Journal of Agricultural Science and Technology ›› 2023, Vol. 25 ›› Issue (7): 43-53.DOI: 10.13304/j.nykjdb.2021.1090
• BIOTECHNOLOGY & LIFE SCIENCE • Previous Articles Next Articles
Jingying JIA(), Yahui LI, Bingzhe FU, Yun MA, Xiaoyan CAI(
)
Received:
2021-12-23
Accepted:
2022-05-06
Online:
2023-07-15
Published:
2023-08-25
Contact:
Xiaoyan CAI
通讯作者:
蔡小艳
作者简介:
贾晶莹 E-mail:jiajingying176@163.com;
基金资助:
CLC Number:
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.
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.
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 |
Table 1 Primer information
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 |
Table 2 Fluorescence quantitative reaction system
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 |
Table 1 Primer information
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 |
品种Variety | miRs数量Number of miRs | 优势表达miRNA(miRs TPM占总TPM百分比) Advantage expressed known miRs (miRs TPM/all miRs TPM) | |
---|---|---|---|
已知Known | 新预测Novel | ||
中苜1号 Zhongmu 1 | 433 | 223 | mtr-miR5213-5p(25.82%), mtr-miR159a(22.40%), mtr-miR396a-5p(11.72%), mtr-miR166e-3p(5.22%) |
新盐52号 Xinyan 52 | 480 | 223 | mtr-miR159a(27.27%), mtr-miR5213-5p(20.07%), mtr-miR396a-5p(14.19%), mtr-miR166e-3p(3.89%) |
Table 3 Advantage expression miRs in Zhongmu 1 andXinyan 52
品种Variety | miRs数量Number of miRs | 优势表达miRNA(miRs TPM占总TPM百分比) Advantage expressed known miRs (miRs TPM/all miRs TPM) | |
---|---|---|---|
已知Known | 新预测Novel | ||
中苜1号 Zhongmu 1 | 433 | 223 | mtr-miR5213-5p(25.82%), mtr-miR159a(22.40%), mtr-miR396a-5p(11.72%), mtr-miR166e-3p(5.22%) |
新盐52号 Xinyan 52 | 480 | 223 | mtr-miR159a(27.27%), mtr-miR5213-5p(20.07%), mtr-miR396a-5p(14.19%), mtr-miR166e-3p(3.89%) |
miRs名称miRs name | 序列Sequence(5’-3’) | TPM值TPM value | |
---|---|---|---|
中苜1号Zhongmu1 | 新盐52号Xinyan52 | ||
miR5743a | TGAGAACTGTTTTCCGCACCTT | 6 019.360 539 | 61.234 256 |
miR5743b | TGAGAACTGTTTTCCGCACCTT | 6 019.360 539 | 61.234 256 |
novel-miR158 | AAAGGAUCAUUGGAUAAGUUC | 1 034.191 978 | 57.554 978 |
miR5754 | TATTGCACTCATCTTCCATGGC | 904.998 534 | 6.803 806 |
novel-miR54 | AAGUCCUUGUGUUGCAUCUC | 645.517 630 | 1 877.094 533 |
miR156f | TTGACAGAAGATAGAGAGCAC | 163.659 584 | 10.655 880 |
miR156e | TTGACAGAAGATAGAGAGCAC | 163.659 584 | 10.655 880 |
miR156h-5p | TTGACAGAAGATAGAGAGCAC | 163.659 584 | 10.655 880 |
Table 4 miRs sequence of differentially expressed
miRs名称miRs name | 序列Sequence(5’-3’) | TPM值TPM value | |
---|---|---|---|
中苜1号Zhongmu1 | 新盐52号Xinyan52 | ||
miR5743a | TGAGAACTGTTTTCCGCACCTT | 6 019.360 539 | 61.234 256 |
miR5743b | TGAGAACTGTTTTCCGCACCTT | 6 019.360 539 | 61.234 256 |
novel-miR158 | AAAGGAUCAUUGGAUAAGUUC | 1 034.191 978 | 57.554 978 |
miR5754 | TATTGCACTCATCTTCCATGGC | 904.998 534 | 6.803 806 |
novel-miR54 | AAGUCCUUGUGUUGCAUCUC | 645.517 630 | 1 877.094 533 |
miR156f | TTGACAGAAGATAGAGAGCAC | 163.659 584 | 10.655 880 |
miR156e | TTGACAGAAGATAGAGAGCAC | 163.659 584 | 10.655 880 |
miR156h-5p | TTGACAGAAGATAGAGAGCAC | 163.659 584 | 10.655 880 |
GO编码 GO ID | 富集层面Enrichment level | 功能Function | 差异表达miR靶基因富集到该通路的数量(百分比/%) Number of differential expressed miR target genes enriched into pathway (Percentage/%) | 全部miR靶基因富集到该通路的数量(百分比/%) Number of all miR target genes enriched into the pathway (Percentage/%) |
