全 文 ：转人α-乳清白蛋白基因烟草中半胱氨酸含量的提高 ?
王逸群
( 福建师范大学生命科学学院发育与神经生物学重点实验室 , 福建 福州 350108 )
摘要 : 将人α-乳清白蛋白基因构建到植物表达载体上 , 通过农杆菌介导采用叶盘法将人α-乳清白蛋白基因
导入到烟草中 , 经过 PCR 和 Southern blot分析表明 : 人α-乳清白蛋白基因已经整合到烟草基因组中 ; 经过
RT-PCR 和 GUS组织化学染色证明 : 人α-乳清白蛋白基因得到了表达。测定了 9 株转基因烟草叶片半胱氨
酸含量 , 大部分植株有着明显的提高 , 最高幅度达到了 318 .02 % , 半胱氨酸含量平均提高了 166 .40 %。
关键词 : 人α-乳清白蛋白基因 ; 转基因烟草 ; 半胱氨酸 ; 提高
中图分类号 : Q 758 文献标识码 : A 文章编号 : 0253 - 2700 (2008) 04 - 457 - 07
Transformation of Human Alpha-lactalbumin Gene into
Tobacco and Improvement of Cysteine Contents
in Transgenic Tobacco Plants
WANG Yi-Qun
( Laboratory of Developmental and Neural Biology, Collegeof Life Sciences, Fujian Normal University, Fuzhou 350108 , China)
Abstract: The plant expression vector containing human alpha-lactalbumin gene was constructed and its gene was trans-
formed into tobacco using leaf disc mediated by AgrobacteriumtumefaciensLBA 4404 as a vehicle . PCR and Southern blot
analysesshowed that human alpha-lactalbumin gene was integrated into the tobacco genome . RT-PCR and histochemical
staining for GUS activitydemonstrated that human alpha-lactalbumin gene was expressed in transgenic tobacco plants . Fur-
ther analysis of the data indicated that the average of cysteine contents in9 transgenic tobacco plantswas greatly increased
by 166.40% besides one of themwhich was increased by 318 .02% .
Key words: Human alpha-lactalbumin gene; Transgenic tobacco plants; Cysteine; Improvement
Human alpha-lactalbumin (α-LA ) is a small ,
acidic, Ca
2 +
binding protein, secreted in the female
mammary gland ( Wim et al. , 1999; Yu et al. ,
2004) . α-LA content is higher in human milk with
about 28 percent of the total protein . It contains rich
tryptophan and cysteine . Tryptophan is one of the es-
sential amino acids which can not be synthesize by hu-
man body itself . Cysteine is the composition of gluta-
thione and can protect cells from being harmed by oxi-
dation . More importantly, α-LA plays an important role
in physiological and health functions, for instance, the
resistance to the most Gram-positive bacteria, killing
the pneumococcus, particularly, inducing apoptosis in
tumor cells ( Walter et al. , 2004; Hakansson et al. ,
2000; Svensson et al. , 1999; Hakansson et al. ,
1995; Kohler et al. , 1999; Pettersson et al. , 2006;
Gustafsson et al. , 2005 ) . Theα-LA genomic DNA has
been transformed into the ratsbymeansof genetic engi-
云 南 植 物 研 究 2008 , 30 (4) : 457～463
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2008.07228
? ?Foundation items: Supported by the scientific and technological program of Fujian Science and Technology Department ( 2004N026) ; the Natural
Science Foundation of Fujian Province (B0410009) and the scientific and technological program of undergraduate students in Fujian Normal
university ( BKL2007062)
Received date: 2007 - 10 - 08 , Accepted date: 2007 - 12 - 03
作者简介 : 王逸群 (1964 - ) 男 , 博士 , 副教授。研究方向 : 植物生物技术。 E-mail : Yiqun20@ yahoo. com. cn
neering and it has been expressed in transgenic rat (Yu
et al. , 2004; Liu et al. , 2004; Fujiwara et al. ,
1997; Fujiwara et al. , 1999; Fujiwara et al. , 2003) .
In comparison with the animal expression system,
the plant expression system has many advantages with
easy operation, planting in field in large scale, low
cost and expressed protein stored in seeds etc ( Wang
et al. , 2002) . Thus, ones pay much more attention to
utilizing plants as the biological reactor to produce the
proteins which human beings need . With the continu-
ous improvement of Chinese people′s food quality and
their living standards, the requirements for food nutri-
tion are being raised daily ( Gao et al. , 2001 ) . This
paper presentsour work on the constructionof theplant
expression vector pBRALA , the expressionof humanα-
LA genein transgenic tobacco plants, and the determi-
nation of the cysteine content of transgenic tobacco pla-
nts . The results could lay down a foundation for study-
ing the production and utilizationof humanα-LA using
themethodof plant genetic engineering .
