全 文 ：热带亚热带植物学报 2005，13(2)：154—158
Jouma／ofTropical and SubtropicalBotany
人工合成的植物化猪0【乳清蛋白基因在
转基因拟兰芥的表达
芦春斌
(暨南大学生殖免疫中心，广州 510632)
摘要：将一个398 bp的植物化的猪d乳清蛋白基因(Lactalbumin，LA)编码区克隆到带有花椰菜花叶病毒的35S启动
子的质粒中。对它们进行PCR检测和序列分析，证实这些阳性克隆是实验预期的重组质粒。随即将 35S启动子-d-乳
清蛋白基因．终止子这一表达单元克隆到双元载体 pCG．CB中，用该重组质粒双元载体pCG—CB-Lact转化农杆菌
V3101后，以农杆菌法进行拟兰芥植物转化，用除草剂 Finale对转化植物进行抗除草剂基因筛选，得到一些抗除草剂
的转化植株。对这些抗除草剂植株进行猪d乳清蛋白基因PCR分析，成功筛选到带有猪d乳清蛋白基因并且可以在
后代稳定遗传的转基因植物。Western blot蛋白质表达分析，表明猪d乳清蛋白在拟兰芥中成功表达，并且猪 d乳清蛋
白被正确加工成天然蛋白。
关键词：猪d乳清蛋白；拟兰芥；转化；蛋白质表达
中图分类号 ：Q943．2 文献标识码：A 文章编号：1005—3395(2005)02-0154—05
Expression of Synthetic Porcine Alpha—Lactalbumin in
Transgenic Arabidopsis
LU Chun—bin
(Centerfor Reproductive Immunology Research，Jinan University，Guangzhou 5 10632，China)
Abstract： A 398一bp plantified encoding fragment ofporcine —lactalbumin， was cloned into plasmid under the
control of the cauliflower mosaic virus 35S promoter． The expression cassette was introduced into A rabidopsis
with Agrobacterium mediated transformation．Some herbicide-resistant lines of transform ed A rabidopsis were
selected．The gene integration of chromosomal DNA level were confirmed in regenerated plants．All of them are
positive for porcine —lactalbumin gene via genomic DNA PCR． The expression at the protein level could be
confirmed only in the transgenic A rabidopsis．The arabidopsis—expressed lactalbumin(LA)migrated in SDS—
PAGE with identical mobility to human LA prepared from human milk． indicating that the LA was correctly pro—
cessed to yield a mature protein in plants
Key words：Porcine OL—lactalbum in；A rabidopsis；Transform ation；Expression
It has been recognized that proteins in seeds from
the commonly grown crop species do not contain a
nutritionally balanced amino acid contentt”．The seed
proteins from cereals are generally deficient in the
amino acids lysine and tryptophan，whereas those
from legumes are deficient in the sulfur amino acids
methionine and cystineg3]．
Although seeds of legume species were the major
food andoil source both forhum an andanimal， their
sulfur amino acids deficit of seed composition is dra．
matically constrained for nutrition valuet=21．Because of
these deficiencies． studies in many laboratories have
focused on the genes that encode seed storage proteins~]．
Methods to improve the nutritional quality of seed
Recieved：2004—06—21 Accepted：2004·10—13
Foundation item：Supported by United Soybean Board Domestic Program Project in USA
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第 2期 芦春斌：人工合成的植物化猪 ot乳清蛋白基因在转基因拟兰芥的表达 155
proteins by genetic engineering are being evaluated．
Up—to date， the technical development of plant trans—
formation makes such experiments feasible． Success—
ful transform ation and modification of seed—specific
proteins in plant seeds were broadly reported[ ．
Therefore，alternation oflegume seed composition
especialy to modify the sulfur level is a very import
aspect for seed protein studies and need to be paid
much more atention．Some animal genes were selected
as transgenes to plants． Vodkin et al reported the
introduction and expression of bovine casein gene into
soybean seeds with Biolistic transform ation『9J．In this
project，Porcine was selected as suitable transgene
because porcine 一lactalbumin is a major component
of sow’s milk and well—characterized amino—acid bal—
anced proteins『8J．It is therefore ideally suited to swine
nutrition．Furtherm ore，it is also a relatively small pro—
tein and the DNA that codes for it can be synthesized
in the laboratory．The LA—expressed transgenic plants
would be good food resource for pig industry．We con—
structed a syn thetic porcine 仅一lactalbumin coding
sequence containing plant—preferred codon and trans
form plants inArabidopsis model system． The resulted
plants have been characterized by DNA level and
western bloting．
1 Materials and Methods
Materials The vector(pGEM．4Z／lacta1)con．