---|---|---|---|---|
GO:0006468 | 生物过程 Biological process | 蛋白质磷酸化Protein phosphorylation | 36(7.16) | 467(6.88) |
GO:0055114 | 氧化还原过程Oxidation-reduction process | 29(5.77) | 440(6.49) | |
GO:0006952 | 防御反应Defense response | 19(3.78) | 683(10.07) | |
GO:0006508 | 蛋白水解作用Proteolysis | 18(3.58) | 169(2.49) | |
GO:0006355 | 转录调控,DNA模板化 Regulation of transcription, DNA-templated | 17(3.38) | 360(5.31) | |
GO:0005975 | 碳水化合物代谢过程 Carbohydrate metabolic process | 15(2.98) | 116(1.71) | |
GO:0055085 | 跨膜运输Transmembrane transport | 13(2.58) | 126(1.86) |
Table 5 GO functional enrichment analysis of miRs target genes
GO编码 GO ID | 富集层面Enrichment level | 功能Function | 差异表达miR靶基因富集到该通路的数量(百分比/%) Number of differential expressed miR target genes enriched into pathway (Percentage/%) | 全部miR靶基因富集到该通路的数量(百分比/%) Number of all miR target genes enriched into the pathway (Percentage/%) |
---|---|---|---|---|
GO:0006468 | 生物过程 Biological process | 蛋白质磷酸化Protein phosphorylation | 36(7.16) | 467(6.88) |
GO:0055114 | 氧化还原过程Oxidation-reduction process | 29(5.77) | 440(6.49) | |
GO:0006952 | 防御反应Defense response | 19(3.78) | 683(10.07) | |
GO:0006508 | 蛋白水解作用Proteolysis | 18(3.58) | 169(2.49) | |
GO:0006355 | 转录调控,DNA模板化 Regulation of transcription, DNA-templated | 17(3.38) | 360(5.31) | |
GO:0005975 | 碳水化合物代谢过程 Carbohydrate metabolic process | 15(2.98) | 116(1.71) | |
GO:0055085 | 跨膜运输Transmembrane transport | 13(2.58) | 126(1.86) |
GO编码 GO ID | 富集层面Enrichment level | 功能Function | 差异表达miR靶基因富集到该通路的数量(百分比/%) Number of differential expressed miR target genes enriched into pathway (Percentage/%) | 全部miR靶基因富集到该通路的数量(百分比/%) Number of all miR target genes enriched into the pathway (Percentage/%) |
---|---|---|---|---|
GO:0043161 | 蛋白酶体介导的泛素依赖性蛋白分解代谢过程Proteasome-mediated ubiquitin-dependent protein catabolic process | 12(2.39) | 26(0.38) | |
GO:0016021 | 细胞组分 Cellular component | 膜的组成部分 Integral component of membrane | 160(31.81) | 2 454(36.18) |
GO:0005634 | 核Nucleus | 85(16.90) | 591(8.71) | |
GO:0005886 | 质膜Plasma membrane | 24(4.77) | 294(4.33) | |
GO:0009506 | 胞间连丝Plasmodesma | 16(3.18) | 122(1.80) | |
GO:0005829 | 胞浆Cytosol | 13(2.58) | 159(2.34) | |
GO:0005622 | 胞内Intracellular | 12(2.39) | 102(1.50) | |
GO:0005737 | 细胞质Cytoplasm | 12(2.39) | 255(3.76) | |
GO:0005524 | 分子功能Molecular function | ATP结合ATP binding | 97(19.28) | 1 227(18.09) |
GO:0003677 | DNA结合DNA binding | 84(16.70) | 502(7.40) | |
GO:0008270 | 锌离子结合Zinc ion binding | 34(6.76) | 317(4.67) | |
GO:0004674 | 蛋白质丝氨酸/苏氨酸激酶活性 Protein serine/threonine kinase activity | 34(6.76) | 394(5.81) | |
GO:0043531 | ADP结合ADP binding | 14(2.78) | 658(9.70) | |
GO:0046872 | 金属离子结合Metal ion binding | 14(2.78) | 267(3.94) | |
GO:0016887 | ATP酶活性ATPase activity | 11(2.19) | 44(0.65) |
Table 5 GO functional enrichment analysis of miRs target genes
GO编码 GO ID | 富集层面Enrichment level | 功能Function | 差异表达miR靶基因富集到该通路的数量(百分比/%) Number of differential expressed miR target genes enriched into pathway (Percentage/%) | 全部miR靶基因富集到该通路的数量(百分比/%) Number of all miR target genes enriched into the pathway (Percentage/%) |
---|---|---|---|---|
GO:0043161 | 蛋白酶体介导的泛素依赖性蛋白分解代谢过程Proteasome-mediated ubiquitin-dependent protein catabolic process | 12(2.39) | 26(0.38) | |
