全 文 ：广 西 植 物 Guihaia 25(4)：349— 352 2005年 7月
拟南芥 AtPSK3基因的克隆及序列分析
林俏慧1，谢秀祯1，2，郭 勇1
(1．华南理工大学生物科学与工程学院，广东广州 510640#2．海南师范大学生物系，海南海 口 571158)
摘 要；根据拟南芥基因组数据库提供的信息，首次以特异引物经 PCR技术克隆到拟南芥硫肽激素一a的一
个前体基因——A￡PSK3，并对其进行了测序 。序列分析表明 ，所获得的 AtPSK3基 因全长为 505 bp，含有一
个内含子和两个没有 3 一或 5 一非转译区的外显子，与数据库提供的序列比较，同源性为 100％。
关键词：AtPSK3基因；基因克隆 ；DNA序列分析
中图分类号：Q813．6 文献标识码 ：A 文章编号 ：1000—3142(2005)04—0349—04
Study on the amplification and sequencing
LIN Qiao—hui ，XIE Xiu—zhenI，2，GUO YongI
(1．Department of Biotechnology，South China University of Technology，Guangzhou 510640，China；
2．Department of Biology。Hainan Normal University，Haikou 571 158，China)
Abstract：A Phytosulfokine-a gene(AtPSK3)was amplified from genomic DNA of Arabidopsis by PCR，based
on the sequence information from Arabidopdis genome database，and its complete DNA sequence was ana-
lyzed．Results indicated that the AtPSK3 gene contained 505 base pairs，consisting of one large intron and two
exons without 3 一or 5 UTR sequence，the sequence of which has fl homology of 100 percent as compared with
the reported sequence．
Key words：AtPSK3 gene；gene clone；DNA sequence analysis
Phytosulfokine—ct(PSK—a)，fl sulfated pen—
tapeptide growth factor universally found in both
monocotyledons and dicotyledons，was originaly i—
solated from conditioned medium(CM )of asparagus
(Asparagus ofifcinalis)mesophyll cell cultures
(Yang et a1．，2000)． PSK—a has gained increasing
attention recently because of its unique biological
activities，such as strongly promoting the prolifera—
tion and differentiation of plant cells in low density
culture at low concentration(M atsubayashi et a1．，
l999；Hanai et a1．，2000)，stimulating somatic em—
bryogenesis and the adventitious bud and root for—
mfltion from callus of plant (Kobayashi et a1．，
1999；Yang et a1．，1999)， and enhancing the
growth and chlorophyl content of seedling (Ya—
makawa et a1．，1999)．
The sequencing and analysis of Arabidopsis
genome were completed at the end of 2000(Rouns—
ley et a1．，2000)．It was reported that four genes
encoding precursors of PSK—a had been identified
from Arabidopsis with the BLAST program using
the amino acid sequence of PSK—a(Yang et a1．。
2001)． Analysis of cDNAs for two of these。At—
PSK2 and AtPSK3．showed that both of them con—
Received date：2004—07—08 Accepted date：2004—12—09
Foundation item：Supported by the Key Technology Research and Development of Guangdong Province(Grant No． A3O LO2O2OI)
First author：LIN Qiao-hui(1981-)，female，born in Sanshui，Guangdong，master candidate Major：Cel and Molecular Biology．E—
mail：Irene-lin@21cn．corn． Corresponding author，E—mail：btyguo@scut．edu．cn
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35O 广 西 植 物 25卷
sist of two exons and one intron．AtPSK2 and At-
PSK3 were expressed demonstrably not only in
cuIt ured cells but also in intact plants，suggesting
that PSK—a may be essential for plant cell prolifer-
ation 竹vivo as wel as in vitro．0verexpression of
either precursor gene allowed the transgenic calli to
grow twice as large as the controls．
In t his paper， the AtPSK3 gene encoding fl
precursor of Phytosulfokine—a was obtained by
PCR from genomic DNA of Arabidopsis for the
first time，using the sequence information from Ar—
abidopsis genome database，and we hope the estab—
lishment of this system could provide fin ideal mod—
el for further studies of its biological activities in
vitro，especially its stimulative functions to plants．
1 M aterials and methods
1．1 Plant materials
Arabidopsis seeds(ecotype Columbia)were
supplied by Professor J ianru Zuo(Institute of Ge—