1 Materials and Methods
1 .1 Materials
1 .1 .1 Plant materials Wild type tobacco Yunyan 85 was
kept in our laboratory .
1 .1 .2 Strains and plamids Escherichia coli DH5α, Agrobac-
terium tumefaciens LBA4404 , plasmids of pCAMBIA-1301 ,
pSPROK and pHALA were stored in our laboratory . The plamid
pHALA contains 375 bpfragment encodedfor human alpha-lactal-
bumin . Plasmid pBRALA was constructed for this experiment .
1 . 1 . 3 Main chemicals Restriction enzymes, T4-DNA li-
gase, Taq DNA polymerase, Takara RNAiso Reagent, Prime-
ScriptTM RT-PCR Kit etc were purchased from the Takara Bio-
technology (Dalian) Co . , Ltd; IPTG, X-gal , X-gluc and DNA
Purification and Recovery Kit were obtained from the Shanghai
Sangon Biological Engineering Technology and Services Co .,
Ltd; and Hygromycin B and DIG High Prime DNA Labeling and
Detection Starter Kit II were used from the Roche Diagnostics
Corporation; DNA marker II was bought fromtheTiangen biotech
(Beijing) Co ., Ltd .
1 . 1 . 4 PCR primers Accordingto a part of DNA sequence of
humanα-LA gene, a pair of the primers were designed : 5′
primer was 5′-CAATGAAAGCACGGAATA-3′and 3′primer was
5′-AACTTCTC ACAAAGCCAC-3′. The length of DNA sequence
between the pairs of primers was 237 bp . The primerswere syn-
thesized by Shanghai Sangon Biological Engineering Technology
and Services Co ., Ltd .
1 . 2 Methods
1 .2 .1 Construction of the plant expression vector Two
fragments, one from pCAMBIA-1301 and the other one from
pSPROK , containing CaMV 35S promoter and nos terminator af-
ter restriction with EcoR I?Hind III and recovery, were ligated
with T4-DNA ligase . Then it was transformed into the competent
cells of Escherichia coli DH5α, andthe E. coli DH5αwasspread
onto LB medium containing kanamycin, IPTG and X-gal . White
colonies were screened and the recombinant plasmid pCBROK
was obtained ( Zhang et al. , 2000 ; Gao et al. , 2001 ) . The
HALA fragment recovered from plasmid pHALA , restricted with
BamH I?Kpn I , was inserted into the plasmid pCBROK cut by
BamH I?Kpn I to obtain the plant expression vector pBRALA .
1. 2.2 Transformation of plant expression vector pBRALA into
Agrobacteriumtumefaciens LBA4404 Theplant expressionvector
pBRALA was introduced into Agrobacterium tumefaciens LBA4404
usingthe freeze-thawmethod (Hofen et al. , 1988) .
1 . 2 . 3 Preparation of axenic tobacco seedlings Tobacco
seedswere surface-sterilized in 70% ethanol for 1 min, rinsed
three times in sterile distilledwater, and then were in 0 .1% sil-
ver nitratefor 5 min, washedthree timeswith sterile distilled wa-
ter . The surface-sterilized tobacco seeds were planted on half-
strength MS medium solidified with 0 .75% agar for germination
to obtain seedlings . The seedlingswere grown in agrowthcham-
ber with a photoperiodof 16 h light?8 hof darkness at atempera-
ture of 25℃ (Nancy and Maud, 1994) .
1 . 2 . 4 Transformation of tobacco When the seedlings were
4 - 6 weeks old, the young leaves were harvested and cut into
small pieces of leaf discs . Then they were immersed in the
Agrobacteriumtumefaciens cell solution for 15 min, dried using
sterile filter paper to remove excessive bacteria solution and
placed on co-culture medium without antibiotics . After co-cul-
tured in the dark for 48 h at 25℃ , Theywere placed on the se-
lectionmedium . When the budswith1 - 2 cmin length appeared
on the selection medium, they were removed carefully from the
explants and transferred into the rooting medium . After tobacco
shoots had rooted, the plantlets were transferred to the pots con-
taining watered soil . The pots with the plantlets were covered
with a plastic bag for 1 week in a growth chamber in order to
keep the humidity at a relatively high level . Then the plantlets
were grown in agrowth chamberwith a photoperiod of 16 h light?