GO:0016021 | 细胞组分 Cellular component | 膜的组成部分 Integral component of membrane | 160(31.81) | 2 454(36.18) |
GO:0005634 | 核Nucleus | 85(16.90) | 591(8.71) | |
GO:0005886 | 质膜Plasma membrane | 24(4.77) | 294(4.33) | |
GO:0009506 | 胞间连丝Plasmodesma | 16(3.18) | 122(1.80) | |
GO:0005829 | 胞浆Cytosol | 13(2.58) | 159(2.34) | |
GO:0005622 | 胞内Intracellular | 12(2.39) | 102(1.50) | |
GO:0005737 | 细胞质Cytoplasm | 12(2.39) | 255(3.76) | |
GO:0005524 | 分子功能Molecular function | ATP结合ATP binding | 97(19.28) | 1 227(18.09) |
GO:0003677 | DNA结合DNA binding | 84(16.70) | 502(7.40) | |
GO:0008270 | 锌离子结合Zinc ion binding | 34(6.76) | 317(4.67) | |
GO:0004674 | 蛋白质丝氨酸/苏氨酸激酶活性 Protein serine/threonine kinase activity | 34(6.76) | 394(5.81) | |
GO:0043531 | ADP结合ADP binding | 14(2.78) | 658(9.70) | |
GO:0046872 | 金属离子结合Metal ion binding | 14(2.78) | 267(3.94) | |
GO:0016887 | ATP酶活性ATPase activity | 11(2.19) | 44(0.65) |
富集通路 Enrichment Pathway | 富集通路类型 Enrichment pathway type | 富集基因数量(百分比/%) Enrichment gene number (Percentage/%) |
---|---|---|
内吞作用Endocytosis | 细胞过程Cellular processes | 6(6.74) |
ABC转运蛋白ABC transporters | 环境信息处理 Environmental information processing | 5(5.62) |
植物激素信号转运体 Plant hormone signal transporters | 3(3.37) | |
RNA转运RNA Transport | 遗传信息处理 Genetic information processing | 6(6.74) |
泛素介导的蛋白质水解作用 Ubiquitin mediated proteolysis | 5(5.62) | |
蛋白酶体Proteasome | 4(4.49) | |
RNA聚合酶RNA polymerase | 4(4.49) | |
核糖体Ribosome | 3(3.37) | |
氨基酸的生物合成 Biosynthesis of amino acids | 新陈代谢 Metabolism | 4(4.49) |
氨基糖和核苷酸糖的代谢 Amino sugar and nucleotide sugar metabolism | 4(4.49) | |
淀粉和蔗糖代谢 Starch and sucrose metabolism | 4(4.49) | |
GPI锚生物合成 Glycosyl phosphatidylinositol(GPI)-anchor biosynthesis | 3(3.37) | |
谷胱甘肽代谢 Glutathione metabolism | 3(3.37) | |
植物-病原互作 Plant-pathogen interaction | 有机系统 Organismal system | 3(3.37) |
Table 6 Target gene KEGG pathway enrichment of differential expressed miRs
富集通路 Enrichment Pathway | 富集通路类型 Enrichment pathway type | 富集基因数量(百分比/%) Enrichment gene number (Percentage/%) |
---|---|---|
内吞作用Endocytosis | 细胞过程Cellular processes | 6(6.74) |
ABC转运蛋白ABC transporters | 环境信息处理 Environmental information processing | 5(5.62) |
植物激素信号转运体 Plant hormone signal transporters | 3(3.37) | |
RNA转运RNA Transport | 遗传信息处理 Genetic information processing | 6(6.74) |
泛素介导的蛋白质水解作用 Ubiquitin mediated proteolysis | 5(5.62) | |
蛋白酶体Proteasome | 4(4.49) | |
RNA聚合酶RNA polymerase | 4(4.49) | |
核糖体Ribosome | 3(3.37) | |
氨基酸的生物合成 Biosynthesis of amino acids | 新陈代谢 Metabolism | 4(4.49) |
氨基糖和核苷酸糖的代谢 Amino sugar and nucleotide sugar metabolism | 4(4.49) | |
淀粉和蔗糖代谢 Starch and sucrose metabolism | 4(4.49) | |
GPI锚生物合成 Glycosyl phosphatidylinositol(GPI)-anchor biosynthesis | 3(3.37) | |
谷胱甘肽代谢 Glutathione metabolism | 3(3.37) | |
植物-病原互作 Plant-pathogen interaction | 有机系统 Organismal system | 3(3.37) |
Fig. 4 Relative expression level of differentially expressed miRsA: Base on RNA-Seq; B: Bsae on RT-qPCR;* and ** indicate signifiant differences between different varieties at P<0.05 and P<0.01 levels, respectively
Fig. 5 Relative expression level of cross-kingdom regulated miRsA: Base on RNA-Seq; B: Bsae on RT-qPCR; ** indicates significant differences between different varieties at P<0.01 level
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