Acta Horticulturae Sinica ›› 2026, Vol. 53 ›› Issue (1): 1-24.doi: 10.16420/j.issn.0513-353x.2024-0564
• Genetic & Breeding·Germplasm Resources·Molecular Biology • Next Articles
WANG Hong1, WAN Yunqing1, QI Xinai1, SHENG Huizi1, REN Yalin1, LIU Mengyu1, LIU Xiaofeng1,*(
), ZHAO Xiaochun1,2,3,*(
)
Received:2025-06-04
Revised:2025-10-24
Online:2026-01-25
Published:2026-01-26
Contact:
LIU Xiaofeng, ZHAO Xiaochun
WANG Hong, WAN Yunqing, QI Xinai, SHENG Huizi, REN Yalin, LIU Mengyu, LIU Xiaofeng, ZHAO Xiaochun. Genome-Wide Identification of CYP71 Subfamily Genes in Citrus sinensis and Functional Analysis of CsCYP2[J]. Acta Horticulturae Sinica, 2026, 53(1): 1-24.
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URL: https://www.ahs.ac.cn/EN/10.16420/j.issn.0513-353x.2024-0564
| 基因名称 Gene name | 基因ID Gene ID | 上游引物(5′-3′) Foward primer | 下游引物(5′-3′) Reverse primer |
|---|---|---|---|
| CsCYP1 | orange1.1t02788 | GCGGATGATCCTACTCTGGAAG | GAGGTGTCAGTTCCAGCAGTTA |
| CsCYP2 | Cs3g07330 | CGTATTCCTTGTGTCACTCCCA | AACTACTACGGTCTGCACAAGG |
| CsCYP3 | orange1.1t02784 | TCTTATTTCGCCGCCGATTTTC | GTACTTCCCAGCAAGAAGAGCT |
| CsCYP4 | Cs1g17420 | CTTCCCTATCGGTCCATCCATG | CAACGGAAGATCCCACAACAAC |
| CsCYP5 | Cs1g17410 | ATACACAGACAAGCAGCTCGAA | AGCCACATAATTTTCGACCCCT |
| CsCYP6 | Cs1g10500 | ATGCAATTCCATGGCTTGCTTT | TCCTGGATTGCTGCCTTAATGT |
| CsCYP7 | Cs5g25880 | TTTGGGCCATGACCTACCTAAC | TTGCCCCCAATCAACGATCTAA |
| CsCYP8 | Cs3g25780 | GGTGCCTATCTTCCTGATCTCG | ATGCAAGTTGCCTATGAAGGGA |
| CsCYP9 | Cs4g09010 | AAGGCACAAGATGAGGTAAGGG | ACACGTCTCTGCACATTCTCTT |
| CsCYP10 | Cs7g29720 | TGTTATCTTTGCATCCAGGCCT | AAAGATTGGACTCTTTTGGCGC |
| CsCYP11 | Cs7g09640 | TGGGCAATTGGAAGAGATTCGA | TTCTACGACCACCTCCAAATGG |
| CsCYP12 | Cs3g01130 | CCCCGAAATCCTCTTTCTGTCA | TTTGGCCAGCTCTTTAGAGGAG |
| CsCYP13 | Cs2g01910 | GGCAGTAACAAGAGATCCCCAA | CCGCAAACCACACCAAATTTTG |
| CsCYP14 | Cs5g09880 | ATTGACTTGGCTGATCCCACAT | ACGCCTTATACCTTGCAAGTCA |
| CsCYP15 | Cs8g09720 | TTCCTTTTCCAGGGGACACAAA | TCATTGTGTTGGTGGGGAAAGA |
| CsCYP16 | orange1.1t04139 | CAGTGATTGGAAACCTCCTCGA | TGATTGTAGGAACAGGCTGCAT |
| CsCYP17 | Cs5g09870 | AACCCGATTGACGAATCAGACA | TATAGCCTGCGACTTGTGTGTT |
| CsCYP18 | orange1.1t01519 | TAAACGGACGGTTGGTGATGAT | CAATTCTTTCACCACGGCTTGT |
| CsCYP19 | Cs7g15460 | GAAAGCAGTGATCTTGGAGGGA | ATCACGTGTCCATCCAATGTGA |
| CsCYP20 | Cs7g15470 | GTGAATTTCATGGTGGCCGAAA | TCAACCTCATCACCATCCACAG |
| CsCYP21 | Cs2g07770 | TGCTGATAACATCACCACAGCT | TATCAGTGGAACCAAGCACTCC |
| CsCYP22 | Cs7g15540 | CGTTTAGGCCTGAGAGGTTCTT | TTCCAAATGAAGCATGGCCAAG |
| CsCYP23 | Cs7g11300 | CCCTAGCTTGCCTTACCTTCAA | TTGGATCATGAGTGATTGCCCA |
| CsCYP24 | orange1.1t00542 | GATACAGTGGCGGTTTTGATCG | GTATCAGTGATGGCTAGACGGG |
| CsCYP25 | Cs1g22200 | TTCACCTCATAAAGCAGCCCAT | GTGAGGAATTTTGGACGGTTGG |
| CsCYP26 | Cs1g22230 | TTGTATCCAACAGCTCCACTCC | TGAATGGCCCATACATTGACCA |
| CsCYP27 | Cs3g15630 | CTTCGGCTGCAACTATGGAATG | CACGTGGAACTTTGAAACCTCC |
| CsCYP28 | Cs3g15590 | CAGGCCTGTCCTTGTTGTATCT | TTTGGTTACCGCTTGCTGAATG |
| CsCYP29 | Cs3g09120 | TGAGTGAGGGAATGAGCTTGAC | ATATAGCGATGCGGAAAGACGT |
| CsCYP30 | Cs1g10540 | TTGGTGGTGTCAGATGCTCTAC | AAGGATGAGTGCCAAGCATGTA |
| CsCYP31 | Cs5g27570 | TTAGACTTGGCAAGCATCCCAT | TGCGTTTTCTGAGCAAAGTGAG |
| CsCYP32 | Cs5g27580 | CTCGGATTCTTCAGGGGTTTGA | TTCAGTTTAGGCAAGGCAATGC |
| CsCYP33 | Cs4g07380 | ATGGGCTATGTCCGAGCTAATG | AGACTGAAGGGTCTCTGCCTAT |
| CsCYP34 | Cs2g08630 | ATCATCTACGGGTTTTGTGGCT | CACCAGCTAAAAGGTCCTGGAT |
| CsCYP35 | orange1.1t02775 | GATCCAACCATGTGGGAAAAGC | CCAAATCTGTCCTCCATGTCCA |
| CsCYP36 | Cs1g17390 | AGGGGTCGAAAATTATGTGGCT | CACTGTTACTGCTGAGCTCTCA |
| CsCYP37 | orange1.1t01321 | ACATTTGCGCCAAACTCGTAAA | CCGGCTCATTATGGCAAGATTG |
| CsCYP38 | Cs5g08070 | CTCATCGGAAACCTCAACCTCA | AATTTTACGAGTTTGGCGCCAA |
| CsCYP39 | Cs5g08200 | TGGCGCCAAACTCGTAAAATTT | ATCCGGCTCATAATGGCAAGAT |
| CsCYP40 | orange1.1t04499 | CATCCTCATCCCTCTGCATCTC | ATGACCCAAATTTGAGCTGCAC |
| CsCYP41 | orange1.1t01327 | TTATTGCCATTTGGAGTGGGGA | ACCATGCAAAAGATTCGCCAAA |
| CsCYP42 | orange1.1t02780 | TGGCCATTTATCGGAAACCTGA | GTACTTCCCAGCAAGAAGAGCT |
| CsCYP43 | Cs5g25060 | TTGGTGCTGGCAGAAGAATTTG | ATTCCTGGAGGCATTTCCCAAT |
| CsCYP44 | Cs5g25070 | TTGGTGCTGGCAGAAGAATTTG | ATTCCTGGAGGCATTTCCCAAT |
| CsCYP45 | Cs1g04220 | CTCCCAATCTGCCTCTTCTTGT | AAAACCCAGACGCAATGACATG |
| CsCYP46 | Cs5g06350 | TGACCAACATTCCGATCCCATT | TGGGGTCATTCCTAATTGGTGG |
| CsCYP47 | Cs1g02540 | CACTTACGCTTAGGCTTAACGC | TTGTTCGCCAATAAGACCCGTA |
| CsCYP48 | Cs7g10570 | TTTGTCCTGGGATTCAGATGGG | TCTCCTTCCTTCATTCCGTTGG |
| CsCYP49 | Cs5g06320 | AAGACCCATGACATCGCTTTCT | TAACGTTCCCGAGATCTTTGCA |
| CsCYP50 | Cs2g17110 | ACAAAATGTCCCTCATCGCTCT | TTTTGTAGCAATGGATCGGGGA |
| CsCYP51 | Cs7g10050 | GGGATGAGGAAGTGAAACGTCT | TTGCGTATTCTTCCTCAGCCTT |
| CsCYP52 | Cs3g21580 | AGAAGATTTTGTCCCGGGACTC | CAGCAGGCATTTCCCAATCAAA |
| CsCYP53 | Cs2g19590 | TAAAACTTGGCCAGCTTGATGC | ATCTGAATGACTGGACACGCTT |
| CsCYP54 | orange1.1t00025 | TGGGCAGAAATCCGACATAACA | GGTCTCTTGAAATGGCCCAGTA |
| CsCYP55 | orange1.1t03363 | CATTGATGGCTCCACACGAATC | CTTCCAATTGCCCAAGCGTTTA |
| CsCYP56 | Cs2g19560 | AAGCATGCAAAGGAAAGAGCAG | TTTGGCCTTAGCTGGTACTCTG |
| CsCYP57 | Cs5g06280 | AGAAATTAGGCGGATTGGGGTT | AACACATCGACCAACCGTATCA |
| CsCYP58 | orange1.1t03357 | CGAGGATTTCGTTGACGTGATG | TGGTGAAGTGTCTATTGCTCCC |
| CsCYP59 | orange1.1t04874 | GGCAATAGGAAGGGACAGTGAA | GCGCATTGCCATTGATATTCCT |
| CsCYP60 | orange1.1t04991 | GGCAATAGGAAGGGACAGTGAA | GCGCATTGCCATTGATATTCCT |
| CsCYP61 | orange1.1t05138 | GGCAATAGGAAGGGACAGTGAA | GCGCATTGCCATTGATATTCCT |
| CsCYP62 | Cs4g09080 | CCCTCATCATCGGCTAAGAGAC | GCTCAAAAGCTCAGACACACAG |
| CsCYP63 | Cs4g09040 | ATTGTTAATGCATGGGCGATCG | ATATTCTCCTGCCAGCACCAAA |
| CsCYP64 | Cs5g06360 | CATCTTGGACATGTTTGCTGCA | ATGGATTCCCTTGGGACTAGGA |
| CsCYP65 | Cs5g06310 | CTTTGATGTTCCTGCACTTGGG | TTTGTTGGTCATGATCGCAACC |
| CsCYP66 | orange1.1t02944 | TCCTTCCGTTGGATTCCTTCAG | AGATCTCCACGCCCTTGAATTT |
| CsCYP67 | Cs2g18790 | AATTGCTTCCATTTGGTTCCGG | TCGAGGTCTTCTAGTTTGGTGC |
| CsCYP68 | Cs7g27010 | TGCTGCCTTTTGTATGGGAGAT | CCATCCTTTCGAAGTTGGAGGA |
| CsCYP69 | Cs4g09050 | TGTTATCTTTGCATCCAGGCCT | AAAGATTGGACTCTTTTGGCGC |
| CsCYP70 | orange1.1t05258 | GGCAATAGGAAGGGACAGTGAA | GCGCATTGCCATTGATATTCCT |
| CsCYP71 | Cs4g09090 | AATGGGTCACTGGATTCAGGTC | CCAGTCCACAGTTGTAGCAGAT |
| CsCYP72 | Cs5g06390 | TACTGAGGATTGCAGGGTAGGA | ATTCTCATCCCTGGTGGCAATT |
| CsCYP73 | orange1.1t03684 | TGATGGGTGGTTTCTATGTGGG | TTCTCAAGCTTGGCATTGAAGC |
| CsCYP74 | Cs4g09110 | GGAGTGAGGCGGAAACTTAAGA | GATGTGTCACTCCCAGCTACAA |
| CsCYP75 | Cs5g22050 | TCGACTCAGGCCGTCAAAATAA | ACACTCTCATCGCAAACTCCAT |
| CsCYP76 | Cs7g09630 | TCATCAGGGAAGAGGAAGTTGC | CCCTCGAAATCTCCTCCCAAAA |
| CsCYP77 | Cs4g09020 | TAAGATTGCACACCCCTCTTCC | AATAGCAGTTGAGCGAGTGGAA |
| CsCYP78 | Cs1g04240 | CTGGCTGGAGGTTTTTCTTTGG | CTGCTGGTCTCTTGAAGCCTTA |
| CsCYP79 | Cs5g13970 | GATTACATCCGGCAGGTACCAT | AGGTTGGCCAACAGATACTCAG |
| CsCYP80 | orange1.1t02083 | GGCTCAATTCCTCCCAAGAGAA | TACCGGCACCAAATGGGATAAA |
| CsCYP81 | Cs5g06380 | ATCCATCCGTGATGAGAAGAGC | CCAATACTCAGGGTCTGTTGCT |
| CsCYP82 | Cs4g09100 | CCTTGTTGGCTGTCTACCTCAT | ATGTCGTGGCTCTTCATCACTT |
| CsCYP83 | Cs5g13940 | AGAATGGGCAATGGCTGAACTA | ATGGTACCTGCCGGATGTAATC |
| CsCYP84 | Cs5g25920 | GTGTCATTTTGGGAGCTGTCAC | GCTGGCCTGCTACAAAATTGAA |
| CsCYP85 | Cs6g15610 | CCATTCGGTTCTGGGAGAAGAA | GAAGCAAAGAGGCAAGACCAAG |
| CsCYP86 | Cs6g15620 | ATGGTGCAGAGTTTTTCCAAGC | GCTTTACCAACATCCCTCTCCA |
| CsCYP87 | Cs9g14170 | TTACCGGTGCTAGGAAACCTTC | AGGCCATAAGAGGTGACCAAAG |
| CsCYP88 | orange1.1t03998 | CTGTGATCGAAAGGTCCCTGAA | AGGTCTTGATTGGCATCGAGTT |
| CsCYP89 | orange1.1t03997 | TATCAAAGGCTTCCACCAGGAC | CAGGGACCTTTCGATCACAGAA |
| CsCYP90 | Cs4g14140 | CAGATTCTGTGAGTTCCCACCA | ATATCGATTGCTTCTCCCGCAT |
| CsCYP91 | orange1.1t04132 | CTGTGATCGAAAGGTCCCTGAA | AGGTCTTGATTGGCATCGAGTT |
| CsCYP92 | Cs4g14130 | CTCCCTGTCTCGACAAAATGGA | AACATCGATTGGTTCACCCGTA |
| CsCYP93 | Cs8g09730 | GAACTACTCCACAACCCTGAGG | GAGGTTTGTCCCATGTGCTTTC |
| CsCYP94 | orange1.1t04372 | CGAATTTGTCCTGGTTTGCCAT | AGGGATAGTACGAAGGGGTTGA |
| CsCYP95 | Cs8g09780 | TGGCAGAACTACTCCACAATCC | GAGTGAGGATTGTCCCATGTGT |
| CsCYP96 | Cs6g15630 | ATGAAGTGGCTCGAGACATGTT | ATGCCTCGGCTTCTTCTACAAA |
| CsCYP97 | Cs8g09480 | GGCGCAAGAAAATCAAAGACCT | TATTGAAAGCAGCTTGGCCAAC |
| CsCYP98 | orange1.1t04771 | CGAATTTGTCCTGGTTTGCCAT | AGGGATAGTACGAAGGGGTTGA |
| CsCYP99 | Cs8g12530 | AAGGTTCTTGGGGTCAAAGGTT | GGTTGAGCCTTCTGTATGGTGA |
| CsCYP100 | Cs8g09390 | GGCGCAAGAAAATCAAAGACCT | GTATTGAAAGCAGCTTGGCCAA |
| CsCYP101 | Cs5g09860 | AACCCGATTGACGAATCAGACA | TATAGCCTGCGACTTGTGTGTT |
| CsCYP102 | Cs7g15500 | TTGCCATTGATTAGGGACCGAA | ATGTTGTATCAGTGCCAGCAGA |
| CsCYP103 | Cs7g15480 | AAAGAAGAAGAGAGCCTCGGTG | ACTTCACCAGATTGGCCATGAT |
| CsCYP104 | Cs7g15430 | TCTCAATCTTCATTGCTGCCCT | TGGCATGAAGCTTTCTGAGGAT |
| CsCYP105 | Cs7g15550 | TACCCACATGTTCAGGAGAAGC | TCATCCTCCATCACCTGTCTCT |
| CsCYP106 | Cs3g22160 | GCCTGGAAAAATGGAAGAACCC | TCGGCTAAACATGAGGCTCTTT |
| CsCYP107 | Cs5g12160 | AGTGTTCTTGGGAAATTTCGCG | CAAGCAACTCATACCCTTCCCT |
| CsCYP108 | Cs7g10680 | TTACCCATGACCCAACCATCTG | AGAGAAAGCTTCAGTGTCTCCG |
| CsCYP109 | Cs5g01500 | ATTTCACTCATGGGGTCTGACC | CCTAGCATGGACCTTCACTGTT |
| CsCYP110 | orange1.1t03325 | GCATCATCGAAGAACACAGACG | TCAGCACCAACTCAGCCATAAT |
| CsCYP111 | Cs1g22210 | CTGATCCCTCACAGATCATCGG | CAAGTAGGCTCATCCCACAACT |
| CsCYP112 | Cs3g15640 | ACGGCATCATCAAAGAGACACT | CACGTGGAACTTTGAAACCTCC |
| CsCYP113 | Cs4g02820 | ATGGGCCATACAAAATGACCCT | GCACTGAATCAATGATCCCAGC |
| CsCYP114 | Cs3g15650 | TTCTGGCACTGTATGTCCTCAC | GTCGCTTGAGGAGGTAGAGATG |
| CsCYP115 | Cs3g09020 | GGAAGAGAGACTCTCAGGAGGT | TTTGTGCCCACCTGAATATCGA |
| CsCYP116 | Cs5g28530 | TTTGCCCTCGGATAACATTCCA | CACTGTAGTGTCATTGCCTCCT |
| CsCYP117 | Cs5g28550 | TGGCAGGATCAAACCAGCTAAT | AGTGCTCGATGAATTCCAAGGT |
| CsCYP118 | Cs3g24170 | CTCTCCAAGTCCTTCACCTGAC | GGGGTGGCCTTAGGAATTGTTA |
| CsCYP119 | Cs3g05310 | AGCTTCCTAGTTTTGATGCCGA | ATGGTGCAGTCTTCCATGGATT |
| CsCYP120 | Cs3g05300 | TGGAGGTTCTCTCAAATCACCG | ACCATCACTGTGCTTCATCCAT |
| CsCYP121 | Cs1g02240 | ACATCTCTTTGGAGGGCAAGAG | ACTAAAGCTCGATTCAAGCCCA |
| CsCYP122 | Cs1g02270 | TGGGCTTGAATCGAGCTTTAGT | AAGCCTTCACTTCAGACTCTCG |
| CsCYP123 | Cs3g24180 | GGATGGCTTGATATCGGTGGAT | TTTTTCCGGCGATGTTCCTCTA |
| CsCYP124 | Cs1g02250 | ATTCAGCGTGATCCAGGAGTTT | GTCCCTTCAGATCAACGTCCTT |
| CsCYP125 | Cs3g08680 | CGACTCAGTGGATGGTGAAGAA | TTGACCTCTCATCACCCCCTAT |
| CsCYP126 | Cs3g09090 | TAGGGGGTGATGAGAGGTCAAT | GTGTTACTGTTGTGGTGTCTGC |
| CsCYP127 | Cs3g09040 | CGACTCAGTGGATGGTGAAGAA | TTGACCTCTCATCACCCCCTAT |
| CsCYP128 | Cs3g09140 | ATGAAGAGATGGCTTGCAGACA | TTGACCTCTCATCACCCCCTAT |
| CsCYP129 | Cs3g09030 | CGACTCAGTGGATGGTGAAGAA | TTGACCTCTCATCACCCCCTAT |
| CsCYP130 | Cs7g29860 | CAACTGGGAAATTGCCAAGGAG | CCTCTGGATTGAAGCCTTCACT |
| CsCYP131 | Cs7g18930 | AGAAGATGGGAAGAGCCAGTTG | CCAGTTCCTTCTTCCATGTCGA |
| CsCYP132 | Cs7g18940 | AGAAGATGGGAAGAGCCAGTTG | CCAGTTCCTTCTTCCATGTCGA |
| CsCYP133 | Cs7g18890 | TGCTTGCTCCTCAGAAATAGCA | CCGTCTTATCTCTTCTGACCGG |
| CsCYP134 | Cs9g13910 | AGCCAAAGAGATTGAGGGTCTG | GCCTCCTCTTCATGTTCCTTCA |
| CsCYP135 | Cs7g18920 | TCTGTCCTGGAACCACATTAGC | AGGAATGAGCCCTAGGAAGAGT |
| CsCYP136 | Cs7g18910 | ACCACCGAGAGAATCCACTAGA | ATCAGCCGAGCCATATGTAAGG |
| CsCYP137 | Cs7g18880 | GGGGAGCTTATGAGGTTGACAA | CCTCACTTCACCAGCTTCATCT |
| CsCYP138 | Cs6g16650 | AGACTGCACCGATCCTCAAAAT | ATTCTTTCCCTTGCTCGTCCAT |
Table 1 Primer of CYP71 gene
| 基因名称 Gene name | 基因ID Gene ID | 上游引物(5′-3′) Foward primer | 下游引物(5′-3′) Reverse primer |
|---|---|---|---|
| CsCYP1 | orange1.1t02788 | GCGGATGATCCTACTCTGGAAG | GAGGTGTCAGTTCCAGCAGTTA |
| CsCYP2 | Cs3g07330 | CGTATTCCTTGTGTCACTCCCA | AACTACTACGGTCTGCACAAGG |
| CsCYP3 | orange1.1t02784 | TCTTATTTCGCCGCCGATTTTC | GTACTTCCCAGCAAGAAGAGCT |
| CsCYP4 | Cs1g17420 | CTTCCCTATCGGTCCATCCATG | CAACGGAAGATCCCACAACAAC |
| CsCYP5 | Cs1g17410 | ATACACAGACAAGCAGCTCGAA | AGCCACATAATTTTCGACCCCT |
| CsCYP6 | Cs1g10500 | ATGCAATTCCATGGCTTGCTTT | TCCTGGATTGCTGCCTTAATGT |
| CsCYP7 | Cs5g25880 | TTTGGGCCATGACCTACCTAAC | TTGCCCCCAATCAACGATCTAA |
| CsCYP8 | Cs3g25780 | GGTGCCTATCTTCCTGATCTCG | ATGCAAGTTGCCTATGAAGGGA |
| CsCYP9 | Cs4g09010 | AAGGCACAAGATGAGGTAAGGG | ACACGTCTCTGCACATTCTCTT |
| CsCYP10 | Cs7g29720 | TGTTATCTTTGCATCCAGGCCT | AAAGATTGGACTCTTTTGGCGC |
| CsCYP11 | Cs7g09640 | TGGGCAATTGGAAGAGATTCGA | TTCTACGACCACCTCCAAATGG |
| CsCYP12 | Cs3g01130 | CCCCGAAATCCTCTTTCTGTCA | TTTGGCCAGCTCTTTAGAGGAG |
| CsCYP13 | Cs2g01910 | GGCAGTAACAAGAGATCCCCAA | CCGCAAACCACACCAAATTTTG |
| CsCYP14 | Cs5g09880 | ATTGACTTGGCTGATCCCACAT | ACGCCTTATACCTTGCAAGTCA |
| CsCYP15 | Cs8g09720 | TTCCTTTTCCAGGGGACACAAA | TCATTGTGTTGGTGGGGAAAGA |
| CsCYP16 | orange1.1t04139 | CAGTGATTGGAAACCTCCTCGA | TGATTGTAGGAACAGGCTGCAT |
| CsCYP17 | Cs5g09870 | AACCCGATTGACGAATCAGACA | TATAGCCTGCGACTTGTGTGTT |
| CsCYP18 | orange1.1t01519 | TAAACGGACGGTTGGTGATGAT | CAATTCTTTCACCACGGCTTGT |
| CsCYP19 | Cs7g15460 | GAAAGCAGTGATCTTGGAGGGA | ATCACGTGTCCATCCAATGTGA |
| CsCYP20 | Cs7g15470 | GTGAATTTCATGGTGGCCGAAA | TCAACCTCATCACCATCCACAG |
| CsCYP21 | Cs2g07770 | TGCTGATAACATCACCACAGCT | TATCAGTGGAACCAAGCACTCC |
| CsCYP22 | Cs7g15540 | CGTTTAGGCCTGAGAGGTTCTT | TTCCAAATGAAGCATGGCCAAG |
| CsCYP23 | Cs7g11300 | CCCTAGCTTGCCTTACCTTCAA | TTGGATCATGAGTGATTGCCCA |
| CsCYP24 | orange1.1t00542 | GATACAGTGGCGGTTTTGATCG | GTATCAGTGATGGCTAGACGGG |
| CsCYP25 | Cs1g22200 | TTCACCTCATAAAGCAGCCCAT | GTGAGGAATTTTGGACGGTTGG |
| CsCYP26 | Cs1g22230 | TTGTATCCAACAGCTCCACTCC | TGAATGGCCCATACATTGACCA |
| CsCYP27 | Cs3g15630 | CTTCGGCTGCAACTATGGAATG | CACGTGGAACTTTGAAACCTCC |
| CsCYP28 | Cs3g15590 | CAGGCCTGTCCTTGTTGTATCT | TTTGGTTACCGCTTGCTGAATG |
| CsCYP29 | Cs3g09120 | TGAGTGAGGGAATGAGCTTGAC | ATATAGCGATGCGGAAAGACGT |
| CsCYP30 | Cs1g10540 | TTGGTGGTGTCAGATGCTCTAC | AAGGATGAGTGCCAAGCATGTA |
| CsCYP31 | Cs5g27570 | TTAGACTTGGCAAGCATCCCAT | TGCGTTTTCTGAGCAAAGTGAG |
| CsCYP32 | Cs5g27580 | CTCGGATTCTTCAGGGGTTTGA | TTCAGTTTAGGCAAGGCAATGC |
| CsCYP33 | Cs4g07380 | ATGGGCTATGTCCGAGCTAATG | AGACTGAAGGGTCTCTGCCTAT |
| CsCYP34 | Cs2g08630 | ATCATCTACGGGTTTTGTGGCT | CACCAGCTAAAAGGTCCTGGAT |
| CsCYP35 | orange1.1t02775 | GATCCAACCATGTGGGAAAAGC | CCAAATCTGTCCTCCATGTCCA |