netics and Developmental Biology，Chinese Acade—
my of Sciences)．
After being immersed in hot water(50~52℃)
for 30 min，Arabidopsis seeds were disinfected
with 70 (v／v)ethanol for 3～5 min，washed three
times with sterile distilled water，transferred to
lO (w／v)NaOCl for 15 min，followed by five rin—
ses with sterile distilled water． The sterile seeds
were cultured on 30 mL solid B5 medium without
hormone in 250 mL flask at 25℃ in the 16 h light／
8 h dark cycles． About 2O—day-old germinated
seedlings of Arabidopsis were prepared for extrac—
tion of genomic DNA．
1．2 Extraction of genomic DNA
The genomic DNA was extracted from Arabi—
dopsis seedlings according to the method of CTAB
(Wang eta1．，2002)．The DNA molecular size was fin—
alyzed by 0．8 9，6(w／v)agarose gel electrophoresis．
1．3 PCR amplification
Primers were designed based on the nucleic
acid sequence of AtPSK3 gene in TAIR(The Ara—
bidopsis Information Resource)Database(TAIR ac—
cession nos．AT3G4978o．1)with the Primer Prem—
ier 5．0 program and synthesized by Shanghai Bioa—
sia Biotechnology Co．，
follows：P1：5’一TCGT
TA AGTTCACAAC 一3’
Ltd． The primers were as
TCTAGA TCAGTATGC~ -
and P2：5’一 C GAGCTC
TTAGGGCTTGTGATTCTGAGT一3’，which had
XbaI site and Sad site，respectively．
PCR amplification was performed using Gene—
Amp PCR system 2 400(Perkin—Elmer)．Total vol—
ume of PCR reaction system was 50 L，including 1
× Taq DNA polymerase buffer，10 L genomic
DNA of Arabidopsis as template，100 nM primer
P1 and P2，respectively，200 M dNTP，1．5 mM
M gC12 and 2．5 U Taq DNA polymerase(TaKaRa)．
Hot start procedure was adopted．The parameters
of the PCR reaction were：96℃ for 5 min，94℃ for
lmin，55℃ for 1 min，72℃ for lmin with 1 cycle，94
℃ for lmin，58℃ for lmin，72℃ for lmin with 29
cycles。and fl final extention for 72℃ for 10 min．
After detected by 1．O 9，6(w／v)agarose gel elec—
trophoresis，the PCR products were purified by
DNA Purification Kit (Dingguo Bioengineering
Co．，Ltd．)，double—digested with XbaI and SacI
(TaKaRa)，followed by gel—purification．Then the
ligation of the DNA fragments with the plasmid
pUCI 9 which was treated by the same methods
was preformed with T4 DNA ligase at 16℃ over
night．The ligation products were used to trans-
form E．coli JM1O9．W hite colonies were selected
on solid LB medium (pre—spread with 40 L of 2O
mg／mL X—Gal and 4 L of 200 mg／mL IPTG)con—
taining 100 mg／L ampicillin．
1．4 Sequence analysis
After identified by PCR amplification，the re—
combinants were sequenced by Shanghai Bioasia Bi—
otechnology Co．，Ltd．
2 Resuhs and discussion
2．1 Extraction of Arabidopsis genomic DNA
An over 21 kb genomic DNA of Arabidopsis
was obtained by the method of CTAB(Plate I—A)，
which was large enough to be used for the AtPSK3
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4期 林俏慧等 ：拟南芥 AtPSK3基因的克隆及序列分析 35l
gene amplification．
2．2 CloningAtPSK3 gene
The genomic DNA from Arabidopsis was used
as template for PCR reaction and a fragment about
530 bp was obtained (Plate I—B，lane 2 and lane
3)．After optimization，we got more AtPSK3 gene
and less nonspecific amplification products(Plate
I—B，lane 2)than before(Plate工一B，lane 3)．We
may draw a conclusion that the specific amplifica—
tion products are consistent with the length of the
reported AtPSK3 gene．
2．3 Identificati0n of the recombinant clones
The products double—digested with XbaI and
Sad were recovered from an agarose gel and liga—
ted into plasmid pUC1 9． Many colonies were ob—
tained after transformation．Plasmids were isolated
from these bacteria and analyzed by PCR． An a—
bout 530 bp DNA fragment was obtained when the