8 h of darkness at a temperature of 25℃ and if necessary, they
were wateredagain . Themediafor transformation of tobacco were
as follows: (1) Theco-culturemedium: MS basal medium+ 0 .5
mg?L 6 - BA + 0 .1 mg?L IAA ; (2 ) The selection medium: MS
basal medium+ 0 .5 mg?L 6 - BA + 0 .1 mg?L IAA + 300 mg?L
cefotaxime+ 20 mg?L Hygromycin B ; and ( 3) The rooting me-
854 云 南 植 物 研 究 30 卷
dium: MS basal medium+ 0 .1 mg?L IAA + 200 mg?L cefotaxime
+ 10 mg?L Hygromycin B (Nancy and Maud, 1994) .
1 .2 .5 PCR analysis Isolation of plant genomic DNA was per-
formed according to Roger et al. (1988 ) . The PCR amplification
was made by using the genomic DNA from transformed tobacco
plants as templates according to Sambrook et al. The PCR reac-
tionconditionwas: after 94℃ for 5 min, 94℃ for 1 min, 50℃ for
1 min, 72℃ for 1 min, 35 cycles, 72℃ for 10 min at theend .
1 . 2 . 6 Southern blotting analysis Analysis of Southern blot-
ting for transformed tobacco plants was conducted according to
Sambrook et al. The tobaccogenomicDNA , after beingrestrict-
ed with BamH I?Kpn I completely and separated on an agarose
gel , was transferred into the positive charged nylon membranes
by acapillary transfer method . DIG-labeledDNA probe, hybrid-
ization and detection were performed according to the instruction
manual of DIG High Prime DNA Labeling and Detection Starter
Kit II (Zhang et al. , 2000; Gao et al. , 2001) .
1 .2 .7 RT-PCR analysis All the vessels for isolation of RNA
weretreatedtobe RNase-free . The glass andmetal wareswrapped
in aluminumfoils andbakedat 180℃ for 12 h . Theplasticwares,
suchas eppendorf tubes and pipette tips, were soaked in freshly
prepared 0 .1% (V?V) diethylpyrocarborate (DEPC) water for 16
h at 37℃ andthenautoclavedfor 30min at 121℃ . Isolation of to-
tal RNAs from the transgenic tobacco leaves was conducted ac-
cording to the instruction manual of Takara RNAiso Reagent .
Analysis of RNA quality was performed by electrophoresis through
1 .2% agarose gel , stained with ethidium bromide to see three
bands of rRNA . RT-PCR was carried out accordingto the instruc-
tionmanual of PrimeScriptTM RT-PCR Kit .
1 . 2 . 8 Histochemical staining of GUS activity Histochemi-
cal staining of GUS activitywas carried out according to Jefferson
et al. (1987) . The leaves of transformedtobacco plants and their
rootsweresoaked in GUS reactionmixturecontaining2 mmol?L X-
Gluc and incubated at 37℃ for 12 h . After staining, the green
color of the leaves was decolorized by ethanol and the roots were
madetransparent by 12% sodium hypochlorite for 40 seconds after
theywere rinsed three times in sterile distilled water . Free-hand
sections of rootswith stained blueweremadeandobservedunder a
light microscope (Zhang et al. , 2000; Gao et al. , 2001) .
1 . 2 . 9 Determination of amino acid content of transgenic
tobacco plants About 20 grams of leaves from each transgenic
tobacco plantwere taken and the content of amino acids was de-
termined at the Institute of Subtropical Forestry, the Chinese
Academy of Forestry Sciences . The data obtained from each
transgenic tobacco plantwere analyzed accordingto the following
formulas:
formula 1: A = B÷C;
formula 2: D = (Ai - Ack ) ÷Ack .
where A is the percentage of the cysteine in total amino acids in
each sample, B is the cysteine contents of the sample, C is the
total amino acid content of the sample, D is thepercentageof the
cysteine improved in transgenic plants, Ai is the percentage of
thecysteine in each transgenic plant, Ack . is the percentage of
thecysteine in non-transformed plants as the control , and i is
used in each transgenic plant, while ck is the non-transformed
plants as the control .
