苜蓿miRs表达谱分析及跨界潜力miRs初步筛选

doi:10.13304/j.nykjdb.2021.1090

摘要/Abstract

摘要：

为探明宁夏苜蓿耐旱新品系‘新盐52号’（XY52）的miRNA表达谱，并筛选有跨界研究价值的microRNA（miR），以‘中苜一号’（ZM1）为参照，对ZM1和XY52进行高通量miRs组学测序和分析（RNA-seq）；通过生物信息学技术筛选差异表达miRs及预测靶基因，并对预测靶基因进行GO和KEGG富集分析；然后运用RT-qPCR技术对10个miRs进行检测和分析。结果表明，①成功构建了2种苜蓿miR表达谱，ZM1和XY52中分别检测到656和703个miRs，其中已知miRs分别为433和480个，新预测miRs数量均为233个；②2种苜蓿中共检测到21个差异表达miRs，其中novel-miR54、miR156f和miR166a表达量较高，并且novel-miR54在XY52中表达量极显著高于ZM1（P<0.01），miR156f在ZM1中的表达量极显著高于XY52（P<0.01）；③5个已知有跨界调控功能miRs中，miR166a在ZM1和XY52中均为表达水平最高的miR；品种之间，miR166a在XY52中的表达量较ZM1极显著上调（P<0.01）；④KEGG通路和GO功能富集分析发现差异表达的21个miRs共预测得到623个靶基因，主要与RNA转运、ABC转运蛋白和泛素介导的蛋白水解等信号通路有密切关系。以上结果为解析苜蓿品种miRs差异表达及研究苜蓿源miRs调控奶牛体内基因奠定初步基础。

关键词: 高通量测序, 中苜1号, 新盐52号, miRs, RT-qPCR, 跨物种调控

Abstract:

To explore miRNA expression profile of ‘Xinyan 52’ （XY52）， a new drought tolerant alfalfa line cultivated in Ningxia， and screen microRNA （miR） with cross-border research value， ‘Zhongmu 1’ （ZM1） was as the reference. ZM1 and XY52 were sequenced and analyzed by high-throughput miRs （RNA-seq）； the bioinformatics technology was used to screen differential expressed miRs and predict target genes， and the predicted target genes were enriched and analyzed by GO and KEGG； then 10 miRs were detected and analyzed by RT-qPCR. The results were as follows： ① the miRs expression profiles of 2 kinds of alfalfa were successfully constructed， and 656 and 703 miRs were detected in ZM1 and XY52， which the number of newly predicted miRs were 233 and the known miRs were 433 and 480； ② a total of 21 differential expressed miRs were detected in the two alfalfa varieties， among which 3 miRs including novel-miR54， miR156f and miR166a were expressed higher， and the expression of novel-miR54 in XY52 was significantly higher than that in ZM1 （P<0.01）， and the expression of miR156f in ZM1 was significantly higher than that in XY52 （P<0.01）； ③ among the five miRs which had cross-border regulatory functions， miR166a had the most expressive level in ZM1 and XY52， while the expression of miR166a in XY52 was significantly higher than that in ZM1 （P<0.01）； ④ a total of 623 target genes were predicted by the KEGG pathway and GO function enrichment analysis of 21 miRs with differential expression， which were mainly related to signaling pathways such as RNA transport， ABC transporter and ubiquitin-mediated proteolysis. These results laid a preliminary foundation for the analysis of miRs in differences of alfalfa and the regulation of genes in dairy cows.

Key words: high-throughput sequencing, Zhongmu 1, Xinyan 52, miRs, RT-qPCR, cross-border regulated

中图分类号:

S551+.7

贾晶莹, 李雅辉, 伏兵哲, 马云, 蔡小艳. 苜蓿miRs表达谱分析及跨界潜力miRs初步筛选[J]. 中国农业科技导报, 2023, 25(7): 43-53.

Jingying JIA, Yahui LI, Bingzhe FU, Yun MA, Xiaoyan CAI. Analysis on miRs Expression Profiles of Alfalfa and Screening of Trans-border Potential miRs[J]. Journal of Agricultural Science and Technology, 2023, 25(7): 43-53.

图/表 13

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miRs名称 miRs name	引物序列 Primer sequence （5’-3’）
5s	F：GCAGACGAAGTCCTTGTGTTG R：AGTGCAGGGTCCGAGGTATT
miR156b-5p	F：CGCAGACAGTTGACAGAAGAGA
	R：AGTGCAGGGTCCGAGGTATT
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTGCTCAC
miR166a	F：CACAGTTCGGACCAGGCTT
	R：AGTGCAGGGTCCGAGGTATT
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGGGAATG
miR167a	F：AAGCTTTGAAGCTGCCAGC
	R：AGTGCAGGGTCCGAGGTATT
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTAGATCAT
miR168a	F：CATGTGTCGCTTGGTGCAG
	R：AGTGCAGGGTCCGAGGTATT
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTCCCGAC
miR319a-3p	F：CGAACGTTTTGGACTGAAGG
	R：AGTGCAGGGTCCGAGGTATT
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGGAGCTC

miRs名称 miRs name	引物序列 Primer sequence （5’-3’）
5s	F：GCAGACGAAGTCCTTGTGTTG R：AGTGCAGGGTCCGAGGTATT
miR156b-5p	F：CGCAGACAGTTGACAGAAGAGA
	R：AGTGCAGGGTCCGAGGTATT
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTGCTCAC
miR166a	F：CACAGTTCGGACCAGGCTT
	R：AGTGCAGGGTCCGAGGTATT
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGGGAATG
miR167a	F：AAGCTTTGAAGCTGCCAGC
	R：AGTGCAGGGTCCGAGGTATT
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTAGATCAT
miR168a	F：CATGTGTCGCTTGGTGCAG
	R：AGTGCAGGGTCCGAGGTATT
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTCCCGAC
miR319a-3p	F：CGAACGTTTTGGACTGAAGG
	R：AGTGCAGGGTCCGAGGTATT
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGGAGCTC

组分Component	体积Volume/μL
预混液 TB Green Premix Ex Taq Ⅱ （Tli RNaseH Plus）（2×）	10.0
PCR上游引物 PCR forward primer （10 μmol·L^-1）	0.4
PCR下游引物 PCR reverse primer （10 μmol·L^-1）	0.4
DNA （100 ng·μL^-1）	2.0
灭菌水dd H₂O	7.2
总计Total	20.0

组分Component	体积Volume/μL
预混液 TB Green Premix Ex Taq Ⅱ （Tli RNaseH Plus）（2×）	10.0
PCR上游引物 PCR forward primer （10 μmol·L^-1）	0.4
PCR下游引物 PCR reverse primer （10 μmol·L^-1）	0.4
DNA （100 ng·μL^-1）	2.0
灭菌水dd H₂O	7.2
总计Total	20.0

miRs名称 miRs name	引物序列 Primer sequence （5’-3’）
miR5754	F：TATTGCACTCATCTTCCATGGC
	R：ATCCAGTGCAGGGTCCGAGG
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCCATG
miR156f	F：CCGTTGACAGAAGATAGAGAGCAC
	R：ATCCAGTGCAGGGTCCGAGG
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTGCTC
miR5743a	F：TGAGAACTGTTTTCCGCACCTT
	R：ATCCAGTGCAGGGTCCGAGG
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAGGTG
Novel-miR54	F：CCAAGTCCTTGTGTTGCATCTC
	R：ATCCAGTGCAGGGTCCGAGG
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAGATG
Novel-miR158	F：GCGCAAAGGATCATTGGATAAGTTc
	R：ATCCAGTGCAGGGTCCGAGG
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAACTT