基于瘤胃转录组探究双峰驼沙漠适应性分子机制

doi:10.11843/j.issn.0366-6964.2021.008

摘要/Abstract

摘要： 旨在比较胚胎期和成年期双峰驼瘤胃在组织形态、编码基因表达上的变化，挖掘影响双峰驼瘤胃发育的关键基因和通路，从消化系统探究双峰驼的沙漠适应性机制。本研究选取3峰10~12岁健康状况良好的成年期阿拉善双峰驼，以及3峰9~10月龄健康状况良好的胚胎期阿拉善双峰驼，采集瘤胃组织样品，制作石蜡组织切片，观察成年期与胚胎期双峰驼瘤胃，比较其组织结构之间的差异。分别提取成年期和胚胎期双峰驼的瘤胃组织总RNA，利用Illumination Hiseq 2000测序平台分别进行转录组测序，对转录组数据进行质控、比对、差异基因筛选、GO、KEGG富集分析。随机选择6个差异表达基因进行荧光定量试验，关联转录组数据与荧光定量试验的表达趋势。结果表明，胚胎期瘤胃组织中上皮细胞和肌纤维细胞清晰可见，分布密集，在成年期的瘤胃组织中，可见明显的肌纤维，肌纤维直径较宽，肌纤维间的空隙较大。转录组测序结果显示，每个样本获得至少10G的数据量，各样本的质控率都在90%以上，Q30数据都在88%以上。对测序数据进行分析，以胚胎期为对照组，成年期为试验组，筛选到1 207个差异表达基因，其中上调基因456个，下调基因751个；对差异表达基因进行层次聚类分析，结果显示，成年期的3个个体（M1、M2、M3）表达模式接近，胚胎期的3个个体（T1、T2、T3）表达模式接近。对差异表达基因进行GO和KEGG富集分析，结果显示，上调差异表达基因显著富集到62条显著的GO条目，下调差异表达基因显著富集到366条显著的GO条目，73条显著的KEGG通路。差异表达基因主要富集在代谢过程的负调控、RNA生物合成过程的负调控、基因表达的负调控等GO条目中，KEGG主要富集到MAPK信号通路、PI3K-Akt信号通路、糖尿病并发症中的AGE-RAGE信号、胰岛素信号通路、醛固酮的合成与分泌等通路中。同时筛选到MAPK12、MAPK13、FABP5、PPARγ、CaMK1等与双峰驼沙漠适应性相关的基因。荧光定量结果显示，差异基因在成年期和胚胎期瘤胃组织中的表达模式与RNA-Seq的结果一致。上述结果表明，双峰驼的瘤胃组织可能与脂肪细胞分化有关。在沙漠环境中，双峰驼可能降低瘤胃组织代谢效率、通过胰岛素抵抗作用提高血糖耐受性以及促进醛固酮的合成与分泌以调节血压平衡，以更好地在沙漠干燥缺水的环境中生存。

