全 文 ：第 50 卷 第 6 期
2 0 1 4 年 6 月
林 业 科 学
SCIENTIA SILVAE SINICAE
Vol. 50，No. 6
Jun．，2 0 1 4
doi:10．11707 / j．1001-7488．20140617
Ｒeceived date: 2014 － 02 － 17; Ｒevised date: 2014 － 04 － 22．
Foundation project: Ministry of Science and Technology of the People’s Ｒepublic of China Project (2009FY210100) ; World Bank Loans Project
Shandong Ecology Afforestation Program ( SEAP － JC － 2) ; Natural Science Foundation of Zhejiang Province (Y3110028) ．
* Corresponding authors: Liu Huixiang． The authors are grateful to Dr． Huanting Liu ( University of St Andrews，UK) and Dr． Shi’en Lu
(Mississippi State University，USA) for help improving the manuscript．
葡萄座腔菌原生质体的制备及 gfp的转化*
陈 亮1 孙庚午1 王洪凯2 吴树敬3 林福呈2 刘会香1
(1．山东农业大学植物保护学院 山东省林业有害生物防控工程技术研究中心 泰安 271018;
2． 浙江大学生物技术研究所 水稻生物学国家重点实验室 杭州 310058;
3． 山东农业大学园艺科学与工程学院 作物生物学国家重点实验室 国家苹果工程技术研究中心 泰安 271018)
摘 要: 葡萄座腔菌是木本植物溃疡类病害的重要病原，研究该病菌的侵染和致病过程有助于揭示病原与
寄主的互作机制。携带 gfp 基因并高效表达的病菌可有效地实时检测和分析病菌的侵染过程，但由于该病菌
在致病和室内培养过程中均不易产孢，因此，制备高质量的原生质体是进行 gfp 基因转化和表达的首要步骤。
通过对酶的种类、酶解液浓度、菌丝年龄、酶解时间、酶解温度和渗透压稳定剂 6 个可能影响原生质体制备效
率的参数进行分析，结果表明 :原生质体最大产量产生的条件是菌龄 42 h，以 1 . 5% 崩溃酶、1 . 5% 葡聚糖在
0 . 7 mol·L － 1 NaCl 的渗透压稳定剂中酶解 3 . 5 h，最适酶解温度 31 ℃ ，制备的原生质体在酵母蛋白胨蔗糖
培养基( YPS)上再生率最高可达 48 . 33% ; 通过 PEG-CaCl2 介导原生质体的遗传转化、gfp 基因 PCＲ 检
测、稳定性检测和荧光显微观察，实现了报告基因 gfp 在葡萄座腔菌转化子内的稳定遗传和高效表达。
关键词: 葡萄座腔菌; 原生质体制备; 再生; 转化; gfp
中图分类号: S718. 81 文献标识码: A 文章编号: 1001 － 7488(2014)06 － 0131 － 07
Protoplast Preparation and gfp Transformation of Botryosphaeria dothidea
Chen Liang 1 Sun Gengwu 1 Wang Hongkai 2 Wu Shujing 3 Lin Fucheng 2 Liu Huixiang 1
(1 ． Shandong Ｒesearch Center for Forestry Harmful Biological Control Engineering and Technology
College of Plant Protection，Shandong Agricultural University Tai’an 271018;
2 ． State Key Laboratory for Ｒice Biology Biotechnology Institute，Zhejiang University Hangzhou 310058;
3 ． National Ｒesearch Center for Apple Engineering and Technology State Key Laboratory of Crop Biology
College of Horticulture Science and Engineering，Shandong Agricultural University Tai’an 271018)
Abstract: Botryosphaeria dothidea is a major important pathogen infecting a wide range of woody plant species．
Understanding infection and pathogenic processes of the pathogen could help reveal the interaction mechanism between the
pathogen and host better． Pathogen with expressed gfp gene can be used as an effective approach to detect and analyze the
infection process． B． dothidea is difficult to produce spores during naturally infecting process and in vitro culture process，
therefore，preparation of high quality protoplasts is essential for gfp gene transformation and expression． In this study，six
parameters influencing protoplast preparation were analyzed，including enzyme species，enzyme concentration，mycelial
age，time and temperature of enzymolysis and osmotic stabilizer． The results showed that optimal condition for gaining
maximum yields of viable protoplasts was of 42-hour-old mycelia age incubated in 0. 7 mol·L － 1 NaCl solution with 1. 5%
driselase and 1. 5% glucanase at 31℃ for 3. 5 h． The prepared protoplasts showed a regeneration efficiency of 48. 33% in
yeast extract peptone sucrose (YPS) medium． A reporter gene gfp conferring green fluorescent protein was transformed
successfully to B． dothidea mediated by PEG-CaCl2 ． Polymerase chain reaction ( PCＲ) analysis，fluorescent microscope
observation and stability test of transformants indicated that the gfp gene was stable in heredity and effective expression．
This protocol was the first report for protoplast preparation and gfp transformation of B． dothidea．
