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中华补血草Syntaxin基因的克隆(英文)



全 文 :Cloning of Syntaxin Gene in Limonium sinense
Kuntzet
Ying ZHANG, Shihua CHEN*, Huiling HAN, Weihong DOU, Haibo YIN, Jiqiang ZHAO, Shanli GUO*
College of Life Sciences, Yantai University, Yantai 264005, China
Supported by National Natural Science Foundation of China (30870199); Shandong
Natural Science Foundation (Y2007D34; ZR2011CM006); Key Projects of Shandong
Natural Science Foundation (2010GNC10937).
*Corresponding author. E-mail: csh-yantai@163.com; gsl@ytu.edu.cn
Received: November 10, 2011 Accepted: December 16, 2011A
Abstract [Objective] The aim was to clone Syntaxin genes in Limonium sinense
Kuntze. [Method] Limonium sinense Kuntze leaves were used as materials and total
RNA was extracted and transcribed reversely. Nested primers were designed based
on EST sequences at 5’ region of Syntaxin, and cDNA obtained through reverse
reaction was taken as the template. Sequences of Syntaxin gene at 3’ region were
obtained through two rounds of PCR amplifications. [Result] DNA fragments (1 096
bp) were obtained. For LsSyntaxin, open reading frame (ORF) was 816 bp and the
encoded amino acids were 271. The relative molecular weight of Syntaxin was 30
254.3 Da and isoelectric point in theory was 5.55. [Conclusion] Syntaxin genes from
Limonium sinense Kuntze were cloned. The research laid foundation for the study
on Syntaxin gene function in Limonium sinense Kuntze and salt-secreted process.
Key words Limonium sinense Kuntze; 3’ Race; Syntaxin gene; Clone
Agricultural Science & Technology, 2012, 13(2): 261-264
Copyright訫 2012, Information Institute of HAAS. All rights reserved Agricultural Biotechnology
S yntaxin owns structural domainof SNARE and regulates vesic-ular transport inside cells (fu-
sion of membrane vesicle with target
membrane)[1]. In animal cells, this kind
of proteins participate berth and fusion
of synaptic vesicles and are related to
release of exocytosis and neurotrans-
mitter. In addition, members of the
family could influence working of im-
mune system and release of insulin
through regulation on vesicle transport
and fusion[2].
In plant, there are a lot of mem-
bers in Syntaxin subfamily. They take
part in formation of cell plate[3], inter-
action with iron channel protein, dis-
ease resistance, and plant gravit-
ropism through vesicular transporta-
tion[4]. However, there is report avail-
able on Sy-ntaxin participation in plant
responses to salt stress.
EST analysis was conducted on
Syntaxin of Limonium sinense Kuntze
(LsSyntaxin) in cDNA expression li-
brary of Limonium sinense Kuntze
constructed under salt stress and the
result indicated that expression of the
gene in Limonium sinense Kuntze was
induced by salt stress. However, re-
search is none on molecular mecha-
nism of the gene responses to salt
stress in plants.
Limonium sinense Kuntze, herba-
ceous plant in Limonium of Plumbage-
naceae[5], grows and distributes in salt
soil in coast area and sandy soil in
Hulun Buir of Inner Mongolia Au-
tonomousRegion of China[6]. The plant
can excrete excessive salt from body
through salt gland to adapt to high-salt
environment. Observation of cellular
level indicated that salt-secreted pro-
cess was related to vesicular transport
and fusion. Nevertheless, research is
few on molecular mechanism of plant
salt-secretion and genes participating
in salt-secretion. We cloned LsSyn-
taxin genes based on primers of EST
sequences of Syntaxin genes, which
laid foundation for research on func-
tion of the genes in Limonium sinense
Kuntze and effect in salt-secretion.
Materials and Methods
Materials
Seedlings of Limonium sinense
Kuntze were cultivated in greenhouse
of College of Life and Sciences, Yantai
University; cloning vectors such as
pMDl8-T, DL2000 Marker, RNAiso
RNA Extraction Kit and PrimeScript Ⅱ
1ST Strand cDNA Synthesis Kit were
all purchased from TaKaRa; agarose
gel Recovery Kits were purchased
from TianGen (Beijing); DH5α (Es-
cheriachia coli) , were preserved in lab
and KOD DNA polymerases were pur-
chased from Baori Biotechnology Co.,
Ltd.; the rest reagents were domestic
analytical reagent; containers for RNA
extraction in the experiment were all
treated with DEPC water for 2 weeks
followed by sterilization and drying in
reserve.