关键词: 双峰驼, 瘤胃, 差异表达基因, 适应性, 转录组测序

Abstract: This study aimed to compare the changes in tissue morphology and the expression of coding genes in the rumen tissues of the Bactrian camel between embryonic and adult stages, to discover the key genes and pathways affecting the development of rumen of the Bactrian camel, and to explore the desert adaptive mechanism of the Bactrian camel from the digestive system. Three adult Alxa Bactrian camels aged 10-12 years old with good health and three embryonic Alxa Bactrian camels aged 9-10 months old with good health were selected to make paraffin tissue sections, observe the rumen of adult and embryonic Bactrian camels, and compare the differences in tissue structure. The total RNA was extracted from rumen tissue of adult and embryonic Bactrian camels, RNA-Seq analysis was performed by Illumination Hiseq 2000 platform, the RNA-Seq data were performed quality-controlled, alignment, differential genes screening, GO and KEGG analysis. Six differentially expressed genes were randomly selected for RT-qPCR, the results of RNA-Seq and RT-qPCR was compared. The results showed that epithelial cells and muscle fiber cells were clearly visible and densely distributed in the rumen of Bactrian camel at embryonic stage. In the rumen of Bactrian camel at adult stage, obvious muscle fibers could be observed, the diameter of the muscle fibers was wider and the space between the muscle fibers was larger. The RNA-Seq sequencing results showed that at least 10G data was obtained from each sample, the clean ratio were more than 90%, and Q30 data were more than 88%. The embryonic rumen was set as the control group and the adult rumen as the experimental group, 1 207 differentially expressed genes were screened, contained 456 up-regulated genes and 751 down-regulated genes. The hierarchical clustering analysis of differentially expressed genes showed that the similar expression pattern was detected among adult individuals(M1, M2, M3) and among embryonic individuals (T1, T2, T3). The GO enrichment analysis results showed that the up-regulated differentially expressed genes were significantly enriched in 62 GO terms, down-regulated differentially expressed genes were significantly enriched in 366 GO terms. GO terms were mainly enriched in negative regulation of metabolic process, negative regulation of RNA biosynthetic process, negative regulation of gene expression, etc. The KEGG pathway analysis of differentially expressed genes showed that 73 significant KEGG pathways were enriched in MAPK signaling pathway, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway in diabetic complications, insulin signaling pathway, aldosterone synthesis and secretion, etc. At the same time, MAPK12, MAPK13, FABP5, PPARγ, CaMK1 and other genes related to desert adaptation of Bactrian camel were screened. The expression pattern of 6 differentially expressed genes detected by RT-qRCR was consistent with the results of RNA-Seq analysis. The above results indicate that the rumen of Bactrian camel may be related to adipocyte differentiation. And in a desert environment, the Bactrian camel might reduce the metabolic efficiency of rumen, improve blood glucose tolerance through insulin resistance, and promote the synthesis and secretion of aldosterone to regulate blood pressure balance to better survive in the dry and water-deficient environment of the desert.

Key words: Bactrian camel, rumen, differentially expressed genes, adaptability, RNA-Seq

中图分类号:

S824.2

方艳, 周俊文, 关伟军, 蒋琳, 浦亚斌, 赵倩君, 何晓红, 马月辉. 基于瘤胃转录组探究双峰驼沙漠适应性分子机制[J]. 畜牧兽医学报, 2021, 52(1): 77-87.

FANG Yan, ZHOU Junwen, GUAN Weijun, JIANG Lin, PU Yabin, ZHAO Qianjun, HE Xiaohong, MA Yuehui. Exploring the Molecular Mechanism of Bactrian Camel's Desert Adaptation Based on Rumen Transcriptome[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 77-87.