Key words: Botryosphaeria dothidea; protoplast preparation; regeneration; transformants; gfp
林 业 科 学 50 卷
Botryosphaeria dothidea is an economically
important pathogen infecting many woody plant hosts
and causing fruit rot， frogeye leaf spot， stem and
branch canker， die-back， gummosis， and in some
cases tree death ( Slippers et al．，2007; Liu et al．，
2009 ) ． It has been reported in Asia， Europe，
America，Oceania and Africa with its infectivity to
poplar，apple，pear，peach，eucalyptus and olive trees
(Phillips et al．，2005; Pitt et al．，2010; Ｒodas et al．，
2009; Yu et al．，2009; Slippers et al．，2007; Tang et
al．，2012 ) ． Attention has been devoted to chemical
and biological control of B． dothidea in China ( Ji et
al．，2008; Yang et al．，2002; Guo et al．，2009)，but
longterm prevention effect should be improved．
Understanding infection and pathogenic process are
critical for efficient control of this pathogen．
Genetic transformation approaches including
electroporation (Chakraborty et al．，1990)，restriction
enzyme-mediated integration (ＲEMI) (Sanchez et al．，
1998 ) and Agrobacterium tumefaciens-mediated
transformation ( de Groot et al．，1998 )，PEG-CaCl2 -
mediated transformation (Kao et al．，1974) have been
widely used to transfer fungi for genetic modification．
Protoplasts of high quantity and quality stand for an
essential premise for genetic transformation，however
no efficient method has been developed so far for
protoplast preparation of B． dothidea． PEG could
cause the protoplasts to clump together ( Fincham，
1989)，which facilitates the trapping of DNA， thus
foreign gene can be easily transformed to the genome of
fungi mediated by this method． Green fluorescent
protein as a reporter gene，has been widely applied in
molecular analysis of fungi，including gene expression，
protein subcellular localization fungal-host interaction
(Lu et al．，2004; Ｒajasekaran et al．，2008 ) ． The
desirable traits of GFP such as convenient detection，
stable fluorescent，real-time observation，nonhazardous
and heterologous cells versatility ( Li et al．，1997 )
provide a convenient approach to study filamentous
fungi in the molecular level (Lorang et al．，2001) ．
In this study， optimization of the preparation
procedure for the protoplast from mycelia of B．
dothidea was conducted and a foreign gene gfp was
transformed and expressed in B． dothidea successfully．
This protocol provides an essential step for establishing
a protoplast-mediated transformation system and a
better approach for further studying the infection and
pathogenic mechanism and functional genomics of B．
dothidea．
1 Materials and methods
1. 1 Strain and culture conditions
Botryosphaeria dothidea strain SDAU11-76 with
high virulence was isolated from the infected apple tree
branches in Qixia，Yantai，Shandong province，China
(120°44. 121E，37°21. 220N)，and it was saved by
this laboratory， stored at 4 ℃ with 30% sterile
glycerin． Plasmid pKO1-HPH containing a positive
selective gene hygromycin phosphotransferase ( hph )
and a reporter gene gfp conferring green fluorescent
protein was stored at － 80 ℃ with 30% sterile
glycerin．
Mycelia of 0. 1 g obtained from pure culture
(5 d) on PDA were added into a complete medium
(CM，D-glucose 10 g·L － 1，peptone 2 g·L － 1，yeast
extract 1 g·L － 1，casamino acid 1g·L － 1，20 × nitrate
salts 50 mL·L － 1，trace elements 1 mL·L － 1，vitamine
solution 1 mL·L － 1 ) broth at 28 ℃ with shaking of
120 r·min － 1 ． Mycelia were collected by 4 layers
sterilized gauze for analysis．
