基于比较基因组学分析的方法定位及注释双峰驼MHC基因

doi:10.3864/j.issn.0578-1752.2018.18.015

摘要/Abstract

摘要：

【目的】定位并注释双峰驼主要组织相容性复合体(major histocompatibility complex,MHC)基因序列,为进一步研究双峰驼MHC基因提供科学依据。【方法】运用比较基因组学方法,提取人类MHC（HLA）基因编码序列和牛MHC（BoLA)基因编码序列并分别与双峰驼转录本进行blastn基因序列比对,识别出相似度较高的scaffolds,通过分析HLA、BoLA基因序列比对在这些scaffolds上的位置顺序,对多条scaffolds进行拼接,得到双峰驼MHC的Pseudo chromosome;再分别提取HLA、BoLA全基因组序列与双峰驼已拼接的scaffolds进行基因组共线性分析,利用lastz建立起的Pseudo chromosome与HLA、BoLA全基因组序列的线性关系判断筛选出的scaffolds是否准确;然后通过分析MHC基因在两物种间的线性关系,在双峰驼参考基因组中提取出MHC基因序列,并对这些序列进行基因注释;最后根据得到的双峰驼MHC基因绘制系统进化树,研究其基因间的进化关系。【结果】通过对HLA、BoLA基因编码序列与双峰驼转录本用blastn进行序列比对,识别出了相似度较高的3条scaffolds,即NW_011511766.1（全长4.1M）、NW_011515227.1（全长1.2M）和NW_011514613.1（全长15K）,对其拼接得到双峰驼MHC的Pseudo chromosome;利用lastz共线性分析,识别出HLA基因序列和BoLA基因序列并比对出其在双峰驼MHC基因的共线性区域。该区域与拼接得到的Pseudo chromosome一致,证明筛选出的scaffolds是准确的。并且发现Class-Ⅰ类和Class-Ⅲ类基因集中分布在NW_011515227.1上,而Class-Ⅱ类基因集中分布在NW_011511766.1和NW_011514613.1上,进一步分析得知Class-Ⅱ类基因主要分布在NW_011511766.1 的3.5—4.1M的位置;将存在共线性区域的序列提取出来,与比对到双峰驼上的MHC基因的编码序列进行blat分析,结果在双峰驼基因组中共识别出24个与牛BoLA基因高度相似的基因,其中Ⅰ类基因1个,Ⅱ类10个, Ⅲ类基因13个。对双峰驼这24个MHC基因进行信息注释并绘制系统进化树,结果显示注释的Class-Ⅰ类和Class-Ⅱ类基因在同一分支。【结论】通过比较基因组学方法定位并注释了双峰驼的MHC基因,将双峰驼MHC基因序列定位到了3条scaffolds上,找到并注释了24个MHC基因,绘制了双峰驼MHC的Pseudo chromosome,为进一步研究双峰驼MHC基因奠定了理论基础。

关键词: 双峰驼, MHC, CBLA, BoLA, HLA, 比较基因组学

Abstract:

【Objective】The objective of this study was to locate and annotate the major histocompatibility complex (MHC) gene sequence of Bactrian camel in order to provide scientific basis for further study on Bactrian camel MHC gene. 【Method】This study used comparative genomics method. The human MHC (HLA) gene coding sequence and bovine MHC (BoLA) gene coding sequence were extracted, compared with Bactrian camel transcripts on the gene sequences through blastn, to identify the scaffolds with higher similarity. By analyzing the sequence of HLA and BoLA gene sequences on their positions on these scaffolds, multiple pieces of scaffolds were spliced to obtain the Pseudo chromosome of Bactrian camel MHC. Then, the human MHC (HLA) gene coding sequence and bovine MHC (BoLA) gene coding sequence were extracted and analyzed with the spliced scaffolds of Bactrian camels through the genomic collinearity analysis. The selected scaffolds could be judged whether or not it was accurate, based on the linear relationship between Pseudo chromosome established by lastz and HLA and BoLA genome sequences; then by analyzing the linear relationship between MHC genes in the two species, MHC gene sequences were extracted from Bactrian camel genomes, and these sequences were genetically annotated; finally, according to the obtained Bactrian camel MHC gene, the phylogenetic tree was drawn to study the evolutionary relationship between their genes. 【Result】By comparing the HLA and BoLA gene coding sequences with the Bactrian camel transcripts through blastn, three scaffolds with high similarity were identified, namely NW_011511766.1 (full-length 4.1M), NW_011515227.1 (full-length 1.2 M) and NW_011514613.1 (15K in total length), and spliced to obtain Bactrian camel MHC Pseudo chromosome; By using the lastz colinear analysis, the HLA gene sequence and the BoLA gene sequence were identified and compared with MHC gene of the Bactrian camel to obtain the colinear region. It was consistent with the spliced Pseudo chromosome, which proved that the selected scaffolds was accurate. It was found that Class-I and Class-III genes were distributed on NW_011515227.1, while Class-II genes were distributed on NW_011511766.1 and NW_011514613.1. Further analysis revealed that Class-II genes were mainly distributed in NW_011511766.1 3.5 to 4.1M position; the sequences that existed in the collinear region were extracted and subjected to blat analysis, namely aligned with the coding sequence of the MHC gene on the Bactrian camel. Results reveal that a total of 24 genes highly similar to bovine BoLA gene were identified in Bactrian camel genome, including 1 of class I gene, 10 of class II gene and 13 of class III gene. The 24 MHC genes of Bactrian camels were annotated and phylogenetic trees were mapped. The results showed that the annotated Class-I and Class-II genes were on the same branch. 【Conclusion】The method of locating and annotating the MHC gene sequence in Bactrian camel was established by comparative genomics. The MHC gene sequence of Bactrian camel was mapped to three scaffolds, 24 MHC genes were found and annotated, and the Pseudo chromosome of the MHC gene of the Bactrian camel was drawn, which laid the foundation for further study of Bactrian camel MHC gene.

Key words: Bactrian camel, MHC, CBLA, BoLA, HLA, comparative genomic

支立康, 额尔敦木图, 安希文, 王超, 王瑞, 包花尔, 王秀珍. 基于比较基因组学分析的方法定位及注释双峰驼MHC基因[J]. 中国农业科学, 2018, 51(18): 3591-3599.

LiKang ZHI, Erdemtu, XiWen AN, Chao WANG, Rui WANG, Huar BAO, XiuZhen WANG. Mapping and Annotating of Bactrian Camel MHC Gene by Using the Comparative Genomic Approach[J]. Scientia Agricultura Sinica, 2018, 51(18): 3591-3599.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2018.18.015