2 Results
2 .1 Vector construction
The multiple cloning site (MCS) of plant expres-
sionvector pCAMBIA-1301 are inside lacZgene . After
insertion of thegene intoMCS, the lacZ gene is inac-
tivated, resulting in obtaining recombinants according
to white colonies . The fragment of P35s-Tnos from
plasmid pSPROK restricted with EcoR I?Hind III was
inserted into theMCS to obtain the recombinants named
pCBROK . The HALA gene in length of 375 bp from
plasmid pHALA cut by BamH I?Kpn I was integrated
into the corresponding site of plasmid pCBROK to ob-
tain the plant expression vector pBRALA , which was
suitable to make the transformationof dicotyls (such as
tobacco) via Agrobacteriumtumefaciens . Theprocedure
for vector construction and the restriction endonuclease
map were shown in Fig . 1 and Fig . 2 respectively .
2 . 2 SProduction of transgenic tobacco plants and
their molecular identifications
2 .2 .1 Transformation of vector pBRALA into to-
bacco via Agrobacterium tumefaciens The young
tobacco leaves were selected as explants for the trans-
formation when the axenic tobacco seedlings were 30 -
40 days old . After infectedwith Agrobacteriumtumefa-
ciens and co-cultured, the explants were grown in the
selectionmediumfor plant regeneration . After 30 days
later, thebuds appeared andwere cultured continuous-
ly till tobacco with shoots and roots formed . After the
rooted plantlets were further developed, they were
transplanted to the large pots until blooming and the
productionof seeds . Finally, the38 transformed tobac-
co plantlets were produced for this experiment .
2. 2. 2 PCR analysis of the transformed tobacco
plants The genomic DNA isolated from the leaves of
transgenic tobacco plants was taken as the PCR template
9544 期 WANG Yi-Qun: Transformation of Human Alpha-lactalbumin Geneinto Tobacco and Improvement . . .
Fig . 1 Construction of plant expression vector pBRALA
Fig . 2 Restriction enzyme analysis of plant
expression plasmid pBRALA
Lane 1 , DNA molecular marker ( 1 kb DNA Ladder) ;
Lane 2 , plasmid pBRALA ; Lane 3 , pBRALA?BamH I
+ Kpn I ; Lane 4 , plasmid pCBROK ; Lane 5 ,
pCBROK?Hind II I + EcoR I
and the synthetic oligodeoxyribonucleotides as primers
were taken together to do PCR . The 15μl PCR product
Fig . 3 PCR analysis of transformed tobacco plants
Lane 1 , DNA molecular marker (λDNA?Hind I II +
EcoR I ) ; Lane 2 , non-transformed tobacco plant DNA
as the negative control ; Lane 3 , plasmid pBRALA as
thepositive control ; Lane 4 - 8 , the amplified products
from transformed tobacco plants
was taken to run electrophoresis of 1 .2% agarose gel .
The result was shown in Fig . 3 . The amplified product
indicated that the size of the specific band with 0.2 kb
fromthe transgenic tobacco plants was the same as the
positive control , but the non-transformed tobacco plants
as thenegative control did not show any amplified bands .
064 云 南 植 物 研 究 30 卷
2 .2 .3 Southern blotting analysis of the trans-
formed tobacco plants The Southern blotting analys-
is of DNA fromsome of transformed tobacco plants was
made using the HALA gene as a probe labeled with
DIG-dUTP and the result was shown in Fig . 4 . It is
indicated that the HALA gene had integrated into the
genome of the transformed tobacco plants .
2. 2. 4 RT-PCR analysis of the transformed tobacco
plants The RT-PCR analysis for some transgenic to-
bacco plants with positive southern blotting was per-
formed and the resultwas shown in Fig . 5 . It is indicat-
ed that the HALA genewas expressed at theRNA level .
2 . 2 .5 Expression of gus gene in transgenic tobac-
co plants The transformed tobacco leaves and young
roots were placed into a GUS reactionmixture . The re-
sults of GUS stainingwere shown in Fig . 6 . It is dem-
onstrated that GUS activity in transformed tobacco leav-
es and young roots was expressed, but in non-trans-
formed tobacco materials as the control was not ex-
pressed .
Fig . 4 Southern blot analysis of transgenic tobacco plants
1 , plasmid pBRALA?BamH I + Kpn I as the positive control ; 2 , 5 ,
genomic DNA of non-transformed tobacco plants digested with BamH I +
Kpn I as thenegative control ; 3 , 4 , 6 , 7 , genomic DNA of transformed to-
bacco plants digested with BamH I + Kpn I
Fig . 5 RT-PCR analysis for transgenic tobacco plants
Lane 1 , DNA molecular marker ( DNA marker I I ) ;
Lane 2 , non-transformed tobacco plant as the negative
control ; Lane 3 - 8 , the amplified products from trans-
formed tobacco plants
1644 期 WANG Yi-Qun: Transformation of Human Alpha-lactalbumin Geneinto Tobacco and Improvement . . .