| CsCYP36 | Cs1g17390 | AGGGGTCGAAAATTATGTGGCT | CACTGTTACTGCTGAGCTCTCA |
| CsCYP37 | orange1.1t01321 | ACATTTGCGCCAAACTCGTAAA | CCGGCTCATTATGGCAAGATTG |
| CsCYP38 | Cs5g08070 | CTCATCGGAAACCTCAACCTCA | AATTTTACGAGTTTGGCGCCAA |
| CsCYP39 | Cs5g08200 | TGGCGCCAAACTCGTAAAATTT | ATCCGGCTCATAATGGCAAGAT |
| CsCYP40 | orange1.1t04499 | CATCCTCATCCCTCTGCATCTC | ATGACCCAAATTTGAGCTGCAC |
| CsCYP41 | orange1.1t01327 | TTATTGCCATTTGGAGTGGGGA | ACCATGCAAAAGATTCGCCAAA |
| CsCYP42 | orange1.1t02780 | TGGCCATTTATCGGAAACCTGA | GTACTTCCCAGCAAGAAGAGCT |
| CsCYP43 | Cs5g25060 | TTGGTGCTGGCAGAAGAATTTG | ATTCCTGGAGGCATTTCCCAAT |
| CsCYP44 | Cs5g25070 | TTGGTGCTGGCAGAAGAATTTG | ATTCCTGGAGGCATTTCCCAAT |
| CsCYP45 | Cs1g04220 | CTCCCAATCTGCCTCTTCTTGT | AAAACCCAGACGCAATGACATG |
| CsCYP46 | Cs5g06350 | TGACCAACATTCCGATCCCATT | TGGGGTCATTCCTAATTGGTGG |
| CsCYP47 | Cs1g02540 | CACTTACGCTTAGGCTTAACGC | TTGTTCGCCAATAAGACCCGTA |
| CsCYP48 | Cs7g10570 | TTTGTCCTGGGATTCAGATGGG | TCTCCTTCCTTCATTCCGTTGG |
| CsCYP49 | Cs5g06320 | AAGACCCATGACATCGCTTTCT | TAACGTTCCCGAGATCTTTGCA |
| CsCYP50 | Cs2g17110 | ACAAAATGTCCCTCATCGCTCT | TTTTGTAGCAATGGATCGGGGA |
| CsCYP51 | Cs7g10050 | GGGATGAGGAAGTGAAACGTCT | TTGCGTATTCTTCCTCAGCCTT |
| CsCYP52 | Cs3g21580 | AGAAGATTTTGTCCCGGGACTC | CAGCAGGCATTTCCCAATCAAA |
| CsCYP53 | Cs2g19590 | TAAAACTTGGCCAGCTTGATGC | ATCTGAATGACTGGACACGCTT |
| CsCYP54 | orange1.1t00025 | TGGGCAGAAATCCGACATAACA | GGTCTCTTGAAATGGCCCAGTA |
| CsCYP55 | orange1.1t03363 | CATTGATGGCTCCACACGAATC | CTTCCAATTGCCCAAGCGTTTA |
| CsCYP56 | Cs2g19560 | AAGCATGCAAAGGAAAGAGCAG | TTTGGCCTTAGCTGGTACTCTG |
| CsCYP57 | Cs5g06280 | AGAAATTAGGCGGATTGGGGTT | AACACATCGACCAACCGTATCA |
| CsCYP58 | orange1.1t03357 | CGAGGATTTCGTTGACGTGATG | TGGTGAAGTGTCTATTGCTCCC |
| CsCYP59 | orange1.1t04874 | GGCAATAGGAAGGGACAGTGAA | GCGCATTGCCATTGATATTCCT |
| CsCYP60 | orange1.1t04991 | GGCAATAGGAAGGGACAGTGAA | GCGCATTGCCATTGATATTCCT |
| CsCYP61 | orange1.1t05138 | GGCAATAGGAAGGGACAGTGAA | GCGCATTGCCATTGATATTCCT |
| CsCYP62 | Cs4g09080 | CCCTCATCATCGGCTAAGAGAC | GCTCAAAAGCTCAGACACACAG |
| CsCYP63 | Cs4g09040 | ATTGTTAATGCATGGGCGATCG | ATATTCTCCTGCCAGCACCAAA |
| CsCYP64 | Cs5g06360 | CATCTTGGACATGTTTGCTGCA | ATGGATTCCCTTGGGACTAGGA |
| CsCYP65 | Cs5g06310 | CTTTGATGTTCCTGCACTTGGG | TTTGTTGGTCATGATCGCAACC |
| CsCYP66 | orange1.1t02944 | TCCTTCCGTTGGATTCCTTCAG | AGATCTCCACGCCCTTGAATTT |
| CsCYP67 | Cs2g18790 | AATTGCTTCCATTTGGTTCCGG | TCGAGGTCTTCTAGTTTGGTGC |
| CsCYP68 | Cs7g27010 | TGCTGCCTTTTGTATGGGAGAT | CCATCCTTTCGAAGTTGGAGGA |
| CsCYP69 | Cs4g09050 | TGTTATCTTTGCATCCAGGCCT | AAAGATTGGACTCTTTTGGCGC |
| CsCYP70 | orange1.1t05258 | GGCAATAGGAAGGGACAGTGAA | GCGCATTGCCATTGATATTCCT |
| CsCYP71 | Cs4g09090 | AATGGGTCACTGGATTCAGGTC | CCAGTCCACAGTTGTAGCAGAT |
| CsCYP72 | Cs5g06390 | TACTGAGGATTGCAGGGTAGGA | ATTCTCATCCCTGGTGGCAATT |
| CsCYP73 | orange1.1t03684 | TGATGGGTGGTTTCTATGTGGG | TTCTCAAGCTTGGCATTGAAGC |
| CsCYP74 | Cs4g09110 | GGAGTGAGGCGGAAACTTAAGA | GATGTGTCACTCCCAGCTACAA |
| CsCYP75 | Cs5g22050 | TCGACTCAGGCCGTCAAAATAA | ACACTCTCATCGCAAACTCCAT |
| CsCYP76 | Cs7g09630 | TCATCAGGGAAGAGGAAGTTGC | CCCTCGAAATCTCCTCCCAAAA |
| CsCYP77 | Cs4g09020 | TAAGATTGCACACCCCTCTTCC | AATAGCAGTTGAGCGAGTGGAA |
| CsCYP78 | Cs1g04240 | CTGGCTGGAGGTTTTTCTTTGG | CTGCTGGTCTCTTGAAGCCTTA |
| CsCYP79 | Cs5g13970 | GATTACATCCGGCAGGTACCAT | AGGTTGGCCAACAGATACTCAG |
| CsCYP80 | orange1.1t02083 | GGCTCAATTCCTCCCAAGAGAA | TACCGGCACCAAATGGGATAAA |
| CsCYP81 | Cs5g06380 | ATCCATCCGTGATGAGAAGAGC | CCAATACTCAGGGTCTGTTGCT |
| CsCYP82 | Cs4g09100 | CCTTGTTGGCTGTCTACCTCAT | ATGTCGTGGCTCTTCATCACTT |
| CsCYP83 | Cs5g13940 | AGAATGGGCAATGGCTGAACTA | ATGGTACCTGCCGGATGTAATC |
| CsCYP84 | Cs5g25920 | GTGTCATTTTGGGAGCTGTCAC | GCTGGCCTGCTACAAAATTGAA |
| CsCYP85 | Cs6g15610 | CCATTCGGTTCTGGGAGAAGAA | GAAGCAAAGAGGCAAGACCAAG |
| CsCYP86 | Cs6g15620 | ATGGTGCAGAGTTTTTCCAAGC | GCTTTACCAACATCCCTCTCCA |
| CsCYP87 | Cs9g14170 | TTACCGGTGCTAGGAAACCTTC | AGGCCATAAGAGGTGACCAAAG |
| CsCYP88 | orange1.1t03998 | CTGTGATCGAAAGGTCCCTGAA | AGGTCTTGATTGGCATCGAGTT |
| CsCYP89 | orange1.1t03997 | TATCAAAGGCTTCCACCAGGAC | CAGGGACCTTTCGATCACAGAA |
| CsCYP90 | Cs4g14140 | CAGATTCTGTGAGTTCCCACCA | ATATCGATTGCTTCTCCCGCAT |
| CsCYP91 | orange1.1t04132 | CTGTGATCGAAAGGTCCCTGAA | AGGTCTTGATTGGCATCGAGTT |
| CsCYP92 | Cs4g14130 | CTCCCTGTCTCGACAAAATGGA | AACATCGATTGGTTCACCCGTA |
| CsCYP93 | Cs8g09730 | GAACTACTCCACAACCCTGAGG | GAGGTTTGTCCCATGTGCTTTC |
| CsCYP94 | orange1.1t04372 | CGAATTTGTCCTGGTTTGCCAT | AGGGATAGTACGAAGGGGTTGA |
| CsCYP95 | Cs8g09780 | TGGCAGAACTACTCCACAATCC | GAGTGAGGATTGTCCCATGTGT |
| CsCYP96 | Cs6g15630 | ATGAAGTGGCTCGAGACATGTT | ATGCCTCGGCTTCTTCTACAAA |
| CsCYP97 | Cs8g09480 | GGCGCAAGAAAATCAAAGACCT | TATTGAAAGCAGCTTGGCCAAC |