https://www.chinaagrisci.com/CN/Y2018/V51/I18/3591

图/表 8

表1

图1

图2

图3

表2

表3

表4

图4

参考文献 31

[1]	亢孝珍, 额尔敦木图, 姜建强, 包花尔, 王瑞, 王秀珍, 李盈. 阿拉善驼与苏尼特驼MHC-DRB3 exon2基因克隆及序列分析. 内蒙古农业大学学报(自然科学版), 2015(5): 5-11.
	KANG X Z, ERDEMTU, JIANG J Q, BAO H, WANG R, WANG X Z, LI Y. Cloning and analysis of sequences of MHC–DRB3 exon2 genes in Alxa bactrian camel and Sunit bactrian camel. Journal of Inner Mongolia Agricultural University(Natural Science Edition), 2015(5): 5-11. (in Chinese)
[2]	李文娟, 李岩, 李美玉, 牟凯, 刘思思, 潘庆杰. MHC基因结构及其功能的研究进展. 青岛农业大学学报(自然科学版), 2014(3): 167-171. doi: 10.3969/J.ISSN.1674-148X.2014.03.003
	LI W J, LI Y, LI M Y, MOU K, LIU S S, PAN Q J.Research Progress of MHC Gene and Its Function. Journal of QingDao Agricultural University(Natural Science),2014(3): 167-171. (in Chinese) doi: 10.3969/J.ISSN.1674-148X.2014.03.003
[3]	KUMANOVICS A, TAKADA T, LINDAHL K F.Genomic organization of the mammalian MHC. Annual Review of Immunology, 2003(21): 629-657.
[4]	CHAVES L D, KRUETH S B, REED K M.Defining the turkey MHC: sequence and genes of the B locus. Journal of Immunology, 2009, 183(10): 6530-6537. doi: 10.4049/jimmunol.0901310 pmid: 19864609
[5]	GARBUZ D G, ASTAKHOVA L N, ZATSEPINA O G, ARKHIPOVA I R, NUDLER E, EVGEN′EV M B. Functional organization of HSP70 cluster in camel(Camelus dromedarius) and other mammals. PLoS One, 2011, 6(11): e27205.
[6]	SEQUENCING T B C G, CONSORTIUM A. Genome sequences of wild and domestic bactrian camels. Nature Communications, 2012, 3: 1202. doi: 10.1038/ncomms2192 pmid: 3868263
[7]	LONGJAM L A, DAS D.Major histocompatibility complex and its importance towards controlling infection. Research Gate, 2017, 8(2): 1-13.
[8]	亢孝珍, 额尔敦木图, 姜建强, 陈泽明, 刘图雅, 伊特格勒图, 沙日扣, 图雅. 主要组织相容性复合体(MHC)基因研究进展. 中国畜牧兽医, 2014, 41(05): 28-33.
	KANG X Z, ERDEMTU, JIANG J Q, CHEN Z M, LIU T Y, YITEGELTU, SHARHU, TU Y. Research progress on major histocompatibility complex (MHC) gene. China Animal Husbandry & Veterinary Medicine, 2014, 41(05): 28-33. (in Chinese)
[9]	KLEIN J.George Snell's first foray into the unexplored territory of the major histocompatibility complex. Genetics, 2001, 159(2): 435-439. pmid: 11606523
[10]	EDWARDS S V, HEDRICK P W.Evolution and ecology of MHC molecules: from genomics to sexual selection. Trends in Ecology & Evolution, 1998, 13(8): 305-311. doi: 10.1016/S0169-5347(98)01416-5 pmid: 21238318
[11]	PLASIL M, MOHANDESAN E, FITAK R R, MUSILOVA P, KUBICKOVA S, BURGERE P A, HORIN P.The major histocompatibility complex in Old World camelids and low poly-morphism of its class Ⅱ genes. BMC Genomics, 2016, 17(1): 167. doi: 10.1186/s12864-016-2500-1 pmid: 4774177
[12]	AVILA F, DAS P J, KUTZLER M, OWENS E, PERELMAN P, RUBES J, HORNAK M, JOHNSON W E, RAUDSEPP T.Development and application of camelid molecular cytogenetic tools. Journal of Heredity, 2014, 105(6): 858-869. doi: 10.1093/jhered/ess067 pmid: 4207859
[13]	DAVIES C J, ANDERSSON L, ELLIS S A, HENSEN E J, LEWIN H A, MIKKO S, MUGGLI-COCKETT N E, POEL J J, RUSSELL G C. Nomenclature for factors of the BoLA system, 1996: report of the IS AG BoLA Nomenclature Committee. Animal Genetics, 2015, 28(3): 159-168.
[14]	BALMUS G, TRIFONOV V A, BILTUEVA L S, O'BRIEN P C M, ALKALAEVA E S, FU B Y, SKIDMORE J A, ALLEN T, GRAPHODATSKY A S, YANG F T, FERGUSON-SMITH M A. Cross-species chromosome painting among camel, cattle, pig and human: further insights into the putative Cetartiodactyla ancestral karyotype. Chromosome Research, 2007, 15(4): 499-514.
[15]	贾震虎, 夏春. 硬骨鱼类MHCⅠ基因结构及表达研究. 中国兽医杂志, 2008, 44(11): 54-55. doi: 10.3969/j.issn.0529-6005.2008.11.030
	JIA Z H, XIA C.Study on the structure and expression of MHCⅠgene in teleosts. Chinese Journal of Veterinary Medicine, 2008, 44(11): 54-55. (in Chinese) doi: 10.3969/j.issn.0529-6005.2008.11.030
[16]	WU H G, GUANG X M, l-FAGEEH M B, CAO J W, PAN S K, ZHOU H M, Zhang L, ABUTARBOUSH M H, XING Y P, XIE Z Y, ALSHANQEETI A S, ZHANG Y R, YAO Q L, AL-SHOMRANI B M, ZHANG D, LI J, MANEE M M, YANG Z L, YANG L F, LIU Y Y, ZHANG J L, ALTAMMAMI M A, WANG S Y, YU L L, ZHANG W B, LIU S Y, BA L, LIU C X, YANG X K, MENG F H, WANG S W, LI L, LI E L, LI X Q, WU K F, ZHANG S, WANG J Y, YIN Y, YANG H M, AL-SWAILEM A M, WANG J. ACamelid genomes reveal evolution and adaptation to desert environments. Nature Communications, 2014, 5(5): 5188. doi: 10.1038/ncomms6188 pmid: 25333821
[17]	ANTCZAK D. Major histocompatibility complex genes of the dromedary camel.2013(2013): BIOP 015.
[18]	SIDDLE H V, DEAKIN J E, COGGILL P, WHILMING L, HARROW J, KAUFMAN J, BECK S, BELOV K.The tammar wallaby major histocompatibility complex shows evidence of past genomic instability. BMC Genomics, 2011, 12(1): 421. doi: 10.1186/1471-2164-12-421 pmid: 3179965
[19]	潘增祥, 许丹, 张金璧, 林飞, 吴宝江, 刘红林. 基于直向同源序列的比较基因组学研究. 遗传, 2009, 31(05): 457-463.
	PAN Z X, XU D, ZHANG J B, LIN F, WU B J, LIU H L.Reviews in comparative genomic research based on orthologs. Hereditas, 2009, 31(05): 457-463. (in Chinese)
[20]	CONSORTIUM T M S. Complete sequence and gene map of a human major histocompatibility complex. Nature, 1999, 401(6756): 921-923.
[21]	SAMBROOK J G, FIGUEROA F, BECK S.A genome-wide survey of Major Histocompatibility Complex (MHC) genes and their paralogues in zebrafish. BMC Genomics, 2005, 6(1): 152. doi: 10.1186/1471-2164-6-152 pmid: 1309616
[22]	KELLEY J, WALTER L, TROWSDALE J.Comparative genomics of major histocompatibility complexes. Immunogenetics, 2005, 56(10): 683-695.
[23]	DIDINGER C, EIMES J A, LILLIE M, WALDM B.Multiple major histocompatibility complex class I genes in Asian anurans: Ontogeny and phylogeny. Developmental & Comparative Immunology, 2017, 70: 69-79.
[24]	CHAVES L D.The Major Histocompatibility Complex of the Turkey.[D]. Twin Cities: University ofMinnesota .2010.
[25]	郭秀丽, 代红星, 李祥龙, 周荣艳, 王立泽. 不同物种MHC-DQA1基因部分序列的生物信息分析. 中国畜牧兽医, 2007, 34(1): 65-67.
	GUO X L, DAI H X, LI X L, ZHOU R Y, WANG L Z.Bioinformatics analysis of part of mhc-dqa1 gene in different species. China Animal Husbandry & Veterinary Medicine, 2007, 34(1): 65-67. (in Chinese)
[26]	BEHL J D, VERMA N K, TYAGI N, MISHRA P, BEHL R, JOSHI B K.The major histocompatibility complex in bovines: A review. ISRN Veterinary Science, 2012, (872710): 1-12.
[27]	LIAN X D, ZHANG X H, DAI Z X, ZHENG Y T.Characterization of classical major histocompatibility complex (MHC) classⅡ genes in northern pig-tailed macaques (Macaca leonina). Infection, Genetics & Evolution, 2017, 56: 26-35. doi: 10.1016/j.meegid.2017.10.015
[28]	JIANLIN H, MBURU D, OCHIENG J, KAUFMANN B, REGE JE O, HANOTTE O.Application of New World Camelidae microsatellite primers for amplification of polymorphic loci in Old World camelids. Animal Genetics, 2010, 31(6): 404-406. doi: 10.1111/j.1365-2052.2000.00683.pp.x pmid: 11167529
[29]	VILA C, SEDDON J, ELLEGREN H.Genes of domestic mammals augmented by backcrossing with wild ancestors. Trends in Genetics, 2005, 21(4): 214-218. doi: 10.1016/j.tig.2005.02.004 pmid: 15797616
[30]	SATO A, FIGUEROA F, O'HUIGIN C, REZNICK D N, KLEIN J.Identification of major histocompatibility complex genes in the guppy, Poecilia reticulata. Immunogenetics, 1995, 43(1-2): 38-49.
[31]	FITAK R R, MOHANDESAN E, CORANDER J, BURGER P A.The de novo genome assembly and annotation of a female domestic dromedary of North African origin. Molecular Ecology Resources, 2016, 16(1): 314-324. doi: 10.1111/1755-0998.12443