raining plantifed 398-bp DNA fragment gene of
porcine alpha lactalbumin is kindly provided by Dr．
Scot(USDA)．The binary vector pCG—CB—ACLA is
provided by Dr．wurtele(Iowa State University)．
Rabbit anti—human LA is kindly provided by Dr．Scot
(USDA)．Human LA(h—LA，Mw1 4．4 kD)was pur-
chased from Sigm a． The protein molecular weight
markers were from Bio—Rad．
cDNA cloning and vector construction The
plasmid DNA of pla~mid pCG—CB—ACLA and plas—
mid pGEM一4Z／lactal were prepared by a modified al—
kaline lysis method En．The 398 bp DNA fragm ent in
plasmid pGEM一4Z／lactal and a 6．5 kb large fragm ent
in binary vector pCG—CB—ACLA,digested with EcoR I／
BamH I，were gel—purifed using QIAEX II Gel Extrac—
tion Kit(QIAGEN Inc．)．The 398 bp DNA fragm ent
was ligated to pCG—CB binary vector to transform E．
coli DH5a． The resulted plasmid with 35S promoter
and target peptide sequence and bar gene selectable
marker was named pCG—CB—alpha—lactalbumin(Fig．
1、．The plasmid pCG—CB—alpha—Lact was introduced
into Agrobacterium tumefaeiens GV3101 by electro—
poration．Th e LA inserts were verified to have the
corect sequence by PCR and DNA sequencing．
The 5’primer for PCR and sequencing(Seq1)is 5’
AAGCAGTTCACCAAGTGC．The 3’primer(Seq2)
js 5’CGAGCCAGTCA ATGC
Fig．1 Binary vector with CaMV 35S promoter alpha lactalbumin
gene expression cassette．The plasmid also contains a
herbicide resistant gene(bar gene)
DNA sequencing The DNA mini prepara—
tion of plasmid DNA was prepared by QIAprep
Miniprep Kit(QIAGEN Inc)．A Perkin Elmer—Applied
Biosystems 373A DNA sequencer was used in combi—
nation with the DyeDeoxy Term inator Cycle
Sequencing kit．
Transformation of Arabidopsis and screening
of transgenic plant Arabidopsis thaliana(Colum—
bia ecotype) transformation using floral dipping of
A robacterium culture to floral buds was according to
the method described by Steve Clough et al[ ”，using
the Agrobacterium binary vector system．Seeds col—
lected from floral dipped plants were planted into soil．
ARer about 1 to 2 days，spray the germ inating seedling
with Finale 1 00 mg L (Bayer，Brand name)．Transfor-
mants will survive and continue to grow， while the
non—transform ants wil die within a few days．Each of
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l56 热带亚热带植物学报 篱 l3卷
seeds collected from individual survived plants were
planted into sol1 and were continuously selected wi血
herbicide Finale(100 mg L-。)．This herbicide screen—
ing was carried through the next two generations．
Those which have 1 00％ resistant progency (the
seedlings are all green and healthy after herbicide
sprayio曲 are stable homozygous seeds based on
MendelIaw
Genomeic DNA extraction and PCR A，
bidopsls leafand seed DNA in regenerated plants were
extracted with DNeasy Plant Mini K(QIAGEN Inc)．
The 5’primer for genomic DNA amplifcation(Seq1)
js 5’AAGCAGT丁CACCAAGTGC The 3’primer
(Seq21 is 5’CGAGCCAGTCAATGC．PCR was per-
formed on MJ PC-200 with Taq DNA polymerase kit
(Promega)for 30 cycles．
Protein extraction and analysis About
100 mg offlesh leaves and seeds were ground in
250 l of extraction bufer(50 mmol／L MOPS pH 7,0，
0．2 mmoFL EDTA．1 romel几 PMSF．1 mineUL amino—
caproic acid,l mmol／Lbeazamidine．5 mmol／L DTT．
and 1 mmol／L ascorbate),respectively．Ground tissue
was transfered into microfugembe and cen~ifuge at
12 000 xg for 5 min． The resulting supemants were
collected and added four volumes of 80％ isopropano1
to pellet proteins at 4℃ for 3 hours Tubes were cen一
~ifuge at 12 000xg for 10 min． The pellets were
washed with 80％ cold acetone twice． The total
amount ofproteins in extracts was estimated using the
Bio-Rad Protein Assay kit wit}1 bOvine serum albumin
(Sigma)as a standard
SDS-PAGE was performed according to Laemm-
li㈤ with 12 5％ ge1．~restern bfot was performed
according to Sambrookt ．Proteins were blotted on a
PVDF membrane(Immobilon—P，Milipore)The LA
