Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (14): 3113-3123.doi: 10.3864/j.issn.0578-1752.2021.14.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Screening, Identification and Functional Analysis of Important LncRNAs for Lactation Traits in Small-Tailed Han Sheep

WANG JiQing(),HAO ZhiYun,SHEN JiYuan,KE Na,HUANG ZhaoChun,LIANG WeiWei,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin   

  1. College of Animal Science and Technology/Gansu Key Laboratory of Herbivorous Animal Biotechnology/Gansu Engineering Lab of Genetic Improvement in Ruminants, Gansu Agricultural University, Lanzhou 730070
  • Received:2020-11-13 Accepted:2020-02-04 Online:2021-07-16 Published:2021-07-26

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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

Table 1

The primers information for RT-qPCR"

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

Fig. 1

The predicted novel lncRNAs in the mammary gland tissues of Small-Tailed Han sheep (A) and the venn diagram of lncRNAs expression in the two groups of Small-Tailed Han ewe (B) ‘HM’ and ‘LM’ represent the mammary gland tissues from high-lactating performance and low-lactating performance Small-tailed Han sheep, respectively. The same as below"

Fig. 2

The statistical distribution of expression levels of lncRNAs in the mammary gland of Small-Tailed Han sheep (A) and volcano plot of the differentially expressed lncRNAs (B) The red and green dots indicate upregulated and downregulated lncRNAs in high-lactating performance Small-Tailed Han sheep, respectively. The black dots indicate the lncRNAs that had no significantly difference in the two groups (P>0.05) "

Fig. 3

The GO enrichment results of the target genes of differentially expressed lncRNAs"

Fig. 4

The KEGG analysis of the target genes of differentially expressed lncRNAs"

Fig. 5

The network of lncRNA-miRNA The red and green triangles indicate up-regulated and down-regulated lncRNAs in mammary gland tissues of high-lactating performance Small-Tailed Han sheep, respectively"

Fig. 6

The RT-qPCR verification of 16 differentially expressed lncRNAs"

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