Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (8): 220-233.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0115
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LIU Bao-cai1,2(), CHEN Jing-ying1,2(), ZHANG Wu-jun1,2, HUANG Ying-zhen1,2, ZHAO Yun-qing1,2, LIU Jian-chao3, WEI Zhi-cheng4
Received:
2023-02-13
Online:
2023-08-26
Published:
2023-09-05
Contact:
CHEN Jing-ying
E-mail:626813844@qq.com;cjy6601@163.com
LIU Bao-cai, CHEN Jing-ying, ZHANG Wu-jun, HUANG Ying-zhen, ZHAO Yun-qing, LIU Jian-chao, WEI Zhi-cheng. Characteristics Analysis of Seed Microrhizome Gene Expression of Polygonatum cyrtonema[J]. Biotechnology Bulletin, 2023, 39(8): 220-233.
Fig. 1 Germination process of P. cyrtonema seeds A: Radicle breaks through seed coat; B: radicle elongation; C: hypocotyl enlargement; D: microrhizomes; E: green microrhizomes; F: seedlings. A, D and E in germination stage were used as experimental materials in this experiment
基因 Gene | 引物 Primer | 序列 Sequence(5'-3') |
---|---|---|
AUX1 | Cluster-68615.91119-F | CCTCTCCTTCTTGGTCCTGTA |
Cluster-68615.91119-R | GGTGGCTCAACTTATGATGCT | |
AUX2 | Cluster-68615.87861-F | TGCTCATCCATCAGTTCATAACC |
Cluster-68615.87861-R | GAATACGATTGCGAGGAACCATA | |
CH3 | Cluster-58608.0-F | AAGAAGACCTCCAGAAGAGCATA |
Cluster-58608.0-R | GACAATCTCCCAGAACAACACAT | |
TF | Cluster-68615.45537-F | CTTCCTGCTGCTTTCTCTTAGTG |
Cluster-68615.45537-R | TGCGGCTGCTCAGATTATTG | |
SnPK2 | Cluster-68615.69985-F | ATGCTGATGACTCTGATTCTGATG |
Cluster-68615.69985-R | GCCTACAACCGATACTTCTGATAC | |
MYC2 | Cluster-68615.81195-F | AGATCAGCTCAGCTTCCATCA |
Cluster-68615.81195-R | CATTCCAGTTCCTTCGCCATT | |
β-glucosidase | Cluster-68615.43996-F | ACAAGGGTTAATGGGACTTCTCT |
Cluster-68615.43996-R | CTGAAGAATCGCCTCCAAGTG | |
Actin | Actin_Cluster-F | CACCGATTGACACAAGGAGAG |
Actin_Cluster-R | AGGATGGCTTACTACATTGACTTC |
Table 1 Gene-specific primers used in RT-qPCR
基因 Gene | 引物 Primer | 序列 Sequence(5'-3') |
---|---|---|
AUX1 | Cluster-68615.91119-F | CCTCTCCTTCTTGGTCCTGTA |
Cluster-68615.91119-R | GGTGGCTCAACTTATGATGCT | |
AUX2 | Cluster-68615.87861-F | TGCTCATCCATCAGTTCATAACC |
Cluster-68615.87861-R | GAATACGATTGCGAGGAACCATA | |
CH3 | Cluster-58608.0-F | AAGAAGACCTCCAGAAGAGCATA |
Cluster-58608.0-R | GACAATCTCCCAGAACAACACAT | |
TF | Cluster-68615.45537-F | CTTCCTGCTGCTTTCTCTTAGTG |
Cluster-68615.45537-R | TGCGGCTGCTCAGATTATTG | |
SnPK2 | Cluster-68615.69985-F | ATGCTGATGACTCTGATTCTGATG |
Cluster-68615.69985-R | GCCTACAACCGATACTTCTGATAC | |
MYC2 | Cluster-68615.81195-F | AGATCAGCTCAGCTTCCATCA |
Cluster-68615.81195-R | CATTCCAGTTCCTTCGCCATT | |
β-glucosidase | Cluster-68615.43996-F | ACAAGGGTTAATGGGACTTCTCT |
Cluster-68615.43996-R | CTGAAGAATCGCCTCCAAGTG | |
Actin | Actin_Cluster-F | CACCGATTGACACAAGGAGAG |
Actin_Cluster-R | AGGATGGCTTACTACATTGACTTC |
样品 Sample | 总读长 Total reads/bp | 总匹配数 Total mapped ones/bp |
---|---|---|
A1 | 40 179 174 | 29 824 074(74.23%) |
A2 | 45 826 078 | 32 824 940(71.63%) |
A3 | 44 560 782 | 32 498 548(72.93%) |
D1 | 45 873 682 | 34 073 516(74.28%) |
D2 | 41 999 498 | 31 537 406(75.09%) |
D3 | 45 760 904 | 33 679 092(73.60%) |
E1 | 41 290 430 | 30 888 404(74.81%) |
E2 | 44 373 676 | 33 739 212(76.03%) |