1. 2 Protoplast preparation
Different enzymes for the cell wall digestion were
driselase from Basidiomycetes sp． ( Sigma Chemical)，
lysing enzymes from Trichoderma harizanum ( Sigma
Chemical) and glucanase ( Grindsted Products AS)，
and which were dissolved in osmotic stabilizes agent
solution， and filter-sterilized through 0. 25 μm
membrane filter and stored at 4 ℃ ．
A total of 1 g of mycelia was incubated with 10
mL of enzyme solutions at the designed temperatures
and incubation times with a shaking speed of 100 r·
min － 1 ． Protoplasts were obtained after filtration with 2-
layer sterilized lens paper and harvested by
centrifugation (3 000 r·min － 1 ) for 10 min． Then the
protoplasts were washed with STC ( Sorbitol 1. 2 mol·
L － 1，Tris-HCl pH 7. 5 10 mmol·L － 1，CaCl2 50 mmol·
L － 1) for 2 times，quantified and maintained with STC
and then stored the protoplasts at － 80 ℃ ．
To establish an efficient B． dothidea protoplast
isolation method，7 kinds enzyme conditions (Tab. 1)，
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第 6 期 陈 亮等: 葡萄座腔菌原生质体的制备及 gfp 的转化
variable enzyme concentrations ( 1. 0%， 1. 5%，
2. 0%，2. 5%，3. 0%，3. 5%，4. 0% )，mycelial age
(18，24，30，36，42，48，54，60 h)，enzymolysis
time ( 1，1. 5，2，2. 5，3，3. 5，4，4. 5，5 h )，
temperature ( 22， 25， 28， 31， 34， 37 ℃ ) and
osmotic stabilizes agent (NaCl，KCl，MgSO4·7H2O，
sorbitol， mannitol and sucrose ) were analyzed
respectively． All the experimental procedures were
triplicated．
1. 3 Protoplast regeneration
The obtained protoplast suspensions were
separately diluted into a concentration of 1 × 103 cell·
mL － 1 with 0. 7 mol·L － 1 NaCl and sterile water was used
as a control． PDA，Czapek-Dox agar (CD，NaNO3 6. 0
g·L － 1，KCl 0. 52 g·L － 1，KH2PO4 1. 52 g·L
－ 1，
glucose 10. 0 g·L － 1，1 mol·L － 1 MgSO4·7H2O 2 mL·
L － 1，trace elements solution 1 mL·L － 1，agar 15 g·
L － 1， final pH was 6. 5 )， yeast extract peptone
dextrose (YPD，yeast extract 3. 5 g·L － 1，peptone 5 g·
L － 1，glucose 10 g·L － 1，agar 15 g·L － 1 ) and YPS
( yeast extract 3. 5 g·L － 1，peptone 5 g·L － 1，1 mol·
L － 1 sucrose，agar 15 g·L － 1 ) media were prepared for
protoplast regeneration． A hundred microliters of
suspension from two kinds of dilution above were
spread on the surface of those plates respectively and
incubated at 28 ℃ for 2 d． Ｒegeneration rate (Ｒ，% )
of protoplasts was calculated as the formula followed:
Ｒ =
NＲ － NC
NT
× 100，
Here NＲ is the number of colonies grown on the
regeneration plate，NC is the number of colonies on
control plate and NT is the total number of protoplasts
inspected．
1 . 4 PEG-CaCl2 -mediated transformation
of protoplasts
Protoplasts of 100 μL (1 × 106 protoplast·mL － 1 )
mixed with plasmid pKO1-HPH of 5 μg were kept on
ice for 25 min， and then gentle mixed with 1 mL
transformation solution ［40% (W /V) PEG 4000，50
mmol·L － 1 CaCl2，50 mmol·L
－ 1 Tris-HCl pH 8. 0］
incubated for 15 min at 25 ℃，followed 5 mL of STC
was added with gentle mixing． Transformed protoplasts
were centrifuged for 10 min with 3 000 r·min － 1 at
4 ℃，resuspended with STC of 100 μL and mixed with
overlay agar ( YPS containing 0. 5%，20 μg·mL － 1
hygromycin B ) plated on underlay agar ( YPS
containing 1. 5% agar，20 μg·mL － 1 hygromycin B) ．
The plates were incubated at 28 ℃ for 7 d and the
colonies were transferred to PDA plates containing
20 μg·mL － 1 hygromycin B (Data of sensitivity of B．
dothidea to hygromycin B were not shown) ．
1. 5 GFP detection of transformants
Five randomly selected transformants grown on
PDA plate for five successive generations were used
