肠道微生物对仔猪胆汁酸谱及胆汁酸代谢的影响

doi:10.13560/j.cnki.biotech.bull.1985.2020-0269

摘要/Abstract

摘要：

旨在研究肠道微生物对仔猪肝脏、空肠内容物和粪便胆汁酸谱及仔猪胆汁酸代谢的影响。随机选择无菌剖腹产所得仔猪11头,分为无菌猪组（GF组,5头）和粪菌移植组（FMT组,6头）。GF组始终保持无菌状态,FMT组在7日龄时口服植入健康猪源粪菌。42日龄时,采集肝脏组织、空肠内容物、新鲜粪便用于胆汁酸定量,肝脏组织与空肠组织进行胆汁酸代谢相关基因定量。结果表明：相比于GF组,FMT组仔猪粪便中的总胆汁酸水平显著高于GF组（P<0.01）;FMT仔猪空肠内容物和粪便中次级胆汁酸占总胆汁酸的比例显著高于GF仔猪（P<0.05）,肝脏中也发现同样的趋势（P<0.05）;相比于GF仔猪,FMT仔猪肝脏中CYP7A1、BAT、FXR等与胆汁酸合成、结合与转运相关基因的下调,肠道中转运蛋白MRP2基因表达上调。由此可见,肠道微生物的转入与定植不仅能够增加仔猪胆汁酸的排泄,提高次级胆汁酸占比,改变仔猪胆汁酸谱,而且还影响胆汁酸的肝肠循环过程。

关键词: 无菌猪, 胆汁酸谱, 胆汁酸代谢

Abstract:

The aim of this study is to investigate the effects of gut microbiota on bile acid profile and bile acid metabolism in liver,jejunal contents and feces of pig models. Eleven piglets from aseptic caesarean section were randomly divided into germ-free（GF）group（5 piglets）and fecal microbiota transplantation（FMT）group（6 piglets）. GF group remained germ-free from beginning to end,and the FMT group was implanted with healthy porcine fecal bacteria at the age of 7 d. Liver,jejunal contents and fresh feces were collected for bile acid quantification at 42 d,as well as liver and jejunal tissues were collected for the quantification of genes related to bile acid metabolism. The results showed that：compared with the GF group,the total bile acid level in the piglet feces of the FMT group was significantly higher than that of the GF group（P<0.01）. The proportion of secondary bile acids in the jejunal contents and feces of the piglets in the FMT group to total bile acids was significantly higher than that in the GF group（P<0.05）,and the same trend was found in the liver（P<0.05）. Compared with the GF group,cholesterol 7-alpha hydroxy-lase gene（CYP7A1）,bile acid amino acid converting enzyme gene（BAT）,and farinol X receptor gene（FXR）,as well as genes related to bile acid synthesis and transport in the FMT group were down-regulated,while the expression of the intestinal transporter MRP2 gene was up-regulated. Thus,the transfer and colonization of gut microbiota can not only increase the bile acid excretion of piglets,increase the proportion of secondary bile acid,change the bile acid profile of piglets,but also affect the hepatointestinal circulation of bile acid.

Key words: germ-free pig, bile acid profile, bile acid metabolism

李梦颖, 周华, 丁玉春, 刘作华, 孙静, 李周权. 肠道微生物对仔猪胆汁酸谱及胆汁酸代谢的影响[J]. 生物技术通报, 2020, 36(10): 49-61.

LI Meng-ying, ZHOU Hua, DING Yu-chun, LIU Zuo-hua, SUN Jing, LI Zhou-quan. Effects of Gut Microbiota on Bile Acid Profile and Bile Acid Metabolism in Piglets[J]. Biotechnology Bulletin, 2020, 36(10): 49-61.

图/表 11

参考文献 35

[1]	Molinaro A, Wahlström A, Marschall H. Role of bile acids in metabolic control[J]. Trends in Endocrinology and Metabolism, 2018,29(1):31-41.
[2]	Ge X, Pan J, Liu Y, et al. Intestinal crosstalk between microbiota and serotonin and its impact on gut motility[J]. Current Pharmaceutical Biotechnology, 2018,19(3):190-195. URL pmid: 29804531
[3]	Li T, Chiang JY. Bile acid signaling in metabolic disease and drug therapy[J]. Pharmacological Reviews, 2014,66(4):948-983. URL pmid: 25073467
[4]	Russell DW. The enzymes, regulation, and genetics of bile acid synjournal[J]. Ann Rev Biochem, 2003,72(1):137-174.
[5]	Hofmann AF, et al. Key discoveries in bile acid chemistry and biology and their clinical applications:history of the last eight decades[J]. J Lipid Res, 2014,55(8):1553-1595.
[6]	Monte MJ, Marin JJ, Antelo A, et al. Bile acids:chemistry, physiology, and pathophysiology[J]. World Journal of Gastroenterology, 2009,15(7):804-816. URL pmid: 19230041
[7]	Wahlström A, Sayin SI, Marschall HU, et al. Intestinal crosstalk between bile acids and microbiota and its impact on host metabolism[J]. Cell Metabolism, 2016,24(1):41-50. URL pmid: 27320064
[8]	Sayin S, Wahlström A, Felin J, et al. Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring fxr antagonist[J]. Cell Metabolism, 2013,17(2):225-235. URL pmid: 23395169
[9]	Li J, Dawson PA. Animal models to study bile acid metabolism[J]. Biochimica Et Biophysica Acta, 2019,1865(5):895-911. URL pmid: 29782919
[10]	Guilloteau P, Zabielski R, et al. Nutritional programming of gastrointestinal tract development. is the pig a good model for man?[J]. Nutrition Research Reviews, 2010,23(1):4-22. URL pmid: 20500926
[11]	Wang M, Donovan SM. Human microbiota-associated swine:current progress and future opportunities[J]. Ilar Journal, 2015,56(1):63-73. URL pmid: 25991699
[12]	孙静, 杜蕾, 丁玉春, 等. 无菌猪的制备与微生物质量控制[J]. 中国实验动物学报, 2017,25(6):699-702.
	Sun J, Du L, Ding YC, et al. Breeding and microbiological quality control of germ-free pigs[J]. Acta Laboratorium Animalis Scientia Sinica, 2017,25(6):699-702.
[13]	Sun J, Du L, Li XL, et al. Identification of the core bacteria in rectums of diarrheic and non-diarrheic piglets[J]. Scientific Reports, 2019,9(1):18675. URL pmid: 31822779
[14]	Kim HB, Isaacson RE. The pig gut microbial diversity:understanding the pig gut microbial ecology through the next generation high throughput sequencing[J]. Veterinary Microbiology, 2015,177:242-251. URL pmid: 25843944
[15]	孙静, 杜蕾, 李晓蕾, 等. 粪便移植实验仔猪模型的构建方法:中国, CN201811024719. 3[P]. 2018-12-8.
	Sun J, Du L, Li XL, et al. Establishment of fecal microbiota transplantation model for piglets:China, CN201811024719. 3[P]. 2018-12-8.
[16]	Hu L, Geng S, Li Y, et al. Exogenous fecal microbiota transplantation from local adult pigs to crossbred newborn piglets[J]. Frontiers in Microbiology, 2018,8(2663):1-16.
[17]	Fang W, Zhang L, Meng Q, et al. Effects of dietary pectin on the profile and transport of intestinal bile acids in young pigs[J]. Journal of Animal Science, 2018,96(11):4743-4754. URL pmid: 30102377
[18]	Guo J, Zhao MH, Liang S, et al. Liver receptor homolog 1 influences blastocyst hatching in pigs[J]. The Journal of Reproduction and Development, 2016,62(3):297-303. URL pmid: 26971889
[19]	Ponsuksili S, Murani E, et al. Identification of functional candidate genes for body composition by expression analyses and evidencing impact by association analysis and mapping[J]. Biochimica Et Biophysica Acta, 2005,1730(1):31-40. doi: 10.1016/j.bbaexp.2005.06.004 URL pmid: 16005530
[20]	Haslewood GA. Bile salts of germ-free domestic fowl and pigs[J]. The Biochemical Journal, 1971,123(1):15-18. URL pmid: 5128663
[21]	Lin S, Yang X, Yuan P, et al. Undernutrition shapes the gut microbiota and bile acid profile in association with altered gut-liver fxr signaling in weaning pigs[J]. Journal of Agricultural and Food Chemistry, 2019,67(13):3691-3701. URL pmid: 30864445
[22]	Hamilton JP, Xie G, Raufman JP, et al. Human cecal bile acids:concentration and spectrum[J]. American Journal of Physiology Gastrointestinal and Liver Physiology, 2007,293(1):256-263.
[23]	Kwekkeboom J, Princen HM, Van Voorthuizen EM, et al. Bile acids exert negative feedback control on bile acid synjournal in cultured pig hepatocytes by suppression of cholesterol 7 alpha-hydroxylase activity[J]. Hepatology, 1990,12(5):1209-1215. doi: 10.1002/hep.1840120522 URL pmid: 2227820
[24]	Sayin SI, Wahlström A, Felin J, et al. Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring fxr antagonist[J]. Cell Metabolism, 2013,17(2):225-235. doi: 10.1016/j.cmet.2013.01.003 URL pmid: 23395169
[25]	Si GL, Yao P, Shi L. Rapid determination of bile acids in bile from various mammals by reversed-phase ultra-fast liquid chromatography[J]. Journal of Chromatographic Science, 2015,53(7):1060-1065. URL pmid: 25520305
[26]	Aldini R, Roda A, Montagnani M, et al. Relationship between structure and intestinal absorption of bile acids with a steroid or side-chain modification[J]. Steroids, 1996,61(10):590-597. doi: 10.1016/s0039-128x(96)00119-5 URL pmid: 8910972
[27]	Mi S, Lim DW, et al. Determination of bile acids in piglet bile by solid phase extraction and liquid chromatography-electrospray tandem mass spectrometry[J]. Lipids, 2016,51(3):359-372.
[28]	Long SL, Gahan CGM, Joyce SA. Interactions between gut bacteria and bile in health and disease[J]. Molecular Aspects of Medicine, 2017,56(8):54-65.
[29]	Dürre P, Andreesen J. Purine and glycine metabolism by purinolytic clostridia[J]. Journal of Bacteriology, 1983,154(1):192-199. URL pmid: 6833177
[30]	Xing M, Wei Y, Zhou Y. Radical-mediated C-S bond cleavage in C2 sulfonate degradation by anaerobic bacteria[J]. Nature Communications, 2019,10(1):1-11.
[31]	Chiang JYL. Bile acids:regulation of synjournal[J]. Journal of Lipid Research, 2009,50(10):1955-1966. URL pmid: 19346330
[32]	Cariello M, Piccinin E, Garcia-irigoyen O, et al. Nuclear receptor fxr, bile acids and liver damage:introducing the progressive familial intrahepatic cholestasis with fxr mutations[J]. Biochimica Et Biophysica Acta. Molecular Basis of Disease, 2018,1864(4):1308-1318.
[33]	Keitel V, Stindt J, Häussinger D. Bile acid-activated receptors:gpbar1(tgr5)and other G protein-coupled receptors[J]. Handbook of Experimental Pharmacology, 2019,256:19-49. URL pmid: 31302759
[34]	Gunness P, Williams BA, et al. Circulating triglycerides and bile acids are reduced by a soluble wheat arabinoxylan via modulation of bile concentration and lipid digestion rates in a pig model[J]. Molecular Nutrition & Food Research, 2016,60(3):642-651. URL pmid: 26694900
[35]	Gunness P, Michiels J, et al. Reduction in circulating bile acid and restricted diffusion across the intestinal epithelium are associated with a decrease in blood cholesterol in the presence of oat β-glucan[J]. FASEB Journal, 2016,30(12):4227-4238. URL pmid: 27630168

简称	英文名	中文名	结合/游离	初级/次级
TDCA	taurodeoxycholate	牛磺脱氧胆酸	T	S
TCA	taurocholic acid	牛磺胆酸	T	P
TCDCA	taurochenodeoxycholate	牛磺鹅脱氧胆酸	T	P
TLCA	taurolithocholate	牛磺石胆酸	T	S
TUDCA	tauroursodeoxycholic acid	牛磺熊脱氧胆酸	T	P
THDCA	taurohyodeoxycholic acid	牛磺猪脱氧胆酸	T	S
TαMCA	tauro α-muricholate	牛磺α鼠胆酸	T	S
TβMCA	tauro β-muricholate	牛磺β鼠胆酸	T	S
TωMCA	tauro ω-muricholate	牛磺ω鼠胆酸	T	S
THCA	taurohyocholate	牛磺猪胆酸	T	P
GCA	glycocholic acid	甘氨胆酸	G	P
GCDCA	glycochenodeoxycholate	甘氨鹅脱氧胆酸	G	P
GLCA	glycolithocholate	甘氨石胆酸	G	S
GUDCA	glycoursodeoxycholic acid	甘氨熊脱氧胆酸	G	S
GHDCA	glycohyodeoxycholate	甘氨猪脱氧胆酸	G	P
GHCA	glycohyocholate	甘氨猪胆酸	G	P
CDCA	chenodeoxycholic acid	鹅脱氧胆酸	U	P
isoLCA	isolithocholic acid	3β-羟基石胆酸	U	S
6-ketoLCA	6-ketolithocholic acid	6酮基石胆酸	U	S
7-ketoLCA	7-ketolithocholic acid	7酮基石胆酸	U	S
12-ketoLCA	12-ketolithocholic acid	12酮基石胆酸	U	S
6,7-diketoLCA	6,7-diketolithocholic acid	6,7-二酮石胆酸	U	S
7,12-diketoLCA	7,12-diketolithocholic acid	7,12-二酮石胆酸	U	S
dehydroLCA	dehydrolithocholic acid	去氢石胆酸	U	S
UDCA	ursodeoxycholic acid	熊脱氧胆酸	U	S
muroCA	murocholic acid	鼠胆酸	U	S
αMCA	α-muricholic acid	α鼠胆酸	U	S
βMCA	β-muricholic acid	β鼠胆酸	U	S
ωMCA	ω-muricholic acid	ω鼠胆酸	U	S
HCA	γ-muricholic acid\hyocholic acid	猪胆酸	U	P
12-DHCA	12-dehydrocholic acid	12-去氢胆酸	U	S
3-DHCA	3-dehydrocholic acid	3-去氢胆酸	U	S
apoCA	apocholic acid	原胆酸	U	S
NorDCA	23-nordeoxycholic acid	23-脱甲脱氧胆酸	U	S
CDCA-24G	Chenodeoxycholic acid 24-Acyl-β-D-glucuronide	鹅去氧胆酸-24-酰基-β-D-葡萄糖醛酸	U	P
UCA	ursocholic acid	熊果胆酸	U	S
βUDCA	3β-Ursodeoxycholic Acid	3β-熊脱氧胆酸	U	S
βHDCA	β-Hyodeoxycholic Acid	β-猪脱氧胆酸	U	S
βCA	3β-Cholic Acid	3β-胆酸	U	S
ACA	Allocholic acid	别胆酸	U	S
NorCA	Nor Cholic acid	正胆酸	U	S
CDCA-3G	Chenodeoxycholic acid-3-β-D-glucuronide	鹅去氧胆酸-3-β-D-葡萄糖醛酸	U	P
βUCA	β-ursocholic acid	β-熊果胆酸	U	S
βCDCA	β-chenodeoxycholic acid	β-鹅脱氧胆酸	U	P
βDCA	β-deoxycholic acid	β-脱氧胆酸	U	S
7-DHCA	7-dehydrocholic acid	7-去氢胆酸	U	S
DCA	deoxycholic acid	脱氧胆酸	U	S
CA	cholic acid	胆酸	U	P
LCA	lithocholic acid	石胆酸	U	S
HDCA	α-hyodeoxycholic acid	猪脱氧胆酸	U	P

简称	英文名	中文名	结合/游离	初级/次级
TDCA	taurodeoxycholate	牛磺脱氧胆酸	T	S
TCA	taurocholic acid	牛磺胆酸	T	P
TCDCA	taurochenodeoxycholate	牛磺鹅脱氧胆酸	T	P
TLCA	taurolithocholate	牛磺石胆酸	T	S
TUDCA	tauroursodeoxycholic acid	牛磺熊脱氧胆酸	T	P
THDCA	taurohyodeoxycholic acid	牛磺猪脱氧胆酸	T	S
TαMCA	tauro α-muricholate	牛磺α鼠胆酸	T	S
TβMCA	tauro β-muricholate	牛磺β鼠胆酸	T	S
TωMCA	tauro ω-muricholate	牛磺ω鼠胆酸	T	S
THCA	taurohyocholate	牛磺猪胆酸	T	P
GCA	glycocholic acid	甘氨胆酸	G	P
GCDCA	glycochenodeoxycholate	甘氨鹅脱氧胆酸	G	P
GLCA	glycolithocholate	甘氨石胆酸	G	S
GUDCA	glycoursodeoxycholic acid	甘氨熊脱氧胆酸	G	S
GHDCA	glycohyodeoxycholate	甘氨猪脱氧胆酸	G	P
GHCA	glycohyocholate	甘氨猪胆酸	G	P
CDCA	chenodeoxycholic acid	鹅脱氧胆酸	U	P
isoLCA	isolithocholic acid	3β-羟基石胆酸	U	S
6-ketoLCA	6-ketolithocholic acid	6酮基石胆酸	U	S
7-ketoLCA	7-ketolithocholic acid	7酮基石胆酸	U	S
12-ketoLCA	12-ketolithocholic acid	12酮基石胆酸	U	S
6,7-diketoLCA	6,7-diketolithocholic acid	6,7-二酮石胆酸	U	S
7,12-diketoLCA	7,12-diketolithocholic acid	7,12-二酮石胆酸	U	S
dehydroLCA	dehydrolithocholic acid	去氢石胆酸	U	S
UDCA	ursodeoxycholic acid	熊脱氧胆酸	U	S
muroCA	murocholic acid	鼠胆酸	U	S
αMCA	α-muricholic acid	α鼠胆酸	U	S
βMCA	β-muricholic acid	β鼠胆酸	U	S
ωMCA	ω-muricholic acid	ω鼠胆酸	U	S
HCA	γ-muricholic acid\hyocholic acid	猪胆酸	U	P
12-DHCA	12-dehydrocholic acid	12-去氢胆酸	U	S
3-DHCA	3-dehydrocholic acid	3-去氢胆酸	U	S
apoCA	apocholic acid	原胆酸	U	S
NorDCA	23-nordeoxycholic acid	23-脱甲脱氧胆酸	U	S
CDCA-24G	Chenodeoxycholic acid 24-Acyl-β-D-glucuronide	鹅去氧胆酸-24-酰基-β-D-葡萄糖醛酸	U	P
UCA	ursocholic acid	熊果胆酸	U	S
βUDCA	3β-Ursodeoxycholic Acid	3β-熊脱氧胆酸	U	S
βHDCA	β-Hyodeoxycholic Acid	β-猪脱氧胆酸	U	S
βCA	3β-Cholic Acid	3β-胆酸	U	S
ACA	Allocholic acid	别胆酸	U	S
NorCA	Nor Cholic acid	正胆酸	U	S
CDCA-3G	Chenodeoxycholic acid-3-β-D-glucuronide	鹅去氧胆酸-3-β-D-葡萄糖醛酸	U	P
βUCA	β-ursocholic acid	β-熊果胆酸	U	S
βCDCA	β-chenodeoxycholic acid	β-鹅脱氧胆酸	U	P
βDCA	β-deoxycholic acid	β-脱氧胆酸	U	S
7-DHCA	7-dehydrocholic acid	7-去氢胆酸	U	S
DCA	deoxycholic acid	脱氧胆酸	U	S
CA	cholic acid	胆酸	U	P
LCA	lithocholic acid	石胆酸	U	S
HDCA	α-hyodeoxycholic acid	猪脱氧胆酸	U	P

饲粮组成/%	含量	营养水平
普通玉米,CP 7.8%	14.10	CP,%	19.00
膨化玉米,CP 7.8%	10.00	DE,Kcal/kg	3 622.00
去皮豆粕,CP 47.13%	13.65	NE,Kcal/kg	2 645.00
膨化大豆,CP 35.5%	7.00	SID Lys,%	1.23
大豆浓缩蛋白,CP 65.2%	5.00	SID Met,%	0.36
乳清粉,CP 3.26%	5.00	SID Thr,%	0.73
鱼粉,65.2%	3.00	SID Trp,%	0.20
血浆蛋白粉,CP 76.66%	3.00	Ca,%	0.70
葡萄糖	2.00	P,%	0.60
大豆油	1.40	AP,%	0.45
玉米淀粉	29.70
纤维	3.00
石灰石	0.45
磷酸氢钙	1.25
L-Lys（78%）	0.29
DL-Met（99%）	0.12
L-Thr（98.5%）	0.13
L-Trp（98%）	0.01
维生素-矿物质预混料	0.50
氯化钠	0.25
氯化胆碱	0.15
总计	100.00

饲粮组成/%	含量	营养水平
普通玉米,CP 7.8%	14.10	CP,%	19.00
膨化玉米,CP 7.8%	10.00	DE,Kcal/kg	3 622.00
去皮豆粕,CP 47.13%	13.65	NE,Kcal/kg	2 645.00
膨化大豆,CP 35.5%	7.00	SID Lys,%	1.23
大豆浓缩蛋白,CP 65.2%	5.00	SID Met,%	0.36
乳清粉,CP 3.26%	5.00	SID Thr,%	0.73
鱼粉,65.2%	3.00	SID Trp,%	0.20
血浆蛋白粉,CP 76.66%	3.00	Ca,%	0.70
葡萄糖	2.00	P,%	0.60
大豆油	1.40	AP,%	0.45
玉米淀粉	29.70
纤维	3.00
石灰石	0.45
磷酸氢钙	1.25
L-Lys（78%）	0.29
DL-Met（99%）	0.12
L-Thr（98.5%）	0.13
L-Trp（98%）	0.01
维生素-矿物质预混料	0.50
氯化钠	0.25
氯化胆碱	0.15
总计	100.00

基因	上游引物（5'-3'）	下游引物（5'-3'）	产物长度/bp	参考文献
GAPDH	CCAGGGCTGCTTTTAACTCTG	GTGGGTGGAATCATACTGGAACAT	104	[16]
CYP7A1	GAAAGAGAGACCACATCTCGG	GAATGGTGTTGGCTTGCGAT	123	[17]
CYP4A21	GATCCAGGAACTGCATTGGGA	CACAATTCCTTGAATAGGAACGGG	128
CYP27A1	ACTGAAGACCGCGATGAAAC	CAAAGGCGAATCAGGAAGGG	106	[17]
FXR	TATGAACTCAGGCGAATGCCTGCT	ATCCAGATGCTCTGTCTCCGCAAA	154	[17]
LRH1	GGTACCACTATGGGCTCCTCAC	TCGGCCCTTACCGCTTCT	193	[18]
OATP1	TGCTTAAAACTCCCACCCCT	GTTTGCTTCATTCACGGCTT	120	[19]
BESP	CGCAGCGTGAAGAAATGTGG	AAAACCGAAACAGTTGAAAGAGGC	136
FGFR4	GCTCAGAGGTGGAGGTCCTA	GCCTGCCAGACAGGTGTATT	74	[17]
BAT	GGCTGATGATCCGAGAAGGG	ATGCCCCCAAACAAGTCGAT	94	[17]
KLβ	GCACCGAGTGGAAGGAGT	TTGCCAGTAGGAAGGATTG	150	[17]
MRP2	GAACAGGTTTGCTGGCGATATT	GCCAGGAGCGCAAAGACA	65	[17]
SHP	ACCATTCTCTTCAACCCTGATGT	GCTGCGAGGAGAACACGAG	149
HNF4α1	GCCTCTCCAAAACCCTGGTC	CTGATGGGGATGTGTCATTGC	126
BACS	CTGGCTCCCTGCCTATGCT	GAACGTGCTTGTGGTCTCCAA	70	[17]
NTCP	TGTCATCAAGGGAGGAACGA	CGAGCATTGAGGCGGAAAAG	189
ASBT	AAGTTCCTGGGGCACGTAAA	CTCCTGGACAGCATCCCATT	161	[17]
OSTα	TGTACAAGAACACTCGCTGC	GAACACACACACTATCGTGGG	80	[17]
FGF19	AAGATGCAAGGGCAGACTCA	AGATGGTGTTTCTTGGACCAGT	101
IBABP	GCAAGGAGTGCGACATAGAGAC	TGGTGGTAGTTGGGGCTGTT	100
MRP3	TGGACAAAGGGACAATAGCTGAGT	TGGCCATCCCGTAGAAGATG	78	[17]
TGR5	TGCTGTCCCTCATCTCATTGG	TGTGTAGCGATGATCACCCAG	80	[17]