| CsCYP98 | orange1.1t04771 | CGAATTTGTCCTGGTTTGCCAT | AGGGATAGTACGAAGGGGTTGA |
| CsCYP99 | Cs8g12530 | AAGGTTCTTGGGGTCAAAGGTT | GGTTGAGCCTTCTGTATGGTGA |
| CsCYP100 | Cs8g09390 | GGCGCAAGAAAATCAAAGACCT | GTATTGAAAGCAGCTTGGCCAA |
| CsCYP101 | Cs5g09860 | AACCCGATTGACGAATCAGACA | TATAGCCTGCGACTTGTGTGTT |
| CsCYP102 | Cs7g15500 | TTGCCATTGATTAGGGACCGAA | ATGTTGTATCAGTGCCAGCAGA |
| CsCYP103 | Cs7g15480 | AAAGAAGAAGAGAGCCTCGGTG | ACTTCACCAGATTGGCCATGAT |
| CsCYP104 | Cs7g15430 | TCTCAATCTTCATTGCTGCCCT | TGGCATGAAGCTTTCTGAGGAT |
| CsCYP105 | Cs7g15550 | TACCCACATGTTCAGGAGAAGC | TCATCCTCCATCACCTGTCTCT |
| CsCYP106 | Cs3g22160 | GCCTGGAAAAATGGAAGAACCC | TCGGCTAAACATGAGGCTCTTT |
| CsCYP107 | Cs5g12160 | AGTGTTCTTGGGAAATTTCGCG | CAAGCAACTCATACCCTTCCCT |
| CsCYP108 | Cs7g10680 | TTACCCATGACCCAACCATCTG | AGAGAAAGCTTCAGTGTCTCCG |
| CsCYP109 | Cs5g01500 | ATTTCACTCATGGGGTCTGACC | CCTAGCATGGACCTTCACTGTT |
| CsCYP110 | orange1.1t03325 | GCATCATCGAAGAACACAGACG | TCAGCACCAACTCAGCCATAAT |
| CsCYP111 | Cs1g22210 | CTGATCCCTCACAGATCATCGG | CAAGTAGGCTCATCCCACAACT |
| CsCYP112 | Cs3g15640 | ACGGCATCATCAAAGAGACACT | CACGTGGAACTTTGAAACCTCC |
| CsCYP113 | Cs4g02820 | ATGGGCCATACAAAATGACCCT | GCACTGAATCAATGATCCCAGC |
| CsCYP114 | Cs3g15650 | TTCTGGCACTGTATGTCCTCAC | GTCGCTTGAGGAGGTAGAGATG |
| CsCYP115 | Cs3g09020 | GGAAGAGAGACTCTCAGGAGGT | TTTGTGCCCACCTGAATATCGA |
| CsCYP116 | Cs5g28530 | TTTGCCCTCGGATAACATTCCA | CACTGTAGTGTCATTGCCTCCT |
| CsCYP117 | Cs5g28550 | TGGCAGGATCAAACCAGCTAAT | AGTGCTCGATGAATTCCAAGGT |
| CsCYP118 | Cs3g24170 | CTCTCCAAGTCCTTCACCTGAC | GGGGTGGCCTTAGGAATTGTTA |
| CsCYP119 | Cs3g05310 | AGCTTCCTAGTTTTGATGCCGA | ATGGTGCAGTCTTCCATGGATT |
| CsCYP120 | Cs3g05300 | TGGAGGTTCTCTCAAATCACCG | ACCATCACTGTGCTTCATCCAT |
| CsCYP121 | Cs1g02240 | ACATCTCTTTGGAGGGCAAGAG | ACTAAAGCTCGATTCAAGCCCA |
| CsCYP122 | Cs1g02270 | TGGGCTTGAATCGAGCTTTAGT | AAGCCTTCACTTCAGACTCTCG |
| CsCYP123 | Cs3g24180 | GGATGGCTTGATATCGGTGGAT | TTTTTCCGGCGATGTTCCTCTA |
| CsCYP124 | Cs1g02250 | ATTCAGCGTGATCCAGGAGTTT | GTCCCTTCAGATCAACGTCCTT |
| CsCYP125 | Cs3g08680 | CGACTCAGTGGATGGTGAAGAA | TTGACCTCTCATCACCCCCTAT |
| CsCYP126 | Cs3g09090 | TAGGGGGTGATGAGAGGTCAAT | GTGTTACTGTTGTGGTGTCTGC |
| CsCYP127 | Cs3g09040 | CGACTCAGTGGATGGTGAAGAA | TTGACCTCTCATCACCCCCTAT |
| CsCYP128 | Cs3g09140 | ATGAAGAGATGGCTTGCAGACA | TTGACCTCTCATCACCCCCTAT |
| CsCYP129 | Cs3g09030 | CGACTCAGTGGATGGTGAAGAA | TTGACCTCTCATCACCCCCTAT |
| CsCYP130 | Cs7g29860 | CAACTGGGAAATTGCCAAGGAG | CCTCTGGATTGAAGCCTTCACT |
| CsCYP131 | Cs7g18930 | AGAAGATGGGAAGAGCCAGTTG | CCAGTTCCTTCTTCCATGTCGA |
| CsCYP132 | Cs7g18940 | AGAAGATGGGAAGAGCCAGTTG | CCAGTTCCTTCTTCCATGTCGA |
| CsCYP133 | Cs7g18890 | TGCTTGCTCCTCAGAAATAGCA | CCGTCTTATCTCTTCTGACCGG |
| CsCYP134 | Cs9g13910 | AGCCAAAGAGATTGAGGGTCTG | GCCTCCTCTTCATGTTCCTTCA |
| CsCYP135 | Cs7g18920 | TCTGTCCTGGAACCACATTAGC | AGGAATGAGCCCTAGGAAGAGT |
| CsCYP136 | Cs7g18910 | ACCACCGAGAGAATCCACTAGA | ATCAGCCGAGCCATATGTAAGG |
| CsCYP137 | Cs7g18880 | GGGGAGCTTATGAGGTTGACAA | CCTCACTTCACCAGCTTCATCT |
| CsCYP138 | Cs6g16650 | AGACTGCACCGATCCTCAAAAT | ATTCTTTCCCTTGCTCGTCCAT |
| 名称 Name | 配方及浓度 Formula and concentration |
|---|---|
| 播种培养基 Seed stripping medium | 4.43 g · L-1 MS培养基,50 g · L-1蔗糖,0.5 g · L-1酸水解酪蛋白,3.5 g · L-1植物凝胶,pH 5.8 4.43 g · L-1 MS medium supplemented,50 g · L-1 sucrose,0.5 g · L-1 acid hydrolyzed casein,3.5 g · L-1 plant gel,pH 5.8 |
| 预培养培养基 Pre-culture medium | 4.43 g · L-1 MS培养基,30 g · L-1 蔗糖,0.5 g · L-1 酸水解酪蛋白,2 mg · L-1 6-苄基腺嘌呤,1 mg · L-1 吲 哚-3-乙酸,pH 5.8 4.43 g · L-1 MS medium supplemented,30 g · L-1 sucrose,0.5 g · L-1 acid hydrolyzed casein,2 mg · L-1 6-BA, 1 mg · L-1 IAA,pH 5.8 |
| 激活培养基 Activation medium | 4.43 g · L-1 MS培养基,30 g · L-1蔗糖,0.5 g · L-1酸水解酪蛋白,pH 5.8 4.43 g · L-1 MS medium supplemented,30 g · L-1 sucrose,0.5 g · L-1 acid hydrolyzed casein,pH 5.8 |
| 共培养培养基 Co-culture medium | 4.43 g · L-1 MS培养基,30 g · L-1蔗糖,0.5 g · L-1酸水解酪蛋白,3.5 g · L-1植物凝胶,2 mg · L-1 6-苄基腺嘌呤,1 mg · L-1 吲哚-3-乙酸,1 mg · L-1 2,4-二氯苯氧乙酸2,4-D,100 μmol · L-1 乙酰丁香酮,pH 5.8 4.43 g · L-1 MS medium supplemented,30 g · L-1 sucrose,0.5 g · L-1 acid hydrolyzed casein,3.5 g · L-1 plant gel,2 mg · L-1 6-BA,1 mg · L-1 IAA,1 mg · L-1 2,4-D,100 μmol · L-1 As,pH 5.8 |
| 筛选培养基 Screening medium | 4.43 g · L-1MS培养基,30 g · L-1蔗糖,0.5 g · L-1酸水解酪蛋白,3.5 g · L-1植物凝胶,2 mg · L-1 6-苄基腺嘌呤,0.5 mg · L-1 吲哚-3-乙酸,50 mg · L-1 卡纳,500 mg · L-1 羧苄青霉素,pH 5.8 4.43 g · L-1 MS medium supplemented,30 g · L-1 sucrose,0.5 g · L-1 acid hydrolyzed casein,3.5 g · L-1 plant gel,0.5 mg · L-1 IAA,50 mg · L-1 Kan,500 mg · L-1 Car,pH 5.8 |
Table 2 Medium formulation
| 名称 Name | 配方及浓度 Formula and concentration |
|---|---|
