Abstract：Tibetan pig (Sus scrofa) is a representative non model animal in plateau habitat. The study of its plateau response mechanism can provide a reference basis for the exploration of hypoxia adaptation mechanism of other animals. In order to improve and optimize the annotation information of Tibetan pig genome, analyze the characteristics of Tibetan pig lung tissue response to hypoxic environment at transcription level, this study performed RNA-seq sequencing of lung tissues in Tibetan pigs at different altitudes to construct cDNA library, and the reassembled sequencing data was compared and analyzed with the reference genome of Tibetan pigs. The results showed that the 5' end and 3' end of 334 genes were extended, and the 5' end of 1 313 genes and the 3' end of 2 734 genes were extended respectively; 809 new transcripts were excavated and 95 new transcripts at different altitudes were differentially expressed (P<0.05). GO analysis results showed that the differential new transcripts were significantly enriched in the process of cell metabolism, enzyme catalytic activity and cellular components (P<0.05); KEGG pathway enrichment results found that the differential new transcripts were significantly enriched (P<0.05) in the pathways such as programmed cell necrosis and nuclear factor-κB (NF-κB) signaling pathway. Nine new transcripts with significant differences were randomly selected for qPCR verification, and the results were consistent with that of RNA-seq. This study provides a useful supplement and reference for improving the annotation information of Tibetan pig genome and a reference basis for further studying the genetic mechanism of Tibetan pig adapting to different altitude environment.
[1] 白玛央宗, 商鹏, 刘金凤. 2012. 藏猪肺组织与高原低氧适应性的初步研究[J]. 中国畜牧兽医文摘, 28(11): 217. (Bai M Y Z, Shang P, Liu J F.2012. Study on the adaptability of Tibetan pig lung tissue to plateau hypoxia[J]. China Animal Husbandry and Veterinary Abstracts, 28(11): 217.) [2] 董坤哲. 2015. 藏猪高海拔环境适应性分子机制探讨[D]. 博士学位论文, 北京畜牧兽医研究所, 导师: 马月辉, pp. 24-50. (Dong K Z.2015. Analysis of molecular mechanisms of high-altitude adaptationin Tibetan pig[D]. Thesis for Ph.D., Beijing Animal Husbandry and Veterinary Research Institute, Supervisor: Ma Y H, pp. 24-50.) [3] 傅芳, 官久强, 曲秀龙, 等. 2020. 不同海拔饲育的麦洼牦牛肺脏组织转录组学分析[J]. 兽类学报, 40(5): 475-484. (Fu F, Guan J Q, Qu X L, et al.2020. Transcriptomics analysis of lungs in yaks breeding at different altitudes[J]. Acta Theriologica Sinica, 40(5): 475-484.) [4] 郭睿, 张璐, 熊翠玲, 等. 2018. 西方蜜蜂的基因注释信息优化及新基因鉴定[J]. 上海交通大学学报(农业科学版), 36(3): 39-44. (Guo R, Zhang L, Xiong C L, et al.2018. Optimization of annotated genes information and identification of novel genes in Apis mellifera[J]. Journal of Shanghai Jiaotong University (Agricultural Sciences), 36(3): 39-44.) [5] 郭英飞, 王玉飞, 龚春丽, 等. 2015. 基于RNA-seq的羊种布鲁氏菌新转录本与非编码RNA鉴定[J]. 中国人兽共患病学报, 31(3): 216-221. (Guo Y F, Wang Y F, Gong C L, et al.2015. Identification of novel transcripts and sRNA of Brucella melitensis by RNA-seq[J]. Chinese Journal of Zoonoses, 31(3): 216-221.) [6] 黄兴. 2019. 牦牛大脑和小脑低氧适应性的转录组研究[D]. 硕士学位论文, 西南民族大学, 导师: 钟金城, pp. 19-70. (Huang X.2019. The Transcriptome analysis of cerebrum and cerebellum reveals the well hypoxic adaptability of yak[D]. Thesis for M.S., Southwest University for Nationalities, Supervisor: Zhong J C, pp. 19-70.) [7] 兰道亮, 熊显荣, 位艳丽, 等. 2014. 基于RNA-seq高通量测序技术的牦牛卵巢转录组研究:进一步完善牦牛基因结构及挖掘与繁殖相关新基因[J]. 中国科学: 生命科学, 44(3): 307-317. (Lan D L, Xiong X R, Wei Y L, et al.2014. Yak ovary transcriptome research based on RNA-seq high-throughput sequencing technology: Further improve the yak gene structure and discover new genes related to reproduction[J]. Science in China: Life Sciences, 44(3): 307-317.) [8] 林宝山. 2015. 麦洼牦牛肺脏比较转录组研究[D]. 硕士学位论文, 西南民族大学, 导师: 李键, pp. 25-51. (Lin B S.2015. Comparative transcriptome analysis of lung in Mai-wa yak[D]. Thesis for M.S., Southwest University for Nationalities, Supervisor: Li J, pp. 25-51.) [9] 李欣, 李洁, 哈小琴. 2019. 低氧诱导因子-1α与自身免疫性疾病的相关研究[J]. 微生物学免疫学进展, 47(3): 86-91. (Li X, Li J, Ha X Q.2019. Correlation between hypoxia-inducible factor 1α and autoimmune diseases[J]. Advances in Microbiology and Immunology, 47(3): 86-91.) [10] 李岩强. 2019. 布氏鲳鰺低氧胁迫转录组分析[D]. 