名称 Name	来源 Source	网址 URL
家养双峰驼参考基因组 Reference genome of domestic Bactrian camels	NCBI	ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/767/855/GCF_000767855.1_Ca_bactrianus_MBC_1.0/GCF_000767855.1_Ca_bactrianus_MBC_1.0_genomic.fna.gz
人类HLA编码序列 Human HLA coding sequence	ensemble	ftp://ftp.ensembl.org/pub/release-90/fasta/homo_sapiens/cdna/
人类HLA全基因组序列 Human HLA whole genome sequence	ensemble	ftp://ftp.ensembl.org/pub/release-90/fasta/homo_sapiens/dna/
人类HLA注释文件 Human HLA annotation file	ensemble	ftp://ftp.ensembl.org/pub/release-90/gtf/homo_sapiens
牛BoLA编码序列 Cattle BoLA coding sequence	ensemble	ftp://ftp.ensembl.org/pub/release-90/fasta/bos_taurus/cdna/
牛BoLA全基因组序列 Cattle BoLA whole genome sequence	ensemble	ftp://ftp.ensembl.org/pub/release-90/fasta/bos_taurus/dna/
牛BoLA注释文件 Cattle BoLA annotation file	ensemble	ftp://ftp.ensembl.org/pub/release-90/gtf/bos_taurus