| 播种培养基 Seed stripping medium | 4.43 g · L-1 MS培养基,50 g · L-1蔗糖,0.5 g · L-1酸水解酪蛋白,3.5 g · L-1植物凝胶,pH 5.8 4.43 g · L-1 MS medium supplemented,50 g · L-1 sucrose,0.5 g · L-1 acid hydrolyzed casein,3.5 g · L-1 plant gel,pH 5.8 |
| 预培养培养基 Pre-culture medium | 4.43 g · L-1 MS培养基,30 g · L-1 蔗糖,0.5 g · L-1 酸水解酪蛋白,2 mg · L-1 6-苄基腺嘌呤,1 mg · L-1 吲 哚-3-乙酸,pH 5.8 4.43 g · L-1 MS medium supplemented,30 g · L-1 sucrose,0.5 g · L-1 acid hydrolyzed casein,2 mg · L-1 6-BA, 1 mg · L-1 IAA,pH 5.8 |
| 激活培养基 Activation medium | 4.43 g · L-1 MS培养基,30 g · L-1蔗糖,0.5 g · L-1酸水解酪蛋白,pH 5.8 4.43 g · L-1 MS medium supplemented,30 g · L-1 sucrose,0.5 g · L-1 acid hydrolyzed casein,pH 5.8 |
| 共培养培养基 Co-culture medium | 4.43 g · L-1 MS培养基,30 g · L-1蔗糖,0.5 g · L-1酸水解酪蛋白,3.5 g · L-1植物凝胶,2 mg · L-1 6-苄基腺嘌呤,1 mg · L-1 吲哚-3-乙酸,1 mg · L-1 2,4-二氯苯氧乙酸2,4-D,100 μmol · L-1 乙酰丁香酮,pH 5.8 4.43 g · L-1 MS medium supplemented,30 g · L-1 sucrose,0.5 g · L-1 acid hydrolyzed casein,3.5 g · L-1 plant gel,2 mg · L-1 6-BA,1 mg · L-1 IAA,1 mg · L-1 2,4-D,100 μmol · L-1 As,pH 5.8 |
| 筛选培养基 Screening medium | 4.43 g · L-1MS培养基,30 g · L-1蔗糖,0.5 g · L-1酸水解酪蛋白,3.5 g · L-1植物凝胶,2 mg · L-1 6-苄基腺嘌呤,0.5 mg · L-1 吲哚-3-乙酸,50 mg · L-1 卡纳,500 mg · L-1 羧苄青霉素,pH 5.8 4.43 g · L-1 MS medium supplemented,30 g · L-1 sucrose,0.5 g · L-1 acid hydrolyzed casein,3.5 g · L-1 plant gel,0.5 mg · L-1 IAA,50 mg · L-1 Kan,500 mg · L-1 Car,pH 5.8 |
| 目的 Objective | 引物名称 Primer name | 引物序列(5′-3′) Primer sequence |
|---|---|---|
| CRISPR/Cas9基因编辑 CRISPR/Cas9 gene editing | CsCYP2-gRNA1-F | GATTGACCAAAAGACCAAGTACGCG |
| CsCYP2-gRNA1-R | AAACCGCGTACTTGGTCTTTTGGTC | |
| CsCYP2-gRNA2-F | GATTGGCTACCTCATCTTCTCTCAC | |
| CsCYP2-gRNA2-R | AAACGTGAGAGAAGATGAGGTAGCC | |
| 阳性植株检测 Positive plant detection | CsCYP2-PCR-F | gtcgaCACAAATTGAACATGGC |
| CsCYP2-PCR-R | gGATCCTTCCCATGTGAGGTG | |
| 编辑位点检测 Editing site detection | CsCYP2-gRNA-T-F | CACTGATGTCACTACGCCTTG |
| CsCYP2-gRNA-T-R | TGAACTTGATCCTCACCACGA |
Table 3 Primer for CsCYP2
| 目的 Objective | 引物名称 Primer name | 引物序列(5′-3′) Primer sequence |
|---|---|---|
| CRISPR/Cas9基因编辑 CRISPR/Cas9 gene editing | CsCYP2-gRNA1-F | GATTGACCAAAAGACCAAGTACGCG |
| CsCYP2-gRNA1-R | AAACCGCGTACTTGGTCTTTTGGTC | |
| CsCYP2-gRNA2-F | GATTGGCTACCTCATCTTCTCTCAC | |
| CsCYP2-gRNA2-R | AAACGTGAGAGAAGATGAGGTAGCC | |
| 阳性植株检测 Positive plant detection | CsCYP2-PCR-F | gtcgaCACAAATTGAACATGGC |
| CsCYP2-PCR-R | gGATCCTTCCCATGTGAGGTG | |
| 编辑位点检测 Editing site detection | CsCYP2-gRNA-T-F | CACTGATGTCACTACGCCTTG |
| CsCYP2-gRNA-T-R | TGAACTTGATCCTCACCACGA |
| 基因 Gene | ID | 氨基酸数 Number of amino acid | 分子量/Da Molecular weight | 等电点 Theoretical pI | 不稳定系数 Instability index | 亲疏水性 GRAVY |
|---|---|---|---|---|---|---|
| CsCYP1 | orange1.1t02788 | 506 | 58 648.45 | 8.95 | 39.74 | -0.295 |
| CsCYP2 | Cs3g07330 | 516 | 58 718.94 | 7.18 | 36.82 | -0.136 |
| CsCYP3 | orange1.1t02784 | 516 | 59 102.82 | 8.54 | 36.81 | -0.184 |
| CsCYP4 | Cs1g17420 | 511 | 58 532.34 | 8.61 | 35.96 | -0.147 |
| CsCYP5 | Cs1g17410 | 512 | 58 031.62 | 8.92 | 32.23 | -0.140 |
| CsCYP6 | Cs1g10500 | 528 | 59 809.65 | 8.85 | 38.37 | -0.164 |
| CsCYP7 | Cs5g25880 | 523 | 60 000.98 | 8.87 | 39.91 | -0.101 |
| CsCYP8 | Cs3g25780 | 504 | 57 866.34 | 7.61 | 42.20 | -0.093 |
| CsCYP9 | Cs4g09010 | 503 | 56 869.33 | 8.71 | 44.37 | -0.133 |
| CsCYP10 | Cs7g29720 | 510 | 57 142.20 | 8.47 | 35.64 | -0.136 |
| CsCYP11 | Cs7g09640 | 509 | 57 471.69 | 8.02 | 42.80 | -0.163 |
| CsCYP12 | Cs3g01130 | 521 | 58 932.97 | 8.88 | 46.84 | -0.199 |
| CsCYP13 | Cs2g01910 | 512 | 58 072.53 | 7.10 | 35.56 | -0.153 |
| CsCYP14 | Cs5g09880 | 499 | 56 391.42 | 7.63 | 37.35 | -0.102 |
| CsCYP15 | Cs8g09720 | 506 | 57 260.85 | 7.64 | 40.69 | -0.031 |
| CsCYP16 | orange1.1t04139 | 501 | 56 711.01 | 8.57 | 41.71 | -0.114 |
| CsCYP17 | Cs5g09870 | 499 | 56 433.49 | 8.61 | 30.49 | -0.166 |
| CsCYP18 | orange1.1t01519 | 508 | 57 480.13 | 9.25 | 36.43 | -0.119 |
| CsCYP19 | Cs7g15460 | 529 | 60 837.13 | 9.17 | 48.01 | -0.162 |
| CsCYP20 | Cs7g15470 | 512 | 59 175.81 | 7.71 | 47.74 | -0.176 |
| CsCYP21 | Cs2g07770 | 508 | 57 241.75 | 8.00 | 49.60 | -0.030 |
| CsCYP22 | Cs7g15540 | 521 | 60 298.13 | 8.50 | 48.55 | -0.215 |
| CsCYP23 | Cs7g11300 | 521 | 58 397.05 | 6.63 | 40.58 | 0.124 |
| CsCYP24 | orange1.1t00542 | 538 | 60 225.22 | 9.43 | 32.60 | 0.041 |
Table 4 Characteristics of CYP71 family members in citrus
| 基因 Gene | ID | 氨基酸数 Number of amino acid | 分子量/Da Molecular weight | 等电点 Theoretical pI | 不稳定系数 Instability index | 亲疏水性 GRAVY |
|---|---|---|---|---|---|---|
| CsCYP1 | orange1.1t02788 | 506 | 58 648.45 | 8.95 | 39.74 | -0.295 |
| CsCYP2 | Cs3g07330 | 516 | 58 718.94 | 7.18 | 36.82 | -0.136 |
| CsCYP3 | orange1.1t02784 | 516 | 59 102.82 | 8.54 | 36.81 | -0.184 |
| CsCYP4 | Cs1g17420 | 511 | 58 532.34 | 8.61 | 35.96 | -0.147 |