Extraction and reverse transcrip-
tion of total RNA
Seedlings of Limonium sinense
Kuntze were treated with 3% of NaCl
for 24 h; 200 mg leaves were taken to
mix with liquid nitrogen; after grinding,
the powders were moved to an EP
tube (1.5 ml) which contained 1 ml of
RNAiso RNA extraction reagents; after
mixed well, the solution was placed at
room temperature for 5 min and cen-
trifuged at 12 000 r/min for 5 min at 4
℃ ; the supernatants were transferred
to a new centrifugal tube (1.5 ml) and
300 ml chloroforms were added fol-
lowed by well mixing (20 -30 times);
after centrifugation (at 12 000 r/min) at
4 ℃ for 10 min, the supernatants were
taken to a new centrifugal tube (1.5
ml); the precooled Isopropyl Alcohol
(double volumes) were added and
mixed well; the solution was placed at
-20℃; after centrifugation (at 12 000
r/min) at 4 ℃ for 10 min, the super-
natant was discarded; 1 ml ethanol
DOI:10.16175/j.cnki.1009-4229.2012.02.031
Agricultural Science & Technology
Agricultural Science & Technology Vol.13, No.2, 2012
2012
(75%) was added to wash sediments;
after centrifugation (at 12 000 r/min) at
4 ℃ for 1 min, the supernatant was
discarded again; the sediments were
placed on ice and then dried in super-
clean bench; 10 -30 ml of RNase-free
water was added to dissolve the pre-
cipitation. After electrophoresis, the
extracted RNA was transcribed and
synthesized to cDNA using Oligo(dT)25-
AP as primer in accordance with in-
structions of PrimeScriptⅡ lST Strand
cDNA Synthesis Kit.
Primer sequence
Based on EST sequences of
Syntaxin at gene 5’ region in cDNA
expression library, nest PCR primers
at 5’ region were designed with Primer
premier 5.0 as follows:
The primers were synthesized by
Beijing Genomic Center (BGI).
Syntaxin gene sequences at 3’ re-
gion cloned through 3’ RACE
The process was under guidance
of BD SMART RACE cDNA AmpIifi-
cation Kit (Clontech) instruction.
1st amplification of Syntaxin gene
sequences at 3’ region cDNA ob-
tained through reverse transcription
was taken as a template; F1 and Oligo
(dT)25-AP were used as upstream and
downstream primers, respectively;
KOD was used for 1st PCR amplifica-
tion. The reaction system (25 μl) was
as follows: 2.5 μl of 10*Buffer; 1 μl of
dNTPs; 1 μl of template (PCRprod-
ucts); 0.5 μl of Taq enzyme; 1 μl of F1; 1
μl Oligo(dT)25-AP; 18 μl of sterile water.
PCR amplification conditions were as
follows: 30 cycles of 94℃ 5 min, 94℃
45 s, 55℃ 45 s, 72℃ 90 s final exten-
sion at 72℃ for 10 min.
2nd amplification of Syntaxin gene
sequences at 3’ region The PCR
products in 1st round were used as
templates and combinations of F2 and
AP were used for 2nd PCR amplifica-
tion (Tm was raised to 56℃). Through
two PCR amplifications, amplification
products through 3’ RACE were ob-
tained. After electrophoresis, PCR
products were purified.
Ligation and transformation
The kits were used to collect and
purify objective bands amplified
through PCR. The bands were then
ligated to pMD18-T vector at 16 ℃
overnight. The reaction system (10 μl)
included: 1 μl of Solution I, 1 μl of
T-Vector, and 8 μl of PCR products.
Ligation products were transformed
into competent cell DH5a for overnight
culture in incubator at 37 ℃ . Single
colonies were chosen for culture. After
enzyme digestion, the positive clones
were chosen and sent to BGI for
sequencing.
Results and Analysis
Clone and sequence analysis of
Syntaxin genes in 3’ region
Nest PCR products in 3’ region in
the 1st round were under electrophore-
sis and the result indicated that PCR
produced non-specific bands distribut-
ed in areas about 1 kb (Fig.1).
PCR products in the 1st round
were taken as templates and combi-
nation of F2 and AP was under 2nd
PCR. Results of electrophoresis indi-
cated that the 2nd round PCR produced
specific products bands distributed in
about 1 kb areas.
Sequencing result
pMD18-T (ligating with PCR prod-
ucts), the positive cloning plasmids,
were under DNA sequence and 1 066
bp nucleotide fragments were obtained
(Fig.2).
Sequence analysis of PCR products
Open reading frames and se-
quences comparison and analysis
of cloned products Open reading
frames (ORF) of Syntaxin were ob-
tained through ORF of NCBI (http://
www.ncbi.nlm.nih.gov/gorf/gorf.html).
ORF contains 816 bp and untranslated
region at 3’s regionwas222bp (Fig.2).
