controlling gene expression in tobacco, because of the presence of significant endogenous /I-galactosidase in this species. However, IacZ may be used as a reporter gene for plants with little or no endogenous P-galactosidase. For example, we found that Gynostemma pentaphyllum, a Cucurbitaceae species, contains no detectable /?-galactosidase activity (unpublished results). The other possible uses of ZacZ as a visible marker include detection and analysis of DNA rearrangements, such as DNA transposition, in plant cells. When a cloned cell line expressing ZacZ at a visually distinguishable level no longer shows blue color on XGal plates, some genetic rearrangement should have taken place around the integrated ZacZ gene. Studies along this line are under way in this laboratory.

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