to determine the existence and stability of the
inserted gfp gene by PCＲ analysis ( Primers: gfp1，
5 -ATGGTGAGCAAGGGCGAGGAG-3 ， gfp2， 5 -
CTTGTACAGCTCGTCCATGCCG-3 ) ． Green
fluorescence of mycelia were analyzed and imaged
under fluorescent microscope ［ Eclipse 80 i
microscope ( Nikon ) equipped with Plan APO VC
50X /1. 40 objective］ with the wild-type strain
SDAU11-76 as a control．
2 Ｒesults
2 . 1 Influence of six parameters on protoplast
preparation
Totally 7 enzyme conditions were analyzed for
protoplast release， the final concentration of enzyme
solutions were all 3% ( enzyme mixture was consisted
of two or three different enzyme all of which were in the
same proportion) ． The results showed the treatments
with each of the three individual enzymes produced
protoplasts the driselase turned out to be the most
efficient ［(2. 80 ± 0. 29) × 108 cell·mL － 1］ while the
lysing enzyme mixture was the weakest one with a yield
of (1. 39 ± 0. 23 ) × 108 cell·mL － 1 ． In comparison
with the reactions containing multiple enzymes， the
combination containing 1. 5% driselase and 1. 5%
glucanase was able to produce the protoplasts of (6. 13
± 0. 32 ) × 108 cell·mL － 1，which was the highest
among the combinations (Tab. 1) ．
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林 业 科 学 50 卷
Tab． 1 Effects of enzyme ( s) on the protoplast yield of B． dothidea strain SDAU11-76①
Enzyme Enzyme combination
Driselase + + + +
Lysing enzymes + + + +
Glucanase + + + +
Yield /( × 108 cell·mL － 1 ) 2. 80 ± 0. 29 c 1. 39 ± 0. 23 d 1. 94 ± 0. 27 d 5. 05 ± 0. 15 a 6. 13 ± 0. 32 a 4. 05 ± 0. 3 b 4. 33 ± 0. 16 b
① + : Presence of the enzyme; Data in this table represent the average ± SEM of three replicates; SEM: Standard error of the mean; a-d:Values in
columns with different superscript letters are significantly different at the 5% level by Duncan’s multiple range test．
The concentration of enzyme mixture ( driselase ∶
glucanase = 1 ∶ 1 ) were further analyzed ranged from
1. 0% to 4. 0% ． The result showed that the protoplast
release efficiency increased as the enzyme
concentration increased from 1% to 3%， and it
reached the plateau at 3%，hereafter it had a slight
decline at 4% (Fig． 1A)
The production efficiency of protoplasts varied greatly
with different growth ages． It increased from the 18 h and
reached a peak at the growth age of 42 h with a yield of
(6. 15 ± 0. 03) × 108 cell·mL －1，and sharply decreased
when the growth age exceeded 48 h (Fig． 1B)．
As shown in Fig. 1C，protoplasts released when
treated with enzymes after 1 hour and the yield reached
its plateau of (6. 17 ± 0. 04 ) × 108 cell·mL － 1 after
3. 5 h incubation，and it showed a slight decline with
extended incubation in the experiments (Fig． 1C) ．
Analysis of six different temperatures showed that
as the incubation temperature rose from 22 ℃，
protoplast release efficiency increased， it reached a
peak at 31 ℃ and then decreased sharply (Fig． 1D) ．
Six substances NaCl， KCl， MgSO4 · 7H2O，
sorbitol，mannitol and sucrose ( each in 0. 7 mol·L － 1)
were used to dissolve enzymes and wash protoplasts
respectively． The results showed that clear variations
and NaCl was the most efficient osmotic stabilizer with
maximum protoplast production of (6. 14 ± 0. 08) ×
108 cell·mL － 1 compared with others (Fig． 1E) ．
Fig． 1 Protoplast releasing from B． dothidea strain SDAU 11-76 on different condition
A. Effect of enzyme concentration on protoplasts yield; B. Influence of hyphal growth stage on protoplasts yield; C. Time course of protoplast