参考文献 40

[1]	哈斯高娃,乌日罕,伊茹汗,等.雌性骆驼繁殖研究进展[J].草食家畜,2019(3):6-12.HASI G W,WU R H,YI R H,et al.Research on female camel breeding[J].Grass-Feeding Livestock,2019(3):6-12.(in Chinese)
[2]	TIBARY A,EL ALLALI K.Dromedary camel:a model of heat resistant livestock animal[J].Theriogenology,2020,154:203-211.
[3]	CAO Y,ZHANG D,ZHOU H M.Key genes differential expressions and pathway involved in salt and water-deprivation stresses for renal cortex in camel[J].BMC Mol Biol,2019,20(1):11.
[4]	曾献春,郑李娟,庄伟伟.骆驼消化道内喹啉降解菌的分离、筛选及降解能力[J].云南大学学报:自然科学版,2017,39(6):1073-1081.ZENG X C,ZHENG L J,ZHUANG W W.Isolation and screening of quinoline-degrading bacteria and their biodegradation capability in the digestive tract of camels[J].Journal of Yunnan University:Natural Sciences Edition,2017,39(6):1073-1081. (in Chinese)
[5]	The Bactrian Camels Genome Sequencing and Analysis Consortium.Genome sequences of wild and domestic Bactrian camels[J].Nat Commun,2012,3:1202.
[6]	GUO F C,SI R D L,HE J,et al.Comprehensive transcriptome analysis of adipose tissue in the Bactrian camel reveals fore hump has more specific physiological functions in immune and endocrine systems[J].Livest Sci,2019,228:195-200.
[7]	凌宇,齐昱,曹俊伟,等.转录组揭示禁水环境下双峰驼结肠组织适应机制[J].畜牧兽医学报,2018,49(11):2359-2370.LING Y,QI Y,CAO J W,et al.Transcriptome reveals the adapting mechanism of colon tissues of Bactrian camel in water-deficient environment[J].Acta Veterinaria et Zootechnica Sinica,2018,49(11):2359-2370.(in Chinese)
[8]	凌宇,齐昱,孟凡华,等.盐胁迫下双峰驼结肠组织环境适应能力研究[J].农业生物技术学报,2018,26(10):1723-1736.LING Y,QI Y,MENG F H,et al.Study on environmental adaptability of Bactrian camel (Camelidae bactrianus) colon tissue under salt stress[J].Journal of Agricultural Biotechnology,2018,26(10):1723-1736.(in Chinese)
[9]	HE J,LI G W,HAI L,et al.An analysis of the forestomach bacterial microbiota in the Bactrian camel[J].J Camel Pract Res,2019,26(1):71-79.
[10]	JIANG Y,XIE M,CHEN W B,et al.The sheep genome illuminates biology of the rumen and lipid metabolism[J]. Science, 2014,344(6188):1168-1173.
[11]	邱名浩,厉洁,刘娟.家鸽石蜡组织切片质量的优化研究[J].德州学院学报,2019,35(4):37-39,43.QIU M H,LI J,LIU J.The study on the quality optimization of paraffin tissue section of pigeon[J].Journal of Dezhou University, 2019, 35(4):37-39,43.(in Chinese)
[12]	董坤哲.藏猪高海拔环境适应性分子机制探讨[D].北京:中国农业科学院,2015.DONG K Z.Analysis of molecular mechanisms of high-altitude adaptation in Tibetan pig[D].Beijing:Chinese Academy of Agricultural Sciences,2015.(in Chinese)
[13]	PATEL R K,JAIN M.NGS QC toolkit:a toolkit for quality control of next generation sequencing data[J].PLoS One,2012, 7(2):e30619.
[14]	TRAPNELL C,PACHTER L,SALZBERG S L.TopHat:discovering splice junctions with RNA-Seq[J].Bioinformatics, 2009, 25(9):1105-1111.
[15]	TRAPNELL C,WILLIAMS B A,PERTEA G,et al.Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J].Nat Biotechnol,2010,28(5):511-515.
[16]	CAMON E,MAGRANE M,BARRELL D,et al.The gene ontology annotation (GOA) database:sharing knowledge in uniprot with gene ontology[J].Nucleic Acids Res,2004,32(S1):D262-D266.
[17]	KANEHISA M,ARAKI M,GOTO S,et al.KEGG for linking genomes to life and the environment[J].Nucleic Acids Res,2008,36(S1):D480-D484.
[18]	REIMAND J,ARAK T,ADLER P,et al.g:profiler-a web server for functional interpretation of gene lists (2016 update)[J].Nucleic Acids Res,2016,44(W1):W83-W89.