| CsCYP5 | Cs1g17410 | 512 | 58 031.62 | 8.92 | 32.23 | -0.140 |
| CsCYP6 | Cs1g10500 | 528 | 59 809.65 | 8.85 | 38.37 | -0.164 |
| CsCYP7 | Cs5g25880 | 523 | 60 000.98 | 8.87 | 39.91 | -0.101 |
| CsCYP8 | Cs3g25780 | 504 | 57 866.34 | 7.61 | 42.20 | -0.093 |
| CsCYP9 | Cs4g09010 | 503 | 56 869.33 | 8.71 | 44.37 | -0.133 |
| CsCYP10 | Cs7g29720 | 510 | 57 142.20 | 8.47 | 35.64 | -0.136 |
| CsCYP11 | Cs7g09640 | 509 | 57 471.69 | 8.02 | 42.80 | -0.163 |
| CsCYP12 | Cs3g01130 | 521 | 58 932.97 | 8.88 | 46.84 | -0.199 |
| CsCYP13 | Cs2g01910 | 512 | 58 072.53 | 7.10 | 35.56 | -0.153 |
| CsCYP14 | Cs5g09880 | 499 | 56 391.42 | 7.63 | 37.35 | -0.102 |
| CsCYP15 | Cs8g09720 | 506 | 57 260.85 | 7.64 | 40.69 | -0.031 |
| CsCYP16 | orange1.1t04139 | 501 | 56 711.01 | 8.57 | 41.71 | -0.114 |
| CsCYP17 | Cs5g09870 | 499 | 56 433.49 | 8.61 | 30.49 | -0.166 |
| CsCYP18 | orange1.1t01519 | 508 | 57 480.13 | 9.25 | 36.43 | -0.119 |
| CsCYP19 | Cs7g15460 | 529 | 60 837.13 | 9.17 | 48.01 | -0.162 |
| CsCYP20 | Cs7g15470 | 512 | 59 175.81 | 7.71 | 47.74 | -0.176 |
| CsCYP21 | Cs2g07770 | 508 | 57 241.75 | 8.00 | 49.60 | -0.030 |
| CsCYP22 | Cs7g15540 | 521 | 60 298.13 | 8.50 | 48.55 | -0.215 |
| CsCYP23 | Cs7g11300 | 521 | 58 397.05 | 6.63 | 40.58 | 0.124 |
| CsCYP24 | orange1.1t00542 | 538 | 60 225.22 | 9.43 | 32.60 | 0.041 |
Fig. 4 Correlation clustering heatmap of the expression of CsCYP71 genes and the relative content of oxygenated compounds in the fruit peel VOC1:p-Cymen-7-ol;VOC2:1,4-Cyclohexadiene-1-carboxaldehyde;VOC3:D-Carvone;VOC4:Perillyl aldehyde;VOC5:(+)-(E)-limonene oxide; VOC6:(Z)-limonene oxide;VOC7:exo-isocitral;VOC8:Isogeranial;VOC9:(Z)-carveol;VOC10:Geranial;VOC11:p-Mentha-1(7),8(10)-dien-9-ol; VOC12:(E,E)-2,4-Decadienal;VOC13:(E)-p-mentha-2,8-dien-1-ol;VOC14:Isogeranial;VOC15:(Z)-sabinene hydrate;VOC16:Linalool VOC17 citronellal;VOC18:1-terpinen-4-ol;VOC19:L-α-Terpineol;VOC20:7-Methyl-3-methylene-6-octen-1-ol;VOC21:Nerol;VOC22:Geraniol;VOC23:(E)-2-Decenal;VOC24:(Z)-p-2-menthen-1-ol;VOC25:(E)-sabinene hydrate;VOC26:exo-2-Hydroxycineole; VOC27:Decanal;VOC28:Citronellol;VOC29:β-sinensal;VOC30:α-sinensal;VOC31:(-)-caryophyllene oxide;VOC32:(1S,4S,4aS)-1-Isopropyl-4,7-dimethyl-1,2,3,4,4a,5-hexahydronaphthalene; VOC33:α-elemol;VOC34:7-epi-trans-sesquisabinene hydrate
Fig. 5 A comparative analysis of cavity numbers,relative expression levels of CsCYP2,and content of volatile terpenoids in the peel and leaf of Huapi kumquat and Rong’an kumquat t-test,*P < 0.05,**P < 0.01
Fig. 8 Genetic transformation of sweet orange CsCYP2 A:Co-cultivation of explants and screening of regenerative bud resistance;B:Growth of positive buds;C:Rooting culture of positive buds;D:Grafting of positive buds on citrus aurantium rootstock;E:Fluorescence detection;F:PCR detection of transgenic plants(M:DL2000)
| 化合物 Compound | 化学式 Chemical formula | OE | KO1 | KO2 |
|---|---|---|---|---|
| α-侧柏烯α-Thujene | C10H16 | 上调Up** | 上调Up*** | 上调Up* |
| 蒎烯 (+)-α-Pinene | C10H16 | 上调Up** | 上调Up** | 上调Up* |
| 桧烯 Sabinene | C10H16 | 上调Up** | 上调Up** | 上调Up* |
| β-蒎烯β-pinene | C10H16 | 上调Up** | 上调Up** | 上调Up* |
| δ-3-蒈烯δ-3-carene | C10H16 | 下调Down* | 上调Up*** | 下调Down* |
| (+)-4-蒈烯 (+)-4-Carene | C10H16 | — | 上调Up* | 上调Up* |
| (Z)-罗勒烯 (Z)-ocimene | C10H16 | — | — | 下调Down* |
| γ-萜品烯 γ-terpinene | C10H16 | 上调Up** | 上调Up* | 上调Up* |
| (Z)-水合桧烯 (Z)-sabinene hydrate | C10H18O | 下调Down** | 上调Up** | 下调Down** |
| 藏红花醛 Safranal | C10H14O | — | — | 下调Down* |
| 萜品油烯 Isoterpinolene | C10H16 | 下调Down* | 上调Up**** | 上调Up* |
| 萜品烯 Terpinolene | C10H16 | 上调Up** | 上调Up**** | 上调Up* |
| 3,5-二叔丁基苯邻二酚 3,5-dimethylcyclobutabenzene | C10H12 | 下调Down** | 下调Down** | 下调Down** |
| 芳樟醇 Linalool | C10H18O | 上调Up** | 上调Up** | 上调Up* |
| 1,4-Cyclohexadiene-1-carboxaldehyde,2,6,6-trimethyl-(9CI) | C10H14O | 上调Up* | 上调Up* | 下调Down** |
| 1,3,8-p-Menthatriene | C10H14 | 上调Up* | 上调Up* | 上调Up* |
| (E)-p-mentha-2,8-dien-1-ol | C10H16O | 下调Down** | 上调Up* | 下调Down** |
| p-Mentha-1,5,8-triene | C10H14 | 上调Up* | 下调Down* | 下调Down* |
| (+)-(E)-柠檬烯氧化物 (+)-(E)-limonene oxide | C10H16O | 上调Up* | 上调Up*** | — |
| 7-甲基-3-亚甲基-6-辛烯醛 Exo-isocitral | C10H16O | 上调Up*** | 上调Up** | 上调Up* |
| 香茅醛 Citronellal | C10H18O | 上调Up** | 上调Up** | 上调Up** |
| 异壬醛 Isoneral | C10H16O | 上调Up*** | 上调Up** | 上调Up** |
| Isogeranial | C10H16O | 上调Up**** | 上调Up** | 上调Up** |
| 4-萜烯醇 Terpinen-4-ol | C10H18O | 下调Down** | 下调Down** | 下调Down** |
| (+)-β-侧柏酮 (+)-β-thujone | C10H16O | 上调Up** | 上调Up* | 上调Up** |
| L-α-松油醇 L-α-Terpineol | C10H18O | 上调Up** | 上调Up** | 上调Up** |
| 檀油醇 Teresantalol | C10H16O | 上调Up* | 上调Up** | 上调Up*** |
| 癸醛 Decanal | C10H20O | 上调Up* | 上调Up* | 上调Up**** |