isolation; D. Influence of temperature on protoplasts release; E. Influence of osmotic stabilizer on protoplasts yield; F. Effect of digestive time on
protoplast regeneration． Ｒesults are calculated based on three replicates experiments，and standard error of the mean ( SEM) is shown．
Tab. 2 Protoplast regeneration of
B． dothidea strain SDAU11-76①
Ｒegeneration medium Ｒegeneration rate (% )
PDA 32 ± 1. 73b
CM 27 ± 3. 05ab
CD 16. 33 ± 0. 67a
YPD 30. 67 ± 2. 41b
YPS 48. 33 ± 1. 76c
① Data in this table represent the average ± SEM of three
replicates; SEM: Standard error of the mean; a － d:Values in columns
with different superscript letters are significantly different at 5% level by
Duncan’s multiple range test．
2. 2 Ｒegeneration of protoplasts
Different media were used in the assays and the
regeneration rates were calculated by colony counting
after incubated at 28 ℃ for 48 h． The results showed that
YPS was the most suitable regeneration medium with the
highest protoplast viability of 48. 33% (Tab. 2) ．
The released protoplasts were round，size ranged
from 6. 72 μm to 32. 35 μm in diameter with an average of
431
第 6 期 陈 亮等: 葡萄座腔菌原生质体的制备及 gfp 的转化
21. 71 μm ( Fig． 2) ． Analysis of the growth ability in
liquid YPS medium showed that the regeneration started
from 2 h incubation and the mycelia were well produced
at 18 h incubation at 28 ℃ (Fig． 3) ．
Further analysis of regeneration of the protoplasts
isolated with different digesting times showed that the
regenerate efficiency was higher for a short digestion
time ( 2. 5 h ) and it decreased as the incubation
extended． Combined with yield and acceptable
regeneration efficiency were obtained with enzyme
digestion period for 3. 5 h (Fig． 1F) ．
2. 3 PEG-CaCl2-mediated transformation of protoplasts
Hygromycin B resistant colonies appeared on the
selective YPS regeneration media after approximately 7
days’culture，and the transformation efficiency about
3 transformants per 1 μg．
Fig． 2 Protoplast examination of B． dothidea strain SDAU
Fig． 3 Ｒegeneration morphology of protoplasts releasing from B． dothidea strain SDAU11-76 following 20 h incubation in culture broths
Fig． 5 Microscopic observations of mycelium samples exposed to ultraviolet and white light
Samples were collected from the transformants G2，G4 and the wild-type SDAU11-76，respectively．
After successive growth of 5 generations，all of the
transformants had no apparent change in morphology
and pathogenicity． PCＲ analysis showed the 0. 72 kb
of gfp was amplified in all 5 transformants，but absence
with the non-transformed recipient DNA template
(Fig． 4 ) ． Green fluorescence detection showed the
high expression of gfp gene in the mycelia of the
transformants，but not in the wild-type (Fig． 5) ． This
result indicates the stability of transformants and the
successful transformation of B． dothidea using A．
tumefaciens with plasmid pKO1-HPH．
Fig． 4 PCＲ analysis of the gfp gene
M:DNA molecular size markers ( in base pair) ． Lanes 1 － 5:
Transformants PLG1-5 respectively． W:Negative control with wild-
type SDAU11-75． P: Positive control with pKO1-HPH．
531
林 业 科 学 50 卷
3 Discussion
Protoplast isolation has been well studied in fungi
( Stasz et al．，1988; Li et al．，2011; Feng et al．，
2012)，while for a specific fungus the optimal method
is required for high quality protoplast． Though spores
always are used as the source in some fungi (Cheng et
al．，2000; Zhang et al．，2013 )， it is experimental
difficult to gather enough spores of B． dothidea for