Table 1 The cysteine percentage and improving percentage in each measured samples
Measured tobacco leaves
ck 1 ?2 =3 4 ?5 6 7 r8 9 T
Cysteine percentage in
total amino acids ( % ) 1 #. 11 2 ?. 72 0 ?. 55 1 v. 90 2 .13 3 X. 61 4 .64 4 :. 30 4 .00 2 .76
Improving percentage
of cysteine ( % ) 0 [145 ?. 05 - 50 9. 45 72 .07 91 . 89 225 }. 23 318 . 02 287 _. 39 260 .36 148 A. 65
where ck was the leaves in non-transformed plants as the control , 1 , 2 , 3⋯⋯were the leaves in transgenic plants
2 .2 .6 Cysteine measurement of transgenic tobac-
co plants The cysteine contents in leaves from 9
transgenic plants with positive reactions of PCR ,
Southern blotting, RT-PCR and GUS stainingwere de-
termined . The cysteine percentage and improved per-
centage in each measured sample was shown in Table
1 .
Table 1 indicated that most of the transgenic to-
bacco plants obviously contain more cysteine contents
than the control . Among them, 6 transgenic plants
showed their cysteine contents were improved by more
than 140% , and one of them was increased by
318 .02% . Data analysis showed that the average of
the improved percentage of cysteine was 166 .40% .
3 Discussions
3 .1 ?Production of the protein in transgenic plants
It was reported that the proteins or amino acids
which human beings needs were produced by transgenic
plants . The human beta-casein gene was transformed
into potato and expressed in transgenic potato ( Chong
et al. , 1997 ) . The soybean ferritin gene was trans-
ferred into rice and the contents of ferritin were in-
creased by three-folds in seeds of T1 transgenic rice
plants . Thus, the contents of iron in transgenic rice
were improved . If peoplehad such transgenic rice, the
iron would be taken into the bodies ( Goto et al. ,
1999) . The gene encoded for respiratory syncytial vir-
us-F protein was introduced into tomato plants and
transgenic tomato plants were produced . The mice had
transgenic tomatoes, their systemic immuneresponse to
respiratory syncytial virus was induced and the specific
antibodywas produced .These results indicated that the
new method of the productionof oral respiratory syncy-
tial virus-F vaccine was developed ( Jagdeep et al. ,
2000) . The lysine-rich protein gene cloned from Pso-
phocarpus tetragonolobus, was transferred into rice and
lysine content in most of the transgenic plants, which
were obviously improved, andoneof themincreased by
16 .04% (Gao et al. , 2001) . These articles suggested
that the proteins and amino acids which human beings
needs were produced via the plant expressed system,
and the ingredients for healthwere improved, thus, the
high quality of theplant food also could beimproveup-
on . In addition, it was significant that the genes en-
coded special proteins rooted from animals or bacteria
were introduced into the plants and the expressed sys-
temsof production of these proteins were enlarged . In
this paper, the human alpha-lactalbumin gene was
transformed into tobacco in order to make some refer-
ence to research into the production and utilization of
humanα-LA .
3 . 2 YThe detection of the human alpha-lactalbu-
min in transgenic tobacco
In this paper, we carried out PCR analysis and
Southern blotting, but didnot doWestern blotting . We
will do it in future in order to study the expression of
the human alpha-lactalbumin gene at the protein level .
It is important to investigate the expression and regula-
tion of the integrated gene at protein level .
3 . 3 JThe function of extrinsic proteins in transgen-
ic tobacco plants
In this paper, transformation of the human alpha-
lactalbumin gene into tobacco was made and transgenic
tobacco plants were obtained, it is not enough . In fu-
ture, the function of the human alpha-lactalbumin ex-
pressed in transgenic tobacco plants would be further
studied, and what is the difference between the pro-
teins synthesized in two expressed systems, i . e ., ani-
mal expressed system and plant expressed system,
would be known and explored .
264 云 南 植 物 研 究 30 卷
Acknowledgement : Theauthor is very grateful to Prof . J ing Yu-
xiang at the Institute of Botany, Chinese Academyof Sciences for
his carefully reading themanuscript andconstructive suggestions .
He expresses his gratitude to Prof . Chen You-qiang and Mr . Li
Wen-mingat theCollege of LifeSciences, FujianNormal Univer-
sity for their kind help .
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