Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (2): 183-196.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0726
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YUAN Xin-yu1,2(), ZHONG Cai-hong2, ZHANG Long2, ZHENG Hao2, LI Ji-tao1(), ZHANG Qiong2()
Received:
2023-07-30
Online:
2024-02-26
Published:
2024-03-13
Contact:
LI Ji-tao, ZHANG Qiong
E-mail:yuanxinyu0618@foxmail.com;ljtyouth@foxmail.com;qiongzhang@wbgcas.cn
YUAN Xin-yu, ZHONG Cai-hong, ZHANG Long, ZHENG Hao, LI Ji-tao, ZHANG Qiong. Identification and Function Analysis of AcMYB88 in Kiwifruit (Actinidia chinensis)[J]. Biotechnology Bulletin, 2024, 40(2): 183-196.
基因名称 Gene name | 正向引物 Forward primer(5'-3') | 反向引物 Reverse primer(5'-3') | 备注 Note |
---|---|---|---|
Actin | TGAGAGATTCCGTTGCCCAGAAGT | TTCCTTACTCATGCGGTCTGCGAT | 实时荧光定量PCR分析所用引物 Primers for real-time RT-qPCR analysis |
Achn033551 | GCATCCATGACATCACCAGC | AGCATAACCGGTGTCCCAAC | |
Achn036201 | TGACCGGTGGTACAATGTGG | CCTGATGGTTCACCCACTCC | |
Achn141271 | GGGCTCTCGCGATATACGAC | TCTCCAACGTGAACGACGAG | |
Achn145641 | TCCATGACATCACCAGCGTC | AGGAAGCATAACTGGCGTCC | |
Achn154591 | ACGACATCACCACCGTCAAT | GCCGGTCCTTCACTTGGTAG | |
Achn172831 | GGAGTGCTCCTCGGATTGTC | CCACTTTCTGCCACCGATCA | |
Achn207851 | CTGTGGTACCCAGAATGCCT | ATGTCGTGGATGCTTGACCG | |
Achn212251 | AAGGCCAACCGTGAATCTCC | AGGCAATCCGACATGGGTTC | |
Achn218481 | AGAAGTACTTCATCCGGCAGC | GGAATTGCGTTCTTGGATTGG | |
Achn258201 | CCCTGGCAACTTATCCCGAG | CTTGCGTCGCTCCTGATCTA | |
Achn365361 | ACGACGGCAGTGATTATCGG | CTTGTCCTCGAACCTCGTCC | |
Achn371351 | ATTGGAGGAGCATATCGCGG | TGGATGCTTGAACGTCTCCC | |
Acc00493(AcMYB10) | GCCATCACCACCATCCAACA | TTCCCCAAAATTGGCTCCTCA | |
Acc19563(AcbHLH42) | TCGTTTAGGGCCGAGTCTAC | TTGAGAGTAGTGCGTGTCGT | |
pSAK-Achn365361 | ACTAGTGGATCCAAAGAATTCATGTATGGCGACGACGGC | AGAAGTACTCTCGAGAAGCTTTACATGGGATAGTTGAAGTTTTGGTACCC | 过表达载体构建所用引物 Primers used for overexpression vector construction |
pSAK-AcMYB10 | ACTAGTGGATCCAAAGAATTCATGGAAAGTGTTACTTTAGGAGTGAGAAAGG | AGAAGTACTCTCGAGAAGCTTTAATCATCACCTAAGAGATCCCAAAGGTTAACA | |
pSAK-AcbHLH42 | ACTAGTGGATCCAAAGAATTCATGGCGGCTCCCCCT | AGAAGTACTCTCGAGAAGCTTCAGGGTATGATTTGGTGTATTGCTCTCT | |
NtActin | AATGGAACTGGAATGGTCAAGGC | TGCCAGATCTTCTCCATGTCATCCCA | 烟草RT-qPCR分析所用引物 Primers for real-time RT-qPCR analysis in tobacco |
AcMYB88 | ACGACGGCAGTGATTATCGG | CTTGTCCTCGAACCTCGTCC | |
AcMYB10 | GCCATCACCACCATCCAACA | TTCCCCAAAATTGGCTCCTCA | |
AcbHLH42 | TCGTTTAGGGCCGAGTCTAC | TTGAGAGTAGTGCGTGTCGT | |
NtC4H | ACAACCCGGATCACTGGAAG | ACTGTCCACCTTTCTCCGTG | |
NtCHS | TTGTTCGAGCTTGTCTCTGC | AGCCCAGGAACATCTTTGAG | |
NtPAL | TTACGCCCTTCGAACATCCC | GAGGCAAGTGCAAGCCTAGT | |
Nt4CL | ACTGCCGAAAGGTGTGATGT | AAATCGCTGCTCCGACTCTC | |
NtCHI | GTCAGGCCATTGAAAAGCTC | CTAATCGTCAATGCCCCAAC | |
NtF3H | CAAGGCATGTGTGGATATGG | TGTGTCGTTTCAGTCCAAGG | |
NtDFR | AACCAACAGTCAGGGGAATG | TTGGACATCGACAGTTCCAG | |
NtANS | TATCCCAAATGCCCCCAACC | ATCTTCTCCTTTGGCGGCTC | |
NtANR | TTCCATCTCTCATGGCTGGTC | ATATGGGCGCGACAAACATC |
Table 1 Primer sequences used in the experiment
基因名称 Gene name | 正向引物 Forward primer(5'-3') | 反向引物 Reverse primer(5'-3') | 备注 Note |
---|---|---|---|
Actin | TGAGAGATTCCGTTGCCCAGAAGT | TTCCTTACTCATGCGGTCTGCGAT | 实时荧光定量PCR分析所用引物 Primers for real-time RT-qPCR analysis |
Achn033551 | GCATCCATGACATCACCAGC | AGCATAACCGGTGTCCCAAC | |
Achn036201 | TGACCGGTGGTACAATGTGG | CCTGATGGTTCACCCACTCC | |
Achn141271 | GGGCTCTCGCGATATACGAC | TCTCCAACGTGAACGACGAG | |
Achn145641 | TCCATGACATCACCAGCGTC | AGGAAGCATAACTGGCGTCC | |
Achn154591 | ACGACATCACCACCGTCAAT | GCCGGTCCTTCACTTGGTAG | |
Achn172831 | GGAGTGCTCCTCGGATTGTC | CCACTTTCTGCCACCGATCA | |
Achn207851 | CTGTGGTACCCAGAATGCCT | ATGTCGTGGATGCTTGACCG | |
Achn212251 | AAGGCCAACCGTGAATCTCC | AGGCAATCCGACATGGGTTC | |
Achn218481 | AGAAGTACTTCATCCGGCAGC | GGAATTGCGTTCTTGGATTGG | |
Achn258201 | CCCTGGCAACTTATCCCGAG | CTTGCGTCGCTCCTGATCTA | |
Achn365361 | ACGACGGCAGTGATTATCGG | CTTGTCCTCGAACCTCGTCC | |
Achn371351 | ATTGGAGGAGCATATCGCGG | TGGATGCTTGAACGTCTCCC | |
Acc00493(AcMYB10) | GCCATCACCACCATCCAACA | TTCCCCAAAATTGