牦牛<i>FTL</i>基因克隆、生物信息学及表达分析

doi:10.7668/hbnxb.20194239

华北农学报 ›› 2023, Vol. 38 ›› Issue (6): 192-200. doi: 10.7668/hbnxb.20194239

所属专题： 畜牧；

• 畜牧·水产·兽医 • 上一篇下一篇

牦牛FTL基因克隆、生物信息学及表达分析

张娟香¹^,², 马小勇², 路建卫³, 仁青吉⁴, 罗胜伟⁵, 肖光峰⁵, 王有鹤⁵, 马晓明¹, 喇永福¹, 郭宪¹, 褚敏¹, 阎萍¹, 梁春年¹

¹ 中国农业科学院兰州畜牧与兽药研究所,甘肃省牦牛繁育工程重点实验室,甘肃兰州 730050
² 甘肃农业大学动物科学技术学院,甘肃兰州 730070
³ 农业农村部青藏高原畜禽遗传育种重点实验室,甘肃兰州 730050
⁴ 合作市佐盖多玛乡畜牧站,甘肃合作 747000
⁵ 合作市动物疫病预防控制中心,甘肃合作 747099

收稿日期:2023-05-05 出版日期:2023-12-28
通讯作者:
梁春年(1973—),男,甘肃武威人,研究员,博士,博士生导师,主要从事牦牛遗传与育种研究。
作者简介:
张娟香(1998—),女,甘肃陇南人,在读硕士,主要从事畜牧研究。
基金资助:
甘肃省科技计划项目(20JR5RA580); 甘肃省科技重大项目(21ZD10NA001); 甘肃省科技重大项目(GZGG-2021-1); 合作市牦牛种质提升与提质增效项目; 现代农业(肉牛牦牛)产业技术体系建设专项资金(CARS-37); 中国农业科学院科技创新工程(25-LZIHPS-01)

Cloning,Bioinformatics Analysis and Expression Analysis of FTL Gene in Yak

ZHANG Juanxiang¹^,², MA Xiaoyong², LU Jianwei³, REN Qingji⁴, LUO Shengwei⁵, XIAO Guangfeng⁵, WANG Youhe⁵, MA Xiaoming¹, LA Yongfu¹, GUO Xian¹, CHU Min¹, YAN Ping¹, LIANG Chunnian¹

¹ Lanzhou Institute of Husbandry and Pharmaceutical Sciences of Chinese Academy of Agricultural Sciences,Key Laboratory of Yak Breeding Engineering of Gansu Province,Lanzhou 730050,China
² College of Animal Science and Technology,Gansu Agricultural University,Lanzhou 730070,China
³ Key Laboratory of Animal Genetics and Breeding on the Tibetan Plateau,Ministry of Agriculture and Rural Affairs,Lanzhou 730050,China
⁴ Animal Husbandry Station of Zogaidoma Township,Hezuo,Hezuo 747000,China
⁵ Hezuo City Animal Disease Prevention and Control Center,Hezuo 747099,China

Received:2023-05-05 Published:2023-12-28

摘要/Abstract

摘要：

旨在了解牦牛铁蛋白轻链(FTL)基因的结构与功能,以及在不同年龄段8个组织中的表达规律。以成年(36月龄)美仁牦牛肝脏组织cDNA为模板,克隆FTL基因的编码区序列,并进行生物信息学分析,利用实时荧光定量(qRT-PCR)技术检测FTL基因在心脏、肝脏、脾脏、肺脏、肾脏、背最长肌、脂肪、睾丸中的相对表达量。结果表明,美仁牦牛FTL基因的CDS区为528 bp,共编码175个氨基酸,与野牦牛和牛的亲缘关系最近(99.8%);FTL蛋白为稳定亲水性分泌蛋白,不存在信号肽和跨膜结构,主要分布在细胞质中,蛋白的二级结构以α-螺旋为主,主要与FTH1、SCARA5、NCOA4等蛋白存在相互作用。qRT-PCR结果显示, FTL基因在成年牛的表达量依次为肝脏、肾脏、睾丸,脾脏、肺脏、脂肪、背最长肌、心脏,在7日龄犊牛的脂肪和背最长肌组织中表达最高,成年牛中肝脏组织表达量最高;通过2个阶段比对发现,脂肪、背最长肌和心脏组织的表达量随美仁牦牛年龄的增长呈下降趋势,而肺脏、脾脏、睾丸、肾脏和肝脏组织的表达量随年龄的增长而升高。

关键词: 美仁牦牛, FTL基因, 克隆, 生物学分析, 组织表达

Abstract:

The aim of this study was to understand the structure and function of ferritin light chain(FTL)gene in yak and its expression pattern in eight tissues of different age groups.The coding sequence(CDS)of FTL gene was cloned from the cDNA of liver tissue of adult(36 months old)Meiren yak,and bioinformatics analysis was carried out.The relative expression of FTL gene in heart,liver,spleen,lung,kidney,longissimus dorsi,fat and testis was detected by Real-time fluorescence quantitative(qRT-PCR).The results showed that the CDS region of FTL gene of Meiren yak was 528 bp,encoding 175 amino acids,and had the closest relationship with wild yak and cattle(99.8%).FTL protein was a stable hydrophilic secretory protein without signal peptide and transmembrane structure.It was mainly distributed in the cytoplasm.The secondary structure of the protein was mainly α-helix,which mainly interacted with FTH1,SCARA5,NCOA4 and other proteins.The results of qRT-PCR showed that the expression levels of FTL gene in adult cattle were liver,kidney,testis,spleen,lung,fat,longissimus dorsi and heart,respectively.The expression levels of FTL gene in fat and longissimus dorsi tissues of 7-day-old calves were the highest,and the expression level of FTL gene in liver tissue of adult cattle was the highest.By comparing the two stages,it was found that the expression levels of fat,longissimus dorsi and heart tissues decreased with the increase of age,while the expression levels of lung,spleen,testis,kidney and liver tissues increased with age.

Key words: Meiren yak, FTL, Gene cloning, Biological analysis, Tissue expression

张娟香, 马小勇, 路建卫, 仁青吉, 罗胜伟, 肖光峰, 王有鹤, 马晓明, 喇永福, 郭宪, 褚敏, 阎萍, 梁春年. 牦牛FTL基因克隆、生物信息学及表达分析[J]. 华北农学报, 2023, 38(6): 192-200. doi: 10.7668/hbnxb.20194239.

ZHANG Juanxiang, MA Xiaoyong, LU Jianwei, REN Qingji, LUO Shengwei, XIAO Guangfeng, WANG Youhe, MA Xiaoming, LA Yongfu, GUO Xian, CHU Min, YAN Ping, LIANG Chunnian. Cloning,Bioinformatics Analysis and Expression Analysis of FTL Gene in Yak[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(6): 192-200. doi: 10.7668/hbnxb.20194239.

http://www.hbnxb.net/CN/Y2023/V38/I6/192

图/表 16

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基因 Gene	引物序列(5'-3') Primer sequences(5'-3')	产物长度/bp Product size	退火温度/℃ Annealing temperature	用途 Application
FTL	F: TTTTCCAGTCGCAACCTTCGG	664	54.9	RT-PCR
	R: GTTAGGTGGGTTCTTCAATGG
FTL	F: ACCTACCTCTCTCTGGGCTT	120	58.0	qRT-PCR
	R: CAGTTTCAAGAGACGCTCCG
GAPDH	F: AATGAAAGGGCCATCACCATC	204	59.0	qRT-PCR
	R: GTGGTTCACGCCCATCACA

基因 Gene	引物序列(5'-3') Primer sequences(5'-3')	产物长度/bp Product size	退火温度/℃ Annealing temperature	用途 Application
FTL	F: TTTTCCAGTCGCAACCTTCGG	664	54.9	RT-PCR
	R: GTTAGGTGGGTTCTTCAATGG
FTL	F: ACCTACCTCTCTCTGGGCTT	120	58.0	qRT-PCR
	R: CAGTTTCAAGAGACGCTCCG
GAPDH	F: AATGAAAGGGCCATCACCATC	204	59.0	qRT-PCR
	R: GTGGTTCACGCCCATCACA

工具Online tools	功能Analysis content	网址Website
ORF Finder	开放阅读框分析	https://www.ncbi.nlm.nih.gov/orffinder/
Blast	同源性比对分析	https://blast.ncbi.nlm.nih.gov/Blast.cgi
ProtParam	蛋白质理化性质分析	https://web.expasy.org/protparam
ProtScale	蛋白质亲疏水性分析	https://web.expasy.org/protscale
SignalP 4.1	蛋白质信号肽分析	https://services.healthtech.dtu.dk/services/SignalP-4.1/
TMHMM 2.0	蛋白质跨膜结构预测	https://services.healthtech.dtu.dk/service.php?TMHMM-2.0
NetPhos 3.1	蛋白磷酸化位点预测	https://services.healthtech.dtu.dk/service.php?NetPhos-3.1
NetNGlyc	蛋白糖基化位点预测	https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0
PSORT Ⅱ	蛋白亚细胞定位预测	https://www.genscript.com/psort.html
SOPMA	蛋白质二级结构预测	https://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html
STRING	蛋白互作网络预测	STRING: functional protein association networks (string-db.org)
SWISS-MODEL	蛋白质三级结构预测	https://swissmodel.expasy.org/interactive

工具Online tools	功能Analysis content	网址Website
ORF Finder	开放阅读框分析	https://www.ncbi.nlm.nih.gov/orffinder/
Blast	同源性比对分析	https://blast.ncbi.nlm.nih.gov/Blast.cgi
ProtParam	蛋白质理化性质分析	https://web.expasy.org/protparam
ProtScale	蛋白质亲疏水性分析	https://web.expasy.org/protscale
SignalP 4.1	蛋白质信号肽分析	https://services.healthtech.dtu.dk/services/SignalP-4.1/
TMHMM 2.0	蛋白质跨膜结构预测	https://services.healthtech.dtu.dk/service.php?TMHMM-2.0
NetPhos 3.1	蛋白磷酸化位点预测	https://services.healthtech.dtu.dk/service.php?NetPhos-3.1
NetNGlyc	蛋白糖基化位点预测	https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0
PSORT Ⅱ	蛋白亚细胞定位预测	https://www.genscript.com/psort.html
SOPMA	蛋白质二级结构预测	https://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html
STRING	蛋白互作网络预测	STRING: functional protein association networks (string-db.org)
SWISS-MODEL	蛋白质三级结构预测	https://swissmodel.expasy.org/interactive

氨基酸 Amino acid	数量/个 Amount	含量/% Content	氨基酸 Amino acid	数量/个 Amount	含量/% Content
丙氨酸Ala (A)	15	8.6	亮氨酸Leu (L)	28	16.0
精氨酸Arg (R)	13	7.4	赖氨酸Lys (K)	10	5.7
天冬酰胺Asn(N)	8	4.6	甲硫氨酸Met(M)	4	2.3
天冬氨酸Asp(D)	12	6.9	苯丙氨酸Phe(F)	8	4.6
半胱氨酸Cys(C)	1	0.6	脯氨酸Pro(P)	3	1.7
谷氨酰胺Gln(Q)	10	5.7	丝氨酸Ser(S)	7	4.0
谷氨酸Glu(E)	15	8.6	苏氨酸Thr(T)	5	2.9
甘氨酸Gly(G)	13	7.4	色氨酸Trp(W)	1	0.6
组氨酸His(H)	6	3.4	酪氨酸Tyr(Y)	5	2.9
异亮氨酸Ile(I)	3	1.7	缬氨酸Val(V)	8	4.6

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牦牛FTL基因克隆、生物信息学及表达分析

Cloning,Bioinformatics Analysis and Expression Analysis of FTL Gene in Yak

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组织 Tissue	心 Heart	肝 Liver	脾 Spleen	肺 Lung	肾 Kidney	背最长肌 Longissiums dorsi	脂肪 Fat	睾丸 Testis
7日龄犊牛	11.71±2.48Bc	0.13±0.11Cd	0.06±0.01Cd	0.22±0.05Cd	0.25±0.03Cd	21.04±2.45Aa	24.46±3.37Ab	0.63±0.13Cd
7-day old
成年牛	0.20±0.06Bc	28.02±0.17Aa	4.52±1.54Bbc	2.34±0.77Bbc	7.56±4.27Bb	0.56±0.25Bc	2.10±0.12Bbc	5.30±2.01Bbc
36 months old

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Cloning,Bioinformatics Analysis and Expression Analysis of FTL Gene in Yak

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