硕士学位论文, 海南大学, 导师: 骆剑, pp. 22-52. (Li Y Q.2019. Transcriptome analysis of hypoxic stress in pompano brasoniana[D]. Thesis for M.S., University Of Hainan, Supervisor: Luo J, pp. 22-52.) [11] 李利. 2010. 低氧胁迫对日本沼虾呼吸代谢、能量代谢和抗氧化能力的影响[D]. 硕士学位论文, 河北大学, 导师: 管越强, pp. 26-44. (Li L.2010. Effects of hypoxic stress on respiratory metabolism, energy metabolism and antioxidant capacity of Macrobrachium nipponense[D]. Thesis for M.S., Hebei University, Supervisor: Guan Y Q, pp. 26-44.) [12] 刘景艳, 修清玉, 张铁锋. 2005. 肺纤维化大鼠肺组织P_(65)蛋白表达及中药的干预作用[J]. 中华中医药杂志, (7): 401-403. (Liu J Y, Xiu Q Y, Zhang T F.2005. Expression of P_(65) protein in lung tissue of rats with pulmonary fibrosis and intervention effect of traditional Chinese medicine[J]. Chinese Journal of Traditional Chinese Medicine, (7): 401-403.) [13] 马芳, 刘哲, 康玉军, 等. 2019. 虹鳟肝组织新转录本分析及基因结构优化[J]. 中国实验动物学报, 27(2): 135-142. (Ma F, Liu Z, Kang Y J, et al.2019. Analysis of novel transcripts and optimization of the gene structure in the liver of rainbow trout[J]. Chinese Journal of Experimental Zoology, 27(2): 135-142.) [14] 王翔宇. 2014. 藏鸡胚胎低氧适应的转录组差异表达分析[D]. 博士学位论文, 中国农业大学, 导师: 鲍海港, pp. 22-55. (Wang X Y.2014. Transcriptomic difference analysis in Tibetan chicken embryo at hypoxia[D]. Thesis for Ph.D., China Agricultural University, Supervisor: Bao H G, pp. 22-55.) [15] 王慧慧. 2019. 青海沙蜥对高原低氧和低温适应性的转录组学研究[D]. 博士学位论文, 兰州大学, 导师: 陈强, pp. 27-75. (Wang H H.2019. A transcriptomics study on the adaptation of Phrynocephalus vlangalii to high altitude hypoxia and low temperature[D]. Thesis for Ph.D., Lanzhou University, Supervisor: Chen Q, pp. 27-75.) [16] 吴晨露, 谢南南, 周伸奥, 等. 2016. 程序性细胞坏死的分子机制及其在炎症中的作用[J]. 中国细胞生物学学报, 38(1): 7-16. (Wu C L, Xie N N, Zhou S A, et al.2016. The molecular mechanisms of necroptosis and its role in inflammation[J]. Chinese Journal of Cell Biology, 38(1): 7-16.) [17] 辛盛鹏, 石达, 晋美加措, 等. 2011. 西藏自治区藏猪遗传资源保护与开发利用研究[J]. 中国牧业通讯, 331(4): 49-51. (Xin S P, Shi D, Jin M J C, et al.2011. Study on the protection, development and utilization of Tibetan pig genetic resources in Tibet Autonomous Region[J]. China Animal Husbandry News, 331(4): 49-51.) [18] 许无恨. 2013. IGFBP-3基因在藏猪, 军牧一号猪种间表达模式的比较研究[D]. 博士学位论文, 吉林大学, 导师: 刘松财, pp. 13-30. (Xu W H.2013. Comparative study on expression patterns of IGFBP-3 gene Tibetan mini-pig and 'Junmu-1' pig[D]. Thesis for Ph.D., Jilin University, Supervisor: Liu S C, pp. 13-30.) [19] Beiki H, Liu H, Huang J, et al.2019. Improved annotation of the domestic pig genome through integration of Iso-seq and RNA-seq data[J]. BMC Genomics, 20(1): 344. [20] Jäger M, Ott C E, Grünhagen J, et al.2011. Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing[J]. BMC Genomics, 12: 158. [21] Lallier F, Truchot J P.1989. Hemolymph oxygen transport during environmental hypoxia in the shore crab, Carcinus maenas[J]. Respiration Physiology, 77(3): 323-336. [22] Li M, Tian S, Jin L, et al.2013. Genomic analyses identify distinct patterns of selection in domesticated pigs and Tibetan wild boars[J]. Nature Genetics, 45(12): 1431-1438. [23] Li Y, Fang C, Fu Y, et al.2018. A survey of transcriptome complexity in Sus scrofa using single-molecule long-read sequencing[J]. DNA Research, 25(4): 421-437. [24] Lv P, Luo H S, Zhou X P, et al.2007. Reversal effect of thalidomide on established hepatic cirrhosis in rats via inhibition of nuclear factor-kappa B/inhibitor of nuclear factor-kappa B pathway[J]. Archives of Medical Research, 38(1): 15-27. [25] Sarada S K S, Veeramohan H P, Titto M, et al .2012. Nifedipine inhibits hypoxia induced transvascular leakage through down regulation of NF-κB[J]. Respiratory Physiology & Neurobiology, 183(1): 26-34. [26] Trapnell C, Williams B A, Pertea G, et al.2010. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation[J]. Nature Biotechnology, 28(5): 511-515. [27] Wang L, Cao C, Ma Q, et al.2014. RNA-seq analyses of multiple meristems of soybean: Novel and alternative transcripts, evolutionary and functional implications[J]. BMC Plant Biology, 14: 169. [28] Wang Z, Gerstein M, Snyder M.2009. RNA-seq: A revolutionary tool for transcriptomics[J]. Nature Reviews Genetics, 10(1): 57-63. [29] Zhao Y, Lu X, Cheng Z, et al.2019. Comparative proteomic analysis of Tibetan pig spermatozoa at high and low altitudes[J]. BMC Genomics, 20(1): 569.
