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遗 传 学 报 Acta Genetica Sinica, September
2006, 33 (9)：808–813
ISSN 0379-4172
The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep
HUANG Zhi-Guo1,2, XIONG Li2, LIU Zhen-Shan1, QIAO Yong1, DAI Rong3, XIE Zhuang1, ①,
LIU Shou-Ren1,3, SHI Guo-Qing3, LIU Guo-Qing1,3,①
1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China;
2. Liquor-making Biotechnology&Application Key Laboratory of Sichuan Province, Sichuan University of Science&Engineering,
Zigong 643000, China;
3. Xinjiang Reclamation Science Institute, Shihezi 832001, China
Abstract: Male Kazak sheep and Xinjiang fine wool sheep, six for each different age group (days 2, 30, 60, 90 and 120), were used
in the present study to investigate the tissue distribution and developmental changes of ghrelin mRNA expression in abomasum;
however, there was no 120-day-old Kazak sheep. After measurement of body weight, the tissues such as hypothalamus, pituitary,
heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle were sampled. And the total RNA of
different tissues was extracted to determine the abundance of ghrelin mRNA by RT-PCR and real-time PCR. The results showed
that (1) for both breeds, body weight among different ages was significantly different (P<0.05). And from day 30 to 90, the body
weight of Kazak was significantly higher than that of Xinjiang (P<0.01); (2) Ghrelin mRNA existed in all the above tissues and was
significantly higher in the abomasum than in other tissues (P<0.05); (3) the temporal patterns of abomasum ghrelin mRNA expression in Kazak and Xinjiang were similar. From day 2 to 60 in Kazak and 2 to 90 in Xinjiang, there was a steady increase in the
ghrelin mRNA level. By day 60 in Kazak and day 90 in Xinjiang, the level reached a plateau and remained steady. These results
also demonstrated that from birth to day 90, ghrelin mRNA level was significantly higher in Kazak than in Xinjiang (P<0.01).
Key words: sheep; ghrelin; abomasum; real-time PCR
Ghrelin, initially found in rat stomach tissue in
1999 by Kojima [1], is a brain-gut peptide containing
28 amino acids and exhibits growth hormone-releasing activities. Ghrelin was originally discovered as the endogenous ligand of growth hormone
secretagogue receptor (GHSR) that could stimulate
the release of growth hormone (GH) after binding to
GHSR. However, it was soon established that ghrelin
had a role in weight regulation because its administration increased food intake and caused fat and
weight gain in rodents. Further studies found that
ghrelin was involved in cardiac and gastrointestinal
functions, cell proliferation, sleep, and so on.
Human prepro-ghrelin displayed 82.9% homol-
ogy with that of rat and both consisted of 117 amino
acids. The described ovine prepro-ghrelin cDNA (520
bp) codes for 116 amino acids, of which 27 amino
acids from the 24th to the 50th residue from the
N-terminus are mature ghrelin sequences [2]. Ghrelin
was secreted primarily by stomach, and in situ hybridization indicated that ghrelin mRNA existed in
the region from the neck to the base of the oxyntic
gland [1]. Other tissues with ghrelin expression included hypothalamus, intestine, pituitary, liver, kidney, placenta, pancreas, testicle, and so on. There are
many articles about the ghrelin gene in humans, rodents, and pigs, but in ruminants, ghrelin gene such
as ovine ghrelin has rarely been reported.
Received: 2005-10-28; Accepted: 2005-12-07
This work was supported by Doctor Foundation of Xinjiang Construction Corps (No. 2003-02).
①
Corresponding author. E-mail: [email protected]; Tel: +86-25-8439 5046; Fax: +86-25-8439 5314.
HUANG Zhi-Guo et al.: The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep
In this article, male Kazak sheep and Xinjiang
fine wool sheep, with different growth rates during
the early growth period, were selected for investigation of the tissue distribution and developmental
changes of ghrelin mRNA expression.
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ment of body weight, animals were slaughtered for tissue sampling, which included hypothalamus, pituitary,
heart, liver, rumen, reticulum, omasum, abomasum,
duodenum, and the longissimus dorsi muscle. The removed samples were snap-frozen in liquid nitrogen and
then stored at −80℃ for total RNA analysis later.
1
Materials and Methods
1. 1
1. 2
Animals
Twenty-four male Kazak sheep and 30 male Xinjiang fine wool sheep, six for each different age group
(days 2, 30, 60, 90, and 120), were selected from the
Ziniquan Sheep Pasture in Shihezi City of Xinjiang
Autonomous Region for the present study; however,
there was no 120-day-old Kazak sheep. After measure-
Primer design
According to the published sequences of ovine
ghrelin and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA, oligonucleotide primer sets for
the two genes were designed using Primer premier 5.0
software and described in detail in Table 1. GAPDH
was used as an internal standard for the determination of
targeted mRNA levels.
Table 1 Parameters of gene-specific primers for ghrelin and GAPDH
Target genes
GenBank accession number
ghrelin
AB060699
Primer sequence
Product size
(bp)
Annealing temperature
205
58
379
58
(℃)
F: 5′-GGAAGTCAGGAGGAAGGTG-3′
R: 5′-GGGAGAACAGACAGGTGGT-3′
F: 5′-ACTTTGGCATCGTGGAGG-3′
GAPDH
AF030943
R: 5′-GAAGAGTGAGTGTCGCTGTTG-3′
were assessed by its optical density ratio at 260/280
2.5 mmol/L Spermidine), and 0.4 mmol/L each of
dNTP. RNA sample, random primer, dNTP, and sterile H2O (final volume 10 µL) were first mixed in a
0.5 mL microcentrifuge tube and incubated at 70℃
for 5 min, and cooled on ice for 2 min. The rest of
the reagents were then added into the reaction tube to
a final volume of 25 µL and incubated at 37℃ for 1 h.
The reaction was terminated by heating at 95℃ for 5
min and quickly cooled on ice. RT products were
nm (OD260/OD280=1.8−2.0) and by electrophoresis
stored at −20℃.
with ethidium-bromide staining.
1. 3. 3 Polymerase chain reaction (PCR)
RT products (0.5 µL) were amplified in a 10 µL
PCR reaction containing 1 U Taq DNA polymerase
(TaKaRa, Inc. Dalian, China), 1 µL of 10× PCR
buffer (100 mmol/L Tris-HCl pH 8.3, 500 mmol/L
KCl), 0.25 mmol/L each of dNTP, 1.25 mmol/L
MgCl2, and 0.5 µmol/L each of gene-specific primers.
The following amplification conditions were
used: one cycle of 1 min at 94℃ followed by 40 PCR
1. 3
Total RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)
1. 3. 1
Total RNA extraction
Total RNA was extracted using the acid-guanidinium
thiocyanate/phenol chloroform extraction method
[3]
.
The extracted RNA was dissolved in DEPC-treated
water and the concentration, purity, and integrity
1. 3. 2 Reverse transcription (RT)
Two micrograms of total RNA was used for reverse transcription in a final volume of 25 µL containing 200 U MMLV reverse transcriptase (Promega,
Inc. Madison, USA), 20 U RNase inhibitor (Promega,
Inc. Madison, USA), 1 µg of random primer, 5 µL of
5× RT buffer (250 mmol/L Tris-HCl pH 8.3, 50
mmol/L MgCl2, 250 mmol/L KCl, 50 mmol/L DTT,
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810
cycles of 30 s at 94℃, 30 s at the annealing temperature of the primers, 30 s at 72℃, and a final extension
for 5 min at 72℃. Correct length of the products was
confirmed on a 8% polyacrylamide gel, which was
subsequently analyzed with a computer flatbed scanner after silver staining.
1. 4 Cloning and sequence analysis of the amplified fragments
PCR products were excised after being confirmed by electrophoresis on a 1% agarose gel and
purified by V-gene DNA Purification Kit (V-gene
Biotechnology Ltd., Hangzhou, China) according to
the manufacturer’s manual and then cloned into
pMD18-T simple vector. Subsequently, the ligation
products were transformed into JM109 cells. Positive
clones based on blue-white selection were picked out
for plasmid extraction by V-gene Kit (V-gene Biotechnology Ltd., Hangzhou, China) according to the
manufacturer’s recommendation and then identified
by PCR using gene-specific primers. Plasmids containing inserts of the right size were sequenced by Invitrogen Biotechnology Co., Ltd. (Shanghai, China).
Acta Genetica Sinica
Vol.33
No.9
2006
The reactions were repeated twice for every
sample. Plasmid DNA with the targeted DNA fragment was diluted to gradient concentrations, which
were used to draw quantitative standard curves.
1. 6
Statistical analyses
Data were described as x ± Sd and statistics
was analyzed using SPSS11.5 For Windows Software.
Differences of body weight and gene expression level
between ages in the same breed, and those at the same
age between the two breeds were analyzed by one-way
ANOVA and independent-samples t-test, respectively.
2
2. 1
Results
Cumulative growth curves of sheep
Results showed that for both breeds, body
weight among different ages was significantly different (P<0.05). And from day 30 to 90, the body weight
of Kazak was significantly higher than that of Xinjiang (P<0.01) (Fig. 1).
1. 5 Real-time polymerase chain reaction (real-time
PCR)
The abundance of ghrelin mRNA was assessed
by real-time reverse transcription polymerase chain
reaction using a fluorescence temperature cycler
(DNA Engine Opticon Real-time PCR Systems, MJ
Research, Inc., Waltham, Massachusetts, USA). The
final reaction volume was 20 µL containing 1 µL of
the RT products, 1 U EX Taq HS DNA polymerase
(TaKaRa Inc., Dalian, China), 4 µL of 5× PCR buffer,
0.3 mmol/L each of dNTP, 3.75 mmol/L MgCl2, 0.5
µmol/L each of primers, and 1 µL of 20× SYBR
green. PCR conditions were as follows: one cycle of
1 min at 95℃; 45 PCR cycles of 10 s at 95℃, 10 s at
the annealing temperature of the primers, 15 s at 72℃,
plate-reading; this was followed by an extension of
10 min at 72℃; plate-reading every other 0.2℃ from
65℃ to 94℃ for drawing melting curves; and then
the reaction was stopped with an extension of 5 min
at 72℃.
Fig. 1
Cumulative growth curves of male Kazak
sheep(HSK) and Xinjiang fine wool sheep(XJXM)
Significant difference is denoted with letters (the capital for
Xinjiang and the small for Kazak) and means without a common superscript indicate significant differences (P<0.05) between ages in the same breed. Double stars (**) indicate extreme differences (P<0.01) between breeds at the same age.
2. 2
RT-PCR of ghrelin and GAPDH genes
Total RNA from the abomasum of a Kazak sheep
was used as an initial sample to amplify ghrelin and
GAPDH genes by RT-PCR, which gave rise to a 205 bp
and a 379 bp cDNA fragment, respectively (Fig. 2).
HUANG Zhi-Guo et al.: The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep
Fig. 2 RT-PCR of ghrelin and GAPDH mRNA in abomasums
1, 2: ghrelin; 3, 4: DNA marker pUC18; 5, 6: GAPDH.
2. 3 Sequence analysis of the amplified fragments
The amplified ghrelin and GAPDH cDNA fragments were then cloned into pMD18-T simple vector.
After ligation, the products were identified by PCR
using gene-specific primers (Fig.3), with those
plasmids containing inserts of the right size being
sequenced. The sequences of the amplified fragments
were aligned by DNAstar software with the corresponding reported sequences according to which the
gene-specific primers were designed. The results
showed that (1) there was 99.51% sequence identity
between the amplified ghrelin gene cDNA fragment
and the published ovine ghrelin gene sequence; (2)
there was 100% sequence identity for the GAPDH
gene. These results indicated that the amplified
cDNA fragments of the two genes were gene-specific
products.
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Fig. 3 PCR of ghrelin-pMD18-T and GAPDH-pMD18-T
1: ghrelin; 2,3: DNA marker pUC18; 4: GAPDH.
2. 4 Distribution of ghrelin mRNA expression in
various tissues
Two-day-old male Kazak sheep were used to
investigate the distribution of ghrelin mRNA expression in various tissues including hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle by
RT-PCR. It was found that ghrelin mRNA existed in
all of the above tissues (Fig. 4). Further study using
real-time PCR (Fig. 5) showed that ghrelin mRNA
level was significantly higher in the abomasum than
in other tissues (P<0.05) (Table 2).
Fig. 4 RT-PCR of ghrelin gene in sheep various tissues
1: pituitary; 2: hypothalami; 3: heart; 4: liver; 5: muscle; 6:
duodenum; 7: rumen; 8: reticulum; 9: omasum; 10: abomasum; 11: DNA marker pUC18.
Fig. 5 The amplification, standard, and melting curves of ghrelin and GAPDH
A, B, and C are the amplification, standard, and melting curves of ghrelin, respectively; D, E, and F are the amplification, standard,
and melting curves of GAPDH, respectively.
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Table 2
Acta Genetica Sinica
Vol.33
No.9
2006
Abundance of ghrelin mRNA in various tissues
Tissues
Rumen
Heart
ghrelin/GAPDH ( x ± Sd )
0.103±0.04
Tissues
Muscle
ghrelin/GAPDH ( x ± Sd )
a
Liver
0.115±0.044
a
Hypothalamus
a
0.263±0.048
0.415±0.05
a
Omasum
0.128±0.041
a
Pituitary
Reticulum
0.142±0.057
a
Duodenum
ab
1.000±0.397
0.138±0.041a
Abomasum
5.852±0.827
b
116.443±13.201c
Note: Significant difference is denoted by letters and means without a common superscript differ significantly between tissues
(P<0.05).
2. 5
Developmental changes of ghrelin mRNA
expression in sheep abomasum
Real-time PCR was used to quantify ghrelin
mRNA levels in the abomasa of Kazak and Xinjiang
fine wool sheep at different ages (Fig. 5). Results
showed that the temporal patterns of abomasum ghrelin mRNA expression in Kazak and Xinjiang were
similar. From day 2 to 60 in Kazak and 2 to 90 in
Xinjiang, there was a steady increase in the ghrelin
mRNA level. By day 60 in Kazak and day 90 in Xinjiang, the level reached a plateau and remained steady.
These results also demonstrated that from birth to day
90 ghrelin mRNA level was significantly higher in
Kazak than in Xinjiang (P<0.01) (Fig.6).
in various sheep tissues, including the hypothalamus,
pituitary, heart, liver, rumen, reticulum, omasum,
abomasum, duodenum, and longissimus dorsi muscle,
although it was expressed primarily in abomasum.
The predominant expression of ghrelin mRNA in
abomasum was consistent with results found in humans, rats, and so on.
An in vitro study of rat showed that ghrelin specifically activated GHSR to stimulate GH release
[4]
and caused significant release of GH-releasing hormone (GHRH), but exerted no effect on the release of
somatostatin (SS) [5]. Nakazato et al.[6] also found that
intravenous administration of ghrelin to freely moving rats caused a dose-dependent increase in GH release. Furthermore, the stimulatory effect of ghrelin
on GH-release was more intense than that of GHRH
and hexarelin
[7]
. Fletcher et al.[8] found that ghrelin
was able to stimulate GH release, but not GH synthesis. Another study discovered that the effects of ghrelin on food intake increase were dose-dependent,
even in GH-deficient rats [9], which suggested that the
effects on food intake were independent of those on
GH secretion. Chronic ghrelin administration has
been shown to increase body fat content in rodents [10],
which suggests that it may be related to adipogenesis
Fig. 6 The developmental changes of abomasum ghrelin
mRNA expression in male Kazak sheep(HSK) and Xinjiang fine wool sheep(XJXM)
Significant difference is denoted by letters (the capital for
Xinjiang and the small for HSK) and means without a common superscript differ significantly (P<0.05) between ages in
the same breed; double stars (**) indicate extreme differences
(P<0.01) between breeds of the same age.
3
Discussion
It was found that ghrelin mRNA was expressed
and storage of energy. These data predict that ghrelin
gene may play an important role in the process of
food intake, weight regulation, and growth.
The present research showed that abomasum
ghrelin mRNA level increased gradually during early
postnatal sheep development, a pattern that was similar to the changes observed in the cumulative growth
curves. It was further found that from birth to day 90,
ghrelin mRNA level in the abomasum of Kazak was
significantly higher than that of Xinjiang, which was
HUANG Zhi-Guo et al.: The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep
in line with the fact that the body weight of Kazak
was significantly higher (P<0.01) than that of Xinjiang during the same period. All these data suggested
that ghrelin gene might be closely related to the
growth and development of sheep. The present research established the basis for further studies on the
effect of ghrelin gene on the growth and development
of sheep and their regulatory mechanisms.
References:
[1] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth hormone releasing acylated peptide from stomach. Nature, 1999, 402(6762) : 656-660.
[2] http://www.ncbi.nlm.nih.gov/entrez/viewer.fegi?db=
protein&val=30140885.
[3] GAO Qin-Xue. Study on the characteristics of histology
and molecular biology of intramuscular adipose in growing Erhualian pigs [Dissertation]. Nanjing Agricultural
University, 2003.
[4] Hayashida T, Murakami K, Mogi K, Nishihara M, Nakazato M, Mondal M S, Horii Y, Kojima M, Kangawa K,
Murakami N. Ghrelin in domestic animals: distribution in
stomach and its possible role. Domest Anim Endocrinol,
2001; 21(1) : 17-24.
[5] Takaya K, Ariyasu H, Kanamoto N, Iwakura H, Yoshi-
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moto A, Harada M, Mori K, Komatsu Y, Usui T, Shimatsu A, Ogawa Y, Hosoda K, Akamizu T, Kojima M,
Kangawa K, Nakao K. Ghrelin strongly stimulates
growth hormone release in humans. J Clin Endocrinol
Metab, 2000, 85(12) : 4908-4911.
[6] Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H,
Kangawa K, Matsukura S. A role for ghrelin in the central
regulation of feeding. Nature, 2001, 409(6817) : 194-198.
[7] Masuda Y, Tanaka T, Inomata N, Ohnuma N, Tanaka S,
Itoh Z, Hosoda H, Kojima M, Kangawa K. Ghrelin
stimulates gastric acid secretion and motility in rats. Biochem Biophys Res Commun, 2000, 276(3) : 905-908.
[8] Fletcher T P, Thomas G B, Clarke I J. Growth hormone-releasing hormone and somatostatin concentrations
in the hypophysial portal blood of conscious sheep during
the infusion of growth hormone-releasing peptide-6. Domest Anim Endocrinol, 1996, 13(3) : 251-258.
[9] Shintani M, Ogawa Y, Ebihara K, Aizawa-Abe M, Miyanaga F, Takaya K, Hayashi T, Inoue G, Hosoda K, Kojima
M, Kangawa K, Nakao K. Ghrelin, an endogenous
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of hypothalamic neuropeptide Y/Y1 receptor pathway.
Diabetes, 2001, 50(2) : 227-232.
[10] Tschöp M, Smiley D L, Heiman M L. Ghrelin induces
adiposity in rodents. Nature, 2000, 407(6806) : 908-913.
绵羊 ghrelin 基因表达的组织分布和发育性变化
黄治国 1,2，熊 俐 2，刘振山 1，乔 永 1，代 蓉 3，谢 庄 1，刘守仁 1,3，石国庆 3，刘国庆 1,3
1. 南京农业大学动物科技学院，南京 210095；
2. 四川理工学院酿酒生物技术及应用四川省重点实验室，自贡 643000；
3. 新疆农垦科学院畜牧兽医研究所，石河子 832000
摘 要：选取2、30、60、90和120日龄的雄性哈萨克羊和新疆细毛羊各6只（无120日龄的哈萨克羊），测体重后屠宰，采下
丘脑、垂体、心脏、肝脏、瘤胃、网胃、瓣胃、皱胃、十二指肠、背最长肌，用RT-PCR和荧光实时定量PCR法检测ghrelin
基因表达的组织分布，及其在皱胃中的发育性变化。研究结果表明：（1）品种内各生长时期的体重差异显著（P<0.05）。
雄性哈萨克羊和新疆细毛羊的体重在2日龄时无显著差异（P>0.05），30～90日龄间，前者的体重极显著高于后者（P<0.01）；
（2）所检测的各组织中都有 ghrelin mRNA 分布，但主要在皱胃中表达，其表达量远高于其他组织（P<0.05）；（3）两品
种绵羊皱胃 ghrelin 基因表达的发育性变化模式基本相似，都随着日龄的增加而呈上升趋势，其中雄性哈萨克羊的表达量
在2～60日龄间持续上升，60日龄后趋于水平；雄性新疆细毛羊的表达量在2～90日龄间持续上升，90日龄后趋于水平。研
究还发现雄性哈萨克羊皱胃 ghrelin 基因的表达量在2～90日龄间极显著高于新疆细毛羊（P<0.01）。
关键词：绵羊；ghrelin；皱胃；荧光实时定量 PCR
作者简介：黄治国（1978-），男，博士，研究方向：分子数量遗传学。E-mail: [email protected]