| 香茅醇 Citronellol | C10H20O | 下调Down* | 下调Down* | 下调Down |
| 橙花醛 Neral | C10H16O | 上调Up** | 上调Up** | 上调Up** |
| 香叶醇 Geraniol | C10H18O | 下调Down* | 下调Down* | 下调Down |
| 牻牛儿醇 Geranial | C10H16O | 上调Up** | 上调Up* | 上调Up*** |
| 萜烯醇 Carveol | C10H16O | — | — | 上调Up** |
| p-Mentha-1(7),8(10)-dien-9-ol | C10H16O | 上调Up* | — | 上调Up* |
| exo-2-Hydroxycineole | C10H18O2 | 上调Up* | 下调Down* | 下调Down* |
| β-榄香烯 β-elemene | C15H24 | 上调Up** | 上调Up* | 上调Up* |
| (+)-7-表-倍半萜烯 (+)-7-epi-sesquithujene | C15H24 | — | 上调Up* | 上调Up* |
| β-倍半水芹烯β-sesquiphellandrene | C15H24 | 上调Up* | 上调Up* | 上调Up** |
| (E)-β-金合欢烯 (E)-β-farnesene | C15H24 | — | 上调Up* | 上调Up** |
| α-蛇麻烯α-humulene | C15H24 | — | 上调Up* | 上调Up* |
| β-古巴烯 β-copaene | C15H24 | 上调Up* | 上调Up* | 上调Up* |
| Di-epi-α-柏木烯 Di-epi-α-cedrene | C15H24 | — | — | 下调Down** |
| 8-Isopropenyl-1,5-dimethyl-cyclodeca-1,5-diene | C15H24 | 上调Up* | — | 上调Up** |
| (+)-δ-杜松烯 (+)-δ-cadinene | C15H24 | 下调Down* | 上调Up** | 上调Up** |
| α-古巴烯α-Copaene | C15H24 | 下调Down* | 下调Down* | 下调Down* |
| α-榄香醇α-elemol | C15H26O | 下调Down* | 下调Down* | 下调Down* |
| 石竹素 (-)-caryophylleneoxide | C15H24O | 上调Up*** | 下调Down* | — |
| trans-Sesquisabinenehydrate | C15H26O | 下调Down* | 下调Down* | 下调Down* |
| Isoaromadendreneepoxide | C15H24O | 下调Down* | 下调Down* | 下调Down* |
| Diepicedrene-1-oxide | C15H24O | 下调Down* | 下调Down* | — |
| β-甜橙醛β-sinensal | C15H22O | 上调Up** | — | 上调Up** |
Table 5 Changes of oxygen-containing compounds contents in CsCYP2 transgenic plants
| 化合物 Compound | 化学式 Chemical formula | OE | KO1 | KO2 |
|---|---|---|---|---|
| α-侧柏烯α-Thujene | C10H16 | 上调Up** | 上调Up*** | 上调Up* |
| 蒎烯 (+)-α-Pinene | C10H16 | 上调Up** | 上调Up** | 上调Up* |
| 桧烯 Sabinene | C10H16 | 上调Up** | 上调Up** | 上调Up* |
| β-蒎烯β-pinene | C10H16 | 上调Up** | 上调Up** | 上调Up* |
| δ-3-蒈烯δ-3-carene | C10H16 | 下调Down* | 上调Up*** | 下调Down* |
| (+)-4-蒈烯 (+)-4-Carene | C10H16 | — | 上调Up* | 上调Up* |
| (Z)-罗勒烯 (Z)-ocimene | C10H16 | — | — | 下调Down* |
| γ-萜品烯 γ-terpinene | C10H16 | 上调Up** | 上调Up* | 上调Up* |
| (Z)-水合桧烯 (Z)-sabinene hydrate | C10H18O | 下调Down** | 上调Up** | 下调Down** |
| 藏红花醛 Safranal | C10H14O | — | — | 下调Down* |
| 萜品油烯 Isoterpinolene | C10H16 | 下调Down* | 上调Up**** | 上调Up* |
| 萜品烯 Terpinolene | C10H16 | 上调Up** | 上调Up**** | 上调Up* |
| 3,5-二叔丁基苯邻二酚 3,5-dimethylcyclobutabenzene | C10H12 | 下调Down** | 下调Down** | 下调Down** |
| 芳樟醇 Linalool | C10H18O | 上调Up** | 上调Up** | 上调Up* |
| 1,4-Cyclohexadiene-1-carboxaldehyde,2,6,6-trimethyl-(9CI) | C10H14O | 上调Up* | 上调Up* | 下调Down** |
| 1,3,8-p-Menthatriene | C10H14 | 上调Up* | 上调Up* | 上调Up* |
| (E)-p-mentha-2,8-dien-1-ol | C10H16O | 下调Down** | 上调Up* | 下调Down** |
| p-Mentha-1,5,8-triene | C10H14 | 上调Up* | 下调Down* | 下调Down* |
| (+)-(E)-柠檬烯氧化物 (+)-(E)-limonene oxide | C10H16O | 上调Up* | 上调Up*** | — |
| 7-甲基-3-亚甲基-6-辛烯醛 Exo-isocitral | C10H16O | 上调Up*** | 上调Up** | 上调Up* |
| 香茅醛 Citronellal | C10H18O | 上调Up** | 上调Up** | 上调Up** |
| 异壬醛 Isoneral | C10H16O | 上调Up*** | 上调Up** | 上调Up** |
| Isogeranial | C10H16O | 上调Up**** | 上调Up** | 上调Up** |
| 4-萜烯醇 Terpinen-4-ol | C10H18O | 下调Down** | 下调Down** | 下调Down** |
| (+)-β-侧柏酮 (+)-β-thujone | C10H16O | 上调Up** | 上调Up* | 上调Up** |
| L-α-松油醇 L-α-Terpineol | C10H18O | 上调Up** | 上调Up** | 上调Up** |
| 檀油醇 Teresantalol | C10H16O | 上调Up* | 上调Up** | 上调Up*** |
| 癸醛 Decanal | C10H20O | 上调Up* | 上调Up* | 上调Up**** |
| 香茅醇 Citronellol | C10H20O | 下调Down* | 下调Down* | 下调Down |
| 橙花醛 Neral | C10H16O | 上调Up** | 上调Up** | 上调Up** |
| 香叶醇 Geraniol | C10H18O | 下调Down* | 下调Down* | 下调Down |
| 牻牛儿醇 Geranial | C10H16O | 上调Up** | 上调Up* | 上调Up*** |
| 萜烯醇 Carveol | C10H16O | — | — | 上调Up** |
| p-Mentha-1(7),8(10)-dien-9-ol | C10H16O | 上调Up* | — | 上调Up* |
| exo-2-Hydroxycineole | C10H18O2 | 上调Up* | 下调Down* | 下调Down* |
| β-榄香烯 β-elemene | C15H24 | 上调Up** | 上调Up* | 上调Up* |
| (+)-7-表-倍半萜烯 (+)-7-epi-sesquithujene | C15H24 | — | 上调Up* | 上调Up* |
| β-倍半水芹烯β-sesquiphellandrene | C15H24 | 上调Up* | 上调Up* | 上调Up** |
| (E)-β-金合欢烯 (E)-β-farnesene | C15H24 | — | 上调Up* | 上调Up** |
| α-蛇麻烯α-humulene | C15H24 | — | 上调Up* | 上调Up* |
| β-古巴烯 β-copaene | C15H24 | 上调Up* | 上调Up* | 上调Up* |
| Di-epi-α-柏木烯 Di-epi-α-cedrene | C15H24 | — | — | 下调Down** |
| 8-Isopropenyl-1,5-dimethyl-cyclodeca-1,5-diene | C15H24 | 上调Up* | — | 上调Up** |
| (+)-δ-杜松烯 (+)-δ-cadinene | C15H24 | 下调Down* | 上调Up** | 上调Up** |
| α-古巴烯α-Copaene | C15H24 | 下调Down* | 下调Down* | 下调Down* |
| α-榄香醇α-elemol | C15H26O | 下调Down* | 下调Down* | 下调Down* |
| 石竹素 (-)-caryophylleneoxide | C15H24O | 上调Up*** | 下调Down* | — |
| trans-Sesquisabinenehydrate | C15H26O | 下调Down* | 下调Down* | 下调Down* |
| Isoaromadendreneepoxide | C15H24O | 下调Down* | 下调Down* | 下调Down* |
| Diepicedrene-1-oxide | C15H24O | 下调Down* | 下调Down* | — |
| β-甜橙醛β-sinensal | C15H22O | 上调Up** | — | 上调Up** |
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