1：
2：
recombinant plasmid pUC-AtPSK3 was used as tern—
plate for PCR reaction(PlateI-C，lane 2)；nothing but
some nonspecific amplification products was got when
the pUC1 9 plasmid was used as template(Plate I-C，
lane 1)．PlateI-C shows that the recombinant plasmid
maybe contains the target gene．
2．4 Analysis of nucleotide sequence of the AtPSK3
gene
To further confirm the inserted fragment，a
DNA sequence analysis was carried out．The result
(Fig．1)proved that the obtained fragment indeed
was the AtPSK3 gene． It contained 505 base
pairs，consisting of one intron(265 bp)and two ex—
ons(1l7 bp and 123 bp)without 3-or 5 一UTR se—
quence．The sequence of the AtPSK3 gene we ob—
tained from the Arabidopsis had a homology of
i 00 as compared with that of the AtPSK3 gene
reported in Arabidopsis genome database．
TCTAGATCAGTATGGGTAAGTTCACAACCATTTTCATCATGGCTCTCCTTCTTTGCTCTA 6O
ATGGGTAAGTTCACAACCATTTTCATCATGGCTCTCCTTCTTTGCTCTA
1：CGCTAACCTACGCAGCAAGGCTGACTCCGACGACAACCACCGCTTTGTCCAGAGAAAACT 1 2O
2：CGCTAACCTACGCAGCAAGGCTGACTCCGACGACAACCACCGCTTTGTCCAGAGAAAACT
1：CCGTCAAGGTTCGTTAACTTCTTTGTCTTTTTCAGTATAGTACTAGTCGAAACATATCTG 18O
2：CCGTCAAG g t t c g t t a a c t t c t t t g t c t t t t t c a g t a t a g t a c t a g t c g a a a c a t a t c t g
1：CAATTGCAAAACAAAGAATTAATCTATCGCAGTATATGTCAAAGTTTCTATATATAGTAC 240
2：c a a t t g c a a a a c a a a g a a t t a a t c t a t c g c a g t a t a t g t c a a a g t t t c t a t a t a t a g t a c
1：AAAACAAAAAACCAAAAAGAGTTTGCATGCATGCTCCTTAAGATTTGTTTCGTGTAATAG 300
2：a a a a c a a a a a a c c a a a a a g a g t t t g c a t g c a t g c t c c t t a a g a t t t g t t t c g t g t a a t a g
1：ATTATATAATATCACACGATTTGTTTATTTGTTACCGCGGTAGTTTAGAAATTAACACCG 360
2：a t t a t a t a a t a t c a c a c g a t t t g t t t a t t t g t t a c c g c g g t a g t t t a g a a a t t a a c a c c g
1：ACGTTCATATGTTGTTGTATATATTATGTATAGGAAATTGAAGGAGACAAGGTTGAAGAA 420
2：a c g t t c a t a t g t t g t t g t a t a t a t t a t g t a t a g GAAATTGAAGGAGACAAGGTTGAAGAA
1：GAAAGCTGCAACGGAATTGGAGAAGAAGAATGTTTGATAAGACGAAGCCTTGTTCTTCAC 480
2：GAAAGCTGCAACGGAATTGGAGAAGAAGAATGTTTGATAAGACGAAGCCTTGTTCTTCAC
1：ACCGATTACATTTATACTCAGAATCACAAGCCCTAA GAGCTC 522
2：ACCGATTACATTTATACTCAGAATCACAAGCCCTAA
Fig． 1 Comparison of nucleotide sequence
1：Sequence of insert fragment(underlined：Xba I and Sac I sites)；2：Sequence of reported AtPSK3 gene(capital：exon，lowercase：intron)．
Anthocyanin is an important kind of dye for
food，makeup and medicine，and it is also a remedy
for many diseases．In recent years，anthocyanin is
obtained by the culture of the Roselle calli to short—
an the yield period，unfortunately however，the
Roselle calli’s growth period is still too long．The
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352 广 西 植 物 25卷
transfer of AtPSK3 gene into Roselle cell is a po—
tential method to stimulate the proliferation of
Roselle cell in low density． In this paper，we have
successfully isolated the AtPSK3 gene from Ara—
bidopsis genomic DNA by PCR，and the sequence
analysis revealed that the obtained AtPSK3 gene is
identical to that in Arabidopsis genome database．
We hope it could be the first step towards con—
structing a transgenic Roselle cell line with rapid
proliferation．
3 Acknowledgements
This research was financially supported by the
Key Technology Research and Development of
Guangdong Province(Grant No．A30102020i)．W e
are grateful to Professor Jianru Zuo at Institute of
Genetics and Dvelopmental Biology(Chinese Acad—
emy of Sciences)for the supply of Arabidopsis
seeds(ecotype Columbia)．
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