基因编码 Gene id	长度 Length	外显子 Exon	位置 Scaffold	起始点 Start	终止点 End	注释信息 Description
XM 010949105.1	2866	5	NW 011511766.1	3837314	3842070	PREDICTED:CBLA-DOA
XM 010949113.1	1189	5	NW 011511766.1	3874933	3878475	PREDICTED:CBLA-DMA
XM 010949114.1	1391	6	NW 011511766.1	3885523	3891578	PREDICTED:CBLA-DMB
XM 010949120.1	1381	6	NW 011511766.1	3965547	3972059	PREDICTED:CBLA-DOB
XM 010949122.1	813	5	NW 011511766.1	4106164	4114129	PREDICTED:CBLA-DRB2
XM 010949125.1	1177	5	NW 011511766.1	4047230	4051352	PREDICTED:CBLA-DQA
XM 010949126.1	1033	4	NW 011511766.1	4031979	4039091	PREDICTED:CBLA-DQB
XM 010949127.1	1307	5	NW 011511766.1	4120230	4124972	PREDICTED:CBLA-DRA
XM 010949153.1	759	4	NW 011511766.1	3992073	3995513	PREDICTED:CBLA-DYA
XM 010958395.1	1315	7	NW 011514613.1	5057	14519	PREDICTED:CBLA-DRB3
XM 010961447.1	1407	9	NW 011515227.1	42577	46241	PREDICTED:PBX2
XM 010961451.1	1602	8	NW 011515227.1	67307	73614	PREDICTED:PPT2
XM 010961470.1	1452	8	NW 011515227.1	175625	182554	PREDICTED:STK19
XM 010961476.1	2773	18	NW 011515227.1	207277	218549	PREDICTED:C2
XM 010961482.1	2376	1	NW 011515227.1	303003	305378	PREDICTED:HSPA1A
XM 010961484.1	2500	2	NW 011515227.1	305769	309435	PREDICTED:HSPA1L
XM 010961490.1	2657	25	NW 011515227.1	340559	355003	PREDICTED:MSH5
XM 010961495.1	1158	6	NW 011515227.1	357933	363242	PREDICTED:CLIC1
XM 010961533.1	668	6	NW 011515227.1	462534	464279	PREDICTED:AIF1
XM 010961536.1	688	4	NW 011515227.1	488003	489391	PREDICTED:LTA
XM 010961538.1	1318	5	NW 011515227.1	499275	507275	PREDICTED:NFKBIL1
XM 010961543.1	1513	9	NW 011515227.1	1073777	1105682	PREDICTED:CBLA-A
XM 010961594.1	6598	30	NW 011515227.1	17926	38510	PREDICTED:NOTCH4
XM 010961596.1	12304	45	NW 011515227.1	105330	155862	PREDICTED:TNXB