[19]	ELMAHDI B,SALLMANN H P,FUHRMANN H,et al.Comparative aspects of glucose tolerance in camels,sheep,and ponies[J]. Comp Biochem Physiol A Physiol,1997,118(1):147-151.
[20]	KASKE M,ELMAHDI B,ENGELHARDT W,et al.Insulin responsiveness of sheep,ponies,miniature pigs and camels:results of hyperinsulinemic clamps using porcine insulin[J].J Comp Physiol B,2001,171(7):549-556.
[21]	FUJII H.PPARs-mediated intracellular signal trans-duction[J].Nihon Rinsho,2005,63(4):565-571.
[22]	SAVAGE D B.PPARγ as a metabolic regulator:insights from genomics and pharmacology[J].Expert Rev Mol Med,2005,7(1):1-16.
[23]	SAKAUE H,OGAWA W,NAKAMURA T,et al.Role of MAPK phosphatase-1(MKP-1) in adipocyte differentiation[J].J Biol Chem,2004,279(38):39951-39957.
[24]	CHOI K,KIM Y B.Molecular mechanism of insulin resistance in obesity and type 2 diabetes[J].Korean J Intern Med,2010, 25(2):119-129.
[25]	LAN D C,XU N G,SUN J,et al.Electroacupuncture mitigates endothelial dysfunction via effects on the Pi3K/Akt signalling pathway in high fat diet-induced insulin-resistant rats[J].Acupunct Med,2018,36(3):162-169.
[26]	WU H G,GUANG X M,AL-FAGEEH M B,et al.Camelid genomes reveal evolution and adaptation to desert environments[J].Nat Commun,2014,5:5188.
[27]	QUAN W,LI Y,JIAO Y,et al.Simultaneous generation of acrylamide,β-carboline heterocyclic amines and advanced glycation ends products in an aqueous Maillard reaction model system[J].Food Chem,2020,332:127387.
[28]	BEDOUI S A,BARBIROU M,STAYOUSSEF M,et al.Identification of novel advanced glycation end products receptor gene variants associated with colorectal cancer in Tunisians:a case-control study[J].Gene,2020,754:144893.
[29]	NEEPER M,SCHMIDT A M,BRETT J,et al.Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins[J].J Biol Chem,1992,267(21):14998-15004.
[30]	YAMAGISHI S I.Role of advanced glycation end products (AGEs) and receptor for AGEs (RAGE) in vascular damage in diabetes[J].Exp Gerontol,2011,46(4):217-224.
[31]	KANWAR Y S,SUN L,XIE P,et al.A glimpse of various pathogenetic mechanisms of diabetic nephro-pathy[J].Annu Rev Pathol Mech Dis,2011,6:395-423.
[32]	CALCUTT N A,COOPER M E,KERN T S,et al.Therapies for hyperglycaemia-induced diabetic complications:from animal models to clinical trials[J].Nat Rev Drug Discov,2009,8(5):417-430.
[33]	BKAILY G,SIMON Y,MENKOVIC I,et al.High salt-induced hypertrophy of human vascular smooth muscle cells associated with a decrease in glycocalyx[J].J Mol Cell Cardiol,2018,125:1-5.
[34]	BEAINI S,SALIBA Y,HAJAL J,et al.VEGF-C attenuates renal damage in salt-sensitive hypertension[J].J Cell Physiol,2019, 234(6):9616-9630.
[35]	KACZENSKY P,ADIYA Y,VON WEHRDEN H,et al.Space and habitat use by wild Bactrian camels in the Transaltai Gobi of southern Mongolia[J].Biol Conserv,2014,169:311-318.
[36]	HATTANGADY N G,OLALA L O,BOLLAG W B,et al.Acute and chronic regulation of aldosterone production[J].Mol Cell Endocrinol,2012,350(2):151-162.
[37]	BOLLAG W B.Regulation of aldosterone synthesis and secretion[J].Compr Physiol,2011,4(3):1017-1055.
[38]	BRÖER S.Amino acid transport across mammalian intestinal and renal epithelia[J].Physiol Rev,2008,88(1):249-286.
[39]	WU G.Intestinal mucosal amino acid catabolism[J].J Nutr,1998,128(8):1249-1252.
[40]	FARAH Z.Camel milk properties and products[M].St. Gallen:SKAT,1996.