Blast (http://www.ncbi.nlm.nih.gov/
BLAST/) was used for comparison and
analysis of nucleotide sequence and
the encoded amino acid sequence
(Fig.3) and the results indicated that
the similarity syntaxins of 271 amino
acids amplified from Limonium sinense
Kuntze shared with that of Glycine max
and Solanum tuberosum was 70%.
Prediction of protein family on the
genes through Pfam 25.0 (http://pfam.
sanger.ac.uk/search/sequence) indi-
cated that this kind of gene was Syn-
taxin in membrane complex super-
BXC-Syntaxin-F1 GTAAAGAAGAGAGAGAGCGACG
BXC-Syntaxin-F2 AAGACAGAGAGGGCAGAAAGA
Oligo(dT)25-AP GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTTTTTTTTTT
AP GACTCGAGTCGACATCG
1 stood for DNA marker 2 000; 2 stood for
PCR products using F1 and Oligo (dT) as
primers; 3 stood for 1 000 objective bands
amplified with F2 and AP as primers.
Fig.1 Results of electrophoresis in two
rounds PCR with KOD enzyme
The underlined ones stood for F1 primer sequence and underlined ones in bold stood for AP
primer sequences; data between ATG in bold and TGA in bold stood for Syntaxin open
reading frame.
Fig.2 PCR products sequencing results using F2 and AP as upstream and downstream
primers
262
Agricultural Science & Technology
Vol.13, No.2, 2012 Agricultural Science & Technology
2012
Responsible editor: Xiaoxue WANG Responsible proofreader: Xiaoyan WU
In abscissa, - srood for hydrophilicity, and + stood for hydrophobicity.
Fig.4 Hydrophobicity profile of Syntaxin from Limonium sinense Kuntze
family. The protein included a single
transmembrane domain (TD) at C ter-
minal, a SNARE domain (H3) and a
regulatory domain at N-terminal (Habc).
Attributive analysis of encoded pro-
tein in cloned products ProtParam
(http://web.expasy.org/protparam/)
and SignalP3.0Server (http://www.cbs.
dtu.dk/services/SignalP/) were used to
analyze molecular weight, isoelectric
point and signal peptide sequence of
proteins encoded by the genes. Pre-
diction result through ProtParam indi-
cated that relative molecular mass of
Lssyntaxin was 30 254.3; isoelectric
point in theory was 5.55; the unstability
index was 38.46; the protein was un-
stable acidic protein. Prediction result
through SignalP3.0Server indicated
that protein sequences encoded by the
genes did not have signal peptide se-
quences. Considering preprotein is
usually guided by signal peptide to ex-
tracellular domain, the Syntaxin were
proved non-secretory protein.
ProScale (http://web.expasy.org/cgi -
bin/protscale/protscale.pl) was used to
conduct hydrophobicity analysis and
the preferences were as per Hphob. /
Kyte & Doolittle’s (Fig.4).
Conclusions and Discus-
sions
The experiment result indicated
that gene sequences of LsSyntaxin in
LsSyntaxin were obtained through 3’
RACE amplification. Primer combina-
tions of F1 and Oligo (dT)25-AP were
used to amplify LsSyntaxin. After PCR
amplification, the dispersed bands
were obtained. PCR specificity was
raised and ideal specific objective
bands were obtained with increase of
Tm and combination of F2 and AP.
Syntaxin is closely related to
vesicular transport of eukaryotic cell.
For animal and plant, Syntaxin be-
longs to SNARE, which is the same as
SNAP-25 and Synaptobrevin (or
Vamp). The protein is closely related
to vesicular transport and fusion. Pre-
sent researches indicate that the Syn-
taxin play roles in neurotransmitter re-
lease, hormone excrine, and vesicular
transport and fusion.
In plant, there are a lot of mem-
bers in syntaxin subfamily. Research
on plant mutants showed that the Syn-
taxin influence plant physiological pro-
cess, such as cell division, disease-re-
sistance, salt-resistance and response
to plant gravitropism through extensive
participation in vesicular transport[4, 7-8].
Plant reaction signals for defending re-
sponse take part in vesicle transloca-
tion (through endocytosis) and target-
ed fusion through participation in syn-
taxin regulation[9-11].
Vesicular transport and fusion in-
side of plant cells are also closely relat-
ed to plant response to salt stress.
Vacuoles can compartment excessive
ion entering cells, and vacuole occur-
rence is closely related to vesicle fu-
sion. Halophytes can secrete salt from
body through structure of salt gland in
response to high salt stress. Analysis
on morphological structure and physi-
ology indicated that salt -secreting is
closely related to intracellular transport
and fusion. However, there is little in-
formation available about salt secre-
tion of plant, syntaxin effect in salt se-
cretion for plant and related influence
in transport and fusion in molecular
aspect. Cloning of syntaxin genes in
Limonium sinense Kuntze has laid
foundation for analysis of cellular lo-
calization, and gene function compari-
son between halophytes and glyco-
phyte.