E3 | 44 063 488 | 33 299 498(75.57%) |
Table 2 Mapped results of sample reads and assembly transcripts
样品 Sample | 总读长 Total reads/bp | 总匹配数 Total mapped ones/bp |
---|---|---|
A1 | 40 179 174 | 29 824 074(74.23%) |
A2 | 45 826 078 | 32 824 940(71.63%) |
A3 | 44 560 782 | 32 498 548(72.93%) |
D1 | 45 873 682 | 34 073 516(74.28%) |
D2 | 41 999 498 | 31 537 406(75.09%) |
D3 | 45 760 904 | 33 679 092(73.60%) |
E1 | 41 290 430 | 30 888 404(74.81%) |
E2 | 44 373 676 | 33 739 212(76.03%) |
E3 | 44 063 488 | 33 299 498(75.57%) |
样品 Sample | FPKM 区间FPKM Interval | ||||||
---|---|---|---|---|---|---|---|
0≤FPKM≤0.1 | 0.1<FPKM≤0.3 | 0.3<FPKM≤3.57 | 3.57<FPKM≤15 | 15<FPKM≤60 | FPKM>60 | ||
A1 | 45 729(25.64%) | 8 928(5.01%) | 84 666(47.48%) | 26 418(14.82%) | 9 731(5.46%) | 2 847(1.60%) | |
A2 | 90 461(50.73%) | 4 155(2.33%) | 46 585(26.12%) | 25 282(14.18%) | 8 776(4.92%) | 3 060(1.72%) | |
A3 | 92 713(51.99%) | 2 373(1.33%) | 42 307(23.73%) | 28 240(15.84%) | 9 676(5.43%) | 3 010(1.69%) | |
D1 | 68 567(38.45%) | 14 059(7.88%) | 64 246(36.03%) | 19 173(10.75%) | 9 466(5.31%) | 2 808(1.57%) | |
D2 | 68 767(38.56%) | 13 366(7.50%) | 64 688(36.28%) | 19 321(10.84%) | 9 411(5.28%) | 2 766(1.55%) | |
D3 | 69 033(38.71%) | 13 351(7.49%) | 64 127(35.96%) | 19 404(10.88%) | 9 570(5.37%) | 2 834(1.59%) | |
E1 | 63 577(35.65%) | 11 564(6.49%) | 70 391(39.47%) | 20 459(11.47%) | 9 569(5.37%) | 2 759(1.55%) | |
E2 | 61 068(34.25%) | 13 276(7.45%) | 72 028(40.39%) | 19 855(11.13%) | 9 390(5.27%) | 2 702(1.52%) | |
E3 | 59 900(33.59%) | 13 120(7.36%) | 73 014(40.95%) | 20 113(11.28%) | 9 453(5.30%) | 2 719(1.52%) |
Table 3 FPKM interval statistics of sample expressed levels
样品 Sample | FPKM 区间FPKM Interval | ||||||
---|---|---|---|---|---|---|---|
0≤FPKM≤0.1 | 0.1<FPKM≤0.3 | 0.3<FPKM≤3.57 | 3.57<FPKM≤15 | 15<FPKM≤60 | FPKM>60 | ||
A1 | 45 729(25.64%) | 8 928(5.01%) | 84 666(47.48%) | 26 418(14.82%) | 9 731(5.46%) | 2 847(1.60%) | |
A2 | 90 461(50.73%) | 4 155(2.33%) | 46 585(26.12%) | 25 282(14.18%) | 8 776(4.92%) | 3 060(1.72%) | |
A3 | 92 713(51.99%) | 2 373(1.33%) | 42 307(23.73%) | 28 240(15.84%) | 9 676(5.43%) | 3 010(1.69%) | |
D1 | 68 567(38.45%) | 14 059(7.88%) | 64 246(36.03%) | 19 173(10.75%) | 9 466(5.31%) | 2 808(1.57%) | |
D2 | 68 767(38.56%) | 13 366(7.50%) | 64 688(36.28%) | 19 321(10.84%) | 9 411(5.28%) | 2 766(1.55%) | |
D3 | 69 033(38.71%) | 13 351(7.49%) | 64 127(35.96%) | 19 404(10.88%) | 9 570(5.37%) | 2 834(1.59%) | |
E1 | 63 577(35.65%) | 11 564(6.49%) | 70 391(39.47%) | 20 459(11.47%) | 9 569(5.37%) | 2 759(1.55%) | |
E2 | 61 068(34.25%) | 13 276(7.45%) | 72 028(40.39%) | 19 855(11.13%) | 9 390(5.27%) | 2 702(1.52%) | |
E3 | 59 900(33.59%) | 13 120(7.36%) | 73 014(40.95%) | 20 113(11.28%) | 9 453(5.30%) | 2 719(1.52%) |
Fig. 2 DvsA different expressed genes A01: Protein phosphorylation. A02: Phosphorylation. A03: Phosphate-containing compound metabolic process. A04: Phosphorus metabolic process. A05: Metabolic process. A06: Oxidation-reduction process. A07: Microtubule-based movement. A08: Cellular carbohydrate metabolic process. A09: Carbohydrate metabolic process. A10: Negative regulation of translation. A11: Negative regulation of cellular amide metabolic process. A12: Cellular polysaccharide metabolic process. A13: Cellular protein modification process. A14: Protein modification process. A15: Microtubule. A16: Tubulin complex. A17: Apoplast. A18: Cell wall. A19: Catalytic activity. A20: Hydrolase activity, acting on glycosyl bonds. A21:Transferase activity. A22: Tetrapyrrole binding, A23: Heme binding. A24: Protein kinase activity. A25: Hydrolase activity, hydrolyzing O-glycosyl compounds. A26: RNA glycosylase activity. A27: rRNA N-glycosylase activity. A28: Hydrolase activity. A29: Phosphotransferase activity, alcohol group as acceptor. A30: Oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen. A31: Microtubule binding. A32: Kinase activity. A33: Transferase activity, transferring acyl groups other than amino-acyl groups. A34: Oxidoreductase activity. A35: Microtubule motor activity. A36: Ion binding. A37: Iron ion binding. A38: Tubulin binding. A39: Xyloglucan: xyloglucosyl transferase activity. A40: Transferase activity, transferring phosphorus-containing groups
Fig. 5 DvsA differentially expressed genes involved in starch and sucrose metabolism The red border of the node contains up-regulated differential genes; the green border of the node contains the down-regulated differential genes; and the yellow border of the node contains the up-regulated and down-regulated differential genes. The same below
Fig. 7 EvsD differently expressed genes B01: Ribosome biogenesis. B02: Translation. B03: Ribonucleoprotein complex biogenesis. B04: Peptide biosynthetic process. B05: Peptide metabolic process. B06: Cellular amide metabolic process. B07: Amide biosynthetic process. B08: Oxidation-reduction process. B09: Organonitrogen compound biosynthetic process. B10: Organonitrogen compound metabolic process. B11: Cellular component biogenesis. B12: Protein metabolic process. B13: Metabolic process. B14: Cellular protein metabolic process. B15: Biosynthetic process. B16: Organic substance biosynthetic process. B17: Cellular macromolecule biosynthetic process. B18: Cellular component organization or biogenesis. B19: Macromolecule biosynthetic process. B20: Cellular nitrogen compound biosynthetic process. B21: Cellular biosynthetic process. B22: Single-organism metabolic process. B23: Obsolete peroxidase reaction. B24: Ribosome. B25: Ribonucleoprotein complex. B26: Non-membrane-bounded organelle. B27: Intracellular non-membrane-bounded organelle. B28: Cytoplasmic part. B29: Cytoplasm. B30: Macromolecular complex. B31: Structural constituent of ribosome. B32: Oxidoreductase activity. B33: Structural molecule activity. B34: Catalytic activity. B35: Antioxidant activity. B36: Oxidoreductase activity, acting on peroxide as acceptor. B37: Pyrophosphatase activity. B38: Peroxidase activity. B39: Nucleoside-triphosphatase activity. B40: GTPase activity.
Fig. 11 Verification of seven selected DEGs by RT-qPCR Comparison of RNA-seq data(blue line chart)with RT-qPCR data(orange bar graph). Different lowercase letters indicate significant differences in the expression levels of the same gene in different morphology of micro-rhizome development at 0.05 level
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