bands were visualized with anti—human LA fdeveloped
in rabbit)and ECL-Plus Immunodection kit rAmet．
sham Pharmacia)(Refer to Amersham Pharmacia Kit
manua1)．
2 Results
2．1 Porcine 一lactalbumin PCR analysis of genomie
DNA in transgenic Arabidopsis
Two sets ofPCR reaction ofDNA template ofthe
binary vector pCG—CB·Lact were set up A 398·bp
fragment encoding a porcine·lactalbumin and 436一bp
bar gone were amplified，respectively，with primers of
porcine ct—lactalbumin and primers of bar gene(data
not shewn1． -
These two bands are the correct size des~ed frag-
ments of porcine＆-lactalbnmin and bar gene The
DNA sequence of porcine d-lactalbumin confirmed
the PCR result．This resuIt showed血at these primers
ofporcine d—lactalbumin and bar gene are highly spe-
cifc for these encoding regions ofthe genes．
Therefore， the two pairs of primers were used
to confirmed porcine d-lactalbumin and bar gone
presented in transgenic plants Genomic DNA oftrans—
genic plants(transformation event Arab 06 and Arabl 51
were ex~acted and used for PCR template A correct
size of 398一bp fragment encoding a porcine-lactal-
bumin and 436一bp bar gent were amplifieck respec—
tively(Fig．2)．
Fig 2 Porcine 0t-lactalbum[n and bar gene PCR analysis of
genomic DNA in transgenic Arabidopsls
M 1 kb plus DNA markeF,【-4：Porcine 一]actalburain amplifica．
tinn with LactSeq；5 8：Bar gene ampfificafion w1th BarSeq；l／5：Ge．
nomic DNA of．Arab 06； 6：Genomic DNA ofArabl5 3 -Genomic
DNAofwild帅 negative control；4 ：Pla．smidDNAofpCG-CB—LACT,
positive control
These resui估 sbowed that porcine ．1actalbumin
gene was successfully in．educed in tabidopsis and
integrated into genomic DNA oftransgenin plants with
Agrobacterium mediated transformation． Th e gene
integration and expression at血e DNA 1evel were also
confirmed with Southern and Nonhem blot(Data pub．
1ished in diferent papers)．
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第 2期 芦春斌：人工合成的植物化猪Ot乳清蚩F_1基因在转基因拟兰芥的表逃
22 Protein expression analysis in regenerated
plants
Protein expression would be the strong and di—
retted evidence for successful plant Wansformation．In
tins research， the expression of plantified porcine
d-lactalbumin gene at the protein level in a transgenic
A rabidopsis was analyzed with SDS—PAGE and West-
em blot．Results showed the expression at the protein
level could be detected both in seeds and leaves of
transgeinc events(Arab 06 and Arab 1 5)．
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Fig 3 Protein analysis ofporcine“一lactedbumin in transgen[c plants
The expressed lactualbumin in transgetfic plants was indicated
[h&flow． Marker：The protein molecular,we ght markers were from
Bio-呲 1：L~avesofArab06；2 SeedsofArab 06；3：Leaves ofArab15
4 Seeds ofArob15
Furthermore，the results of protein analysis
showed that the expression in transgenic plants is
non—tissue specific expression． This reason is that
cauliflower mosaic virus 35S promoter is universal
promoter in engineered plants The feature of
cauliflower mosaic vires 35S promoter is its strong
promoter efect in plants． But this tmiversaI promoter
is poor spatial and tissue-specific expression of trans-
geinc genes． 35S controlled protein expression in
transgenic plants can not be occurred in the specific
tissue or organs such as protein body in seeds． The
arabidopsis—expressed LA migrated in SDS-PAGE
with identical mobility"to LA prepared from human
milk，indicating that the LA woA correctly processed to
yield a mature protein in transgenic plants
m ¨ }I l 』 1
I 一 ●■●
Fig 4 W estern blot ofporcine d一[actalbtmin in transgenic
pl~ts expresion
Control：S0 s ofwildqa．0e idop．~ts negative control；1 Leaves
ofArab06；2：SeedsofArab06；3：Leaves ofArab1置 Seeds ofArab15：
h·LA Human a-laetalbumin．positive control
3 Discussion
Successml transformations in plant seeds were
broadly reported and used in other researches ．
Recently plant transformation was used for express
system for some valuable animal genes，especially for
development ofedible vaccine．
Alternation of legume seed composition is very
important for seed protein studies and need to be paid
much more atention．Ooe of such researches is modi—
fication ofseed-specific proteins ofplant seeds， espe—
cially to modify the sulfur】evel[ ．O1．
Porcine Q—lactalbumin is a protein ideal1Y suited
to swine nutrition The porcine(3l—lactalbumin DNA
can be easily synthesized in the laboratory Therefore，
porcine —lactalbumin was selected as a suitable Wals—
gen e
Generally speaking，it is much more dificult that
original native porcine tx—lactalbumin gene can be
correctly~anscfibed and translated both at DNA level
and protein level in transgenic plants 31, The main
reason is that there are diferent preferred codon for
diferent species Prefered codons in animal are
much different from 山OSC 1n plants． The codon bias
resulted in the transcription and translation problems
ofnative porcine 一Iactalbumin in plants
Porcine —lactalbumin gone could be plantified to
be plant-preferred coding sequence of porcine d·
lactalbumin with site—directed mutation． The planti．
fled animal gene is a good way to overcome such
transctiption and translation problems in plants．In this
research，a 398·bp plantified porcine d—lactalbumin，
was cloned into a binary vector under the contro】of
the cauliflower mosaic viils 35S promoter．Aflerward．
plant transformation ofA robidopsis model system was
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158 热带亚热带植物学报 第 13卷
successfully done with Agrobaterium mediated trans—
formation．Seeds ofal transgenic plants were harvested
together and then do screening of transgenic plants
containing porcine—lactalbumin encoding region with
herbicide Finale application． The herbicide—resistant
plants have been characterized by DNA level and
Western bloting．All of transgenic events are PCR
positive of porcine —lactalbumin gene． The gene
chromosomal integration and expression at the DNA
level were confirm ed in regenerated plants． Progeny
analysis of herbicide selection and screening in puta—
tive transform ants showed that the transform ation of
transgenic stability and heritability were occurred in
regenerated plants． These results confirm ed the hy-
pothesis that plantification of animal genes is a feasi—
ble method to be correctly translated and expressed in
plants．
Th e expression at the protein level could be
detected in transgenic A rabidopsis．The arabidopsis—
expressed LA migrated in SDS—PAGE with identical
mobility to h—LA prepared from human milk,indicating
that the LA was corectly processed to yield a mature
protein in plants．Results of Western blot is coincident
results that the arabidopsis—expressed LA was corectly
expressed and processed in plants．The results suggested
that synthetic transgene can be corectly translated and
expressed in plants． The protein expression of trans—
genic plants was observed both in seeds and leaves of
transgenic plants． It was showed that 35S promoter
regualetd expression in plants is non—tissue specific．It
was broadly reported that 35S promoter of cauliflower
mosaic virus is a universal and highly strong promoter
for its regulation in many species of plants． But 35S
promoter is poor spatial and tissue—specific expression
of tran sgenes．
A rabidopsis is an ideal model system for the
transform ation ofplantified porcine 一lactalbumin due
to its small genomic DNA and relative short life span．
In this research， the arabidopsis transform ation can
only asset feasibility of our strategy to develop trans—
genic plants with plantified animal gene and has less
application value． The most important part of the re—
search is that we are also developing transgenic soy-
beans containing porcine —lactalbumin in the seeds．
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