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263
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Agricultural Science & Technology Vol.13, No.2, 2012
2012
仙人掌多糖结构分析及抗凝血活性研究
蔡为荣 1,谢亮亮 1,陶鹏飞 2,崔武卫 3* (1.安徽工程大学生化与化学工程学院,安徽芜湖 241000;2.南陵县质量技术监督局,安徽南陵 242400;
3.加拿大农业与农业-食品部圭尔夫食品研究中心,加拿大安大略湖州 N1G5C9)
摘 要 [目的]分析仙人掌多糖组分和结构,并研究其抗凝血活性。[方法] 以米邦塔仙人掌为试验材料,通过热水浸提、醇沉制得粗多糖 CP,经交
换层析和凝胶层析纯化,研究其单糖组成和重均分子质量分布与主链结构,并对多糖组分抗凝血活性进行了初步研究。[结果] 对仙人掌粗多糖分
离纯化后共得到 3个多糖组分WSP1、WSP2a和WSP3,重均分子量分别为 2.32×106、1.24×106和 7.92×106。对氨基苯甲酸(PMP)衍生化高效液相色
谱法检测表明,WSP1主要成分为半乳糖;WSP2a由鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖、半乳糖、葡萄糖醛酸和半乳糖醛酸组成,含量分别为
6.3%、32.3%、12.9%、1.5%、4.8%、37.1%、0.64%和 4.4%;WSP3主要成分为阿拉伯糖、半乳糖和木糖以及少量鼠李糖、甘露糖、糖醛酸。WSP2能延长
活化部分凝血酶原时间 (APTT) 和凝血酶时间 (TT) ,而对凝血酶原时间 (PT) 的影响不显著,说明其是通过影响内源性凝血系统而发挥抗凝血
作用。纤维蛋白原转化纤维蛋白的抑制试验初步表明WSP2a和WSP3具有抗凝血活性。甲基化分析显示WSP2a含有 1,4-连接的半乳糖酸,1,2-
连接的鼠李糖和 1,2,4-连接的鼠李糖构成的主链。[结论] 该研究为仙人掌多糖资源优化和深度开发提供了试验依据。
关键词 米邦塔仙人掌;抗凝血;多糖结构
基金项目 国家自然科学基金资助项目(31171753),安徽省国际科技合作计划项目(10080703035)。
作者简介 蔡为荣(1963-) ,男,安徽芜湖人,教授,博士,主要从事天然产物功能因子的研究。E-mail; weirongcai0781@sina.com。*通讯作者,教授,
主要从事多糖结构与功能。
收稿日期 2011-11-15 修回日期 2011-12-04
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
Responsible editor: Na LI Responsible proofreader: Xiaoyan WU
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et al. Chemical characterization of the
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(Continued from page 260)
中华补血草 Syntaxin基因的克隆
张莹,陈世华 *,韩会玲,窦伟红,尹海波,赵吉强,郭善利 * (烟台大学生命科学学院,山东烟台 264005)
摘 要 [目的] 对中华补血草 Syntaxin基因进行克隆。[方法] 以中华补血草叶片为材料,提取总 RNA并进行反转录反应。依据已知 Syntaxin基
因 5’端 EST序列信息设计巢式引物,以反转录得到的 cDNA为模板,利用 2轮 PCR扩增 cDNA 3’末端(3’RACE),获得 Syntaxin基因 3’端序列。
[结果]获得 1 090 bp的 DNA片段。分析表明,该片段编码 LsSyntaxin全长基因,其中开放阅读框长 816 bp,编码 271个氨基酸。该基因编码的
Syntaxin蛋白相对分子量为 30 254.3 Da,理论等电点为 5.55。[结论] 为研究 Syntaxin基因在补血草中的功能及其在补血草泌盐过程的作用奠定
了基础。
关键词 中华补血草;3’Race;Syntaxin基因;克隆
基金项目 国家自然科学基金(30870199);山东省自然科学基金 (Y2007D34);山东科技攻关项目(2010GNC10937)。
作者简介 张莹(1985-),女,山东济宁人,在读硕士,研究方向:植物抗逆分子生物学。*共同通讯作者,副教授,博士,从事植物发育分子生物学
研究(E-mail: csh-yantai@163.com);郭善利,教授,博士,从事植物发育分子生物学与基因工程研究,E-ail:gsl@ytu.edu.cn。
收稿日期 2011-11-10 修回日期 2011-12-16

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