小尾寒羊泌乳性状重要lncRNAs的筛选、鉴定及功能分析

doi:10.3864/j.issn.0578-1752.2021.14.016

摘要/Abstract

摘要：

【目的】长链非编码RNA（long non-coding RNAs,lncRNAs）是一类长度大于200 nt的非编码RNA分子,它对奶牛和奶山羊的乳腺发育和泌乳过程发挥了重要调控作用,然而在绵羊上研究甚少。为此开展lncRNAs对绵羊泌乳性能的调控作用研究,为解析绵羊泌乳性能的分子机理提供理论基础。【方法】选取高泌乳性能（高泌乳量、高乳脂率）和低泌乳性能（低泌乳量、低乳脂率）小尾寒羊各3只,采集泌乳期乳腺组织,用RNA-Seq构建lncRNAs表达谱,研究差异表达lncRNAs靶基因的GO和KEGG富集通路,最后用实时荧光定量PCR（reverse transcription-quantitative PCR, RT-qPCR）对16个差异表达lncRNAs进行验证。【结果】在小尾寒羊乳腺组织中共鉴定出7 239个表达的lncRNAs,包括2 262个已知lncRNAs和4 977个新的lncRNAs,大部分lncRNAs呈低丰度表达。在两组小尾寒羊中发现120个差异表达lncRNAs,其中68个lncRNAs在高泌乳性能小尾寒羊中上调表达,52个lncRNAs下调表达。差异表达lncRNAs的靶基因显著富集在硫化物代谢过程、硫酯生物合成过程、酰基辅酶A生物合成过程、Rap1信号通路、粘附连接等通路上。LncRNA-miRNA网络分析发现,MSTRG.125242.6、MSTRG.59580.8等6个最显著差异表达lncRNAs的靶向miRNAs海绵体在家畜乳腺发育和泌乳过程中发挥了重要作用。RT-qPCR结果表明,16个lncRNAs的表达趋势与RNA-Seq结果完全吻合,证实了RNA-Seq测序结果的准确性和真实性。【结论】筛选的差异表达lncRNAs参与了绵羊乳腺发育及泌乳性能的调控,该结果将为解析绵羊泌乳性状的分子遗传机制提供理论参考。

关键词: 绵羊, 乳腺, 长链非编码RNA（lncRNA）, 泌乳期

Abstract:

【Objective】Long non-coding RNAs (lncRNAs) are a type of non-coding RNAs with >200 nt in length, which have been shown to regulate mammary gland development and lactation process in dairy cows and dairy goats. However, little is known about the effect of lncRNAs on milk traits in sheep. The aim of the study was to analyze the effect of lncRNAs on milk performance and then provided a theoretical basis for elucidating molecular mechanism of lactation performance in sheep.【Method】Three high-lactating yield and high-milk-fat-content Small-Tailed Han sheep and three low-lactating yield and low-milk-fat-content Small-Tailed Han sheep were selected to profile the expression of lncRNAs in the mammary gland tissues during lactation using RNA-Seq. The enrichment analysis was performed using GO and KEGG databases for the target genes of differentially expressed lncRNAs between the two groups. The expression levels of 16 differentially expressed lncRNAs were verified using reverse transcription-quantitative PCR (RT-qPCR).【Result】A total of 7 239 expressed lncRNAs were identified in the mammary gland tissues of Small-Tailed Han sheep, including 2 262 known lncRNAs and 4 977 novel lncRNAs. The most of lncRNAs were expressed at low levels. The 120 differentially expressed lncRNAs were found between the two groups of Small-Tailed Han sheep, of which 68 lncRNAs were up-regulated in high-lactating performance Small-Tailed Han sheep, while 52 lncRNAs were down-regulated. The target genes of differentially expressed lncRNAs were significantly enriched in sulfur compound metabolic process, thioester biosynthesis process, acyl-CoA biosynthetic process, Rap1 signal pathway and adhesion junction. The lncRNA- miRNA network showed that some target miRNAs of the six most differentially expressed lncRNAs including MSTRG.125242.6 and MSTRG.59580.8, play important roles in mammary gland development and lactation in domestic animals. The RT-qPCR results showed that the expression tendency of 16 lncRNAs was consistent with the RNA-Seq results, which confirmed the accuracy and authenticity of the RNA-Seq data.【Conclusion】The differentially expressed lncRNAs screened were involved in the regulation of mammary gland development and milk performance in sheep and the results will provide a theoretical basis for analyzing the molecular genetic mechanism of lactation performance in sheep.

Key words: sheep, mammary gland, long non-coding RNA (lncRNA), lactation period

王继卿,郝志云,沈继源,柯娜,黄兆春,梁维炜,罗玉柱,胡江,刘秀,李少斌. 小尾寒羊泌乳性状重要lncRNAs的筛选、鉴定及功能分析[J]. 中国农业科学, 2021, 54(14): 3113-3123.

WANG JiQing,HAO ZhiYun,SHEN JiYuan,KE Na,HUANG ZhaoChun,LIANG WeiWei,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin. Screening, Identification and Functional Analysis of Important LncRNAs for Lactation Traits in Small-Tailed Han Sheep[J]. Scientia Agricultura Sinica, 2021, 54(14): 3113-3123.

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https://www.chinaagrisci.com/CN/Y2021/V54/I14/3113

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引物名称 Name	正向序列（5'→3'） Forward sequence (5'→3')	反向序列（5'→3'） Reverse sequence (5'→3')
MSTRG.59580.8	ACCAGGTTGCCTAAGGAGGC	TGGAGTACAGTGGCTATTCACAA
MSTRG.59580.9	ACCAGGTTGCCTAAGGAGG	TGGAGTACAGTGGCTATTCACA
MSTRG.59580.10	TCGGGTGTCCGCACTAAG	GTTGCCCAGGCTGGAGTA
MSTRG.38680.1	GGTCTGCCTTCTTGGGTTC	TGGAGATGGAGCAGGGATC
MSTRG.47064.1	GCCGACTAAGGTCCATCT	GGCACTCAGCCTTCTTCA
MSTRG.137650.1	GTGCCGAAGAATAGATGC	TTCCTCCAAAGAAATCCC
MSTRG.59580.12	ACCAGGTTGCCTAAGGAGG	TGGAGTACAGTGGCTATTCACA
MSTRG.80056.21	CACTTCACTTAGCACGAT	CAACAGATAAATGGACGA
MSTRG.125242.6	GCACGAACAGCGACATCAG	CAGGGTCTCAGCAAGTTAGGAG
MSTRG.119809.14	TGTAGTCCTTTCCCAGTT	TGAATACCAGACCACCTTA
MSTRG.59580.14	GGCTGGAGGATCGCTTGA	TTGACCTGCTCCGTTTCC
MSTRG.59580.11	CAGCCTGGGCAACATAGC	CCGAACTTAGTGCGGACAC
MSTRG.114625.4	TGATCGCCAGGGTTGATT	GGATGGTCGTCCTCTTCG
MSTRG.59580.3	ACCAGGTTGCCTAAGGAGG	TGGAGTACAGTGGCTATTCACA
MSTRG.106261.2	TGTCACCCTCCACCAATG	CAGGCTGAAGTCCCAAAA
rna-XR_003586216.1	TACACGGTATTTCCTCCAA	CCAATTCTAAGATGCGATT
GAPDH	ATCTCGCTCCTGGAAGATG	TCGGAGTGAACGGATTCG