processing， the method established in this study
provides a simple and efficient way to isolate
protoplasts using mycelia of B． dothidea for genetic
transformation．
In general，the process of protoplast isolation is to
remove cell wall while keeping integrity of other
components， especially the cytoplasm membrane．
Enzymatic digestion of cell walls and osmotic stabilizers
represent two major factors affecting protoplast isolation
efficiency． The former removes the cell wall while the
later keeps the cell stable after its cell wall is removed．
In this study，driselase showed a high efficiency in
protoplast production of B． dothidea in comparison with
other two enzymes． It is worth noting that in this study
we considered the driselase as one unit，however，it
actually consisted of multiple enzymes including
laminarinase，xylanase，cellulase and protease，which
could effectively digest cell walls． Combination of
enzymes often acts synergistically to enhance cell wall
degradation and increases protoplast yield ( Tilburn et
al．，1983; Solis et al．，1996)，the similar results were
obtained when driselase mixed with lysing enzymes or
mixed with glucanase in the present study．
Osmotic stabilizer is another important factor
during protoplast generation． It was proved that 0. 7
mol·L － 1 NaCl acted as a good osmotic stabilizer for a
large amount of protoplast isolation of B． dothidea． Li
et al．(2011) also used 0. 7 mol·L － 1 NaCl as osmotic
stabilizer for protoplast preparations． MgSO4，sorbitol，
mannitol and sucrose were used as stabilizers in
protoplast isolations for various reasons and these
substances had showed important influence on
protoplast productions of many different organisms．
However，they were not as good as 0. 7 mol·L － 1 NaCl
for B． dothidea in this study due to unknown reasons．
The protoplasts isolated in the present study were
regenerated well especially in YPS medium， and
regeneration rate is much higher of the protoplasts
obtained with a short period of digestion than these with
a longer digestion ( Fig． 1F)，which is similar to that
reported previously ( Feng et al．，2012 ) ． The exact
reason is unclear，and it is possible that the decrease is
likely caused by the damage of cytoplasm membrane
proteins as the cell-wall digesting enzyme mix usually
contains proteases，which remains to be investigated．
PEG-CaCl2 -mediated transformation as an efficient
tool is commonly used for transformation of different
species of fungi ( Ｒobinson et al．，2001; Lin et al．，
2008 )， In this study， a foreign gene gfp was
transformed and expressed successfully in B． dothidea
strain SDAU11-76 mediated by PEG-CaCl2，and the
obtained transformants has no apparent change in
morphology and pathogenicity， further more the
transformants could express GFP stably． The efficiency
of transformation can be influenced by many reasons
such as the status of protoplasts and conditions of
transformation，and that still need to be investigated in
the future study． The transformants with high expressed
GFP can be used as a vital tool for invasion，
colonization，localization and interaction study．
In conclusion， this paper provides an efficient
protocol for protoplast preparation and gfp
transformation of B． dothidea as the first time． All the
results in this research will help for further exploring
the infection and pathogenic mechanism and functional
genomics of B． dothidea．
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第 6 期 陈 亮等: 葡萄座腔菌原生质体的制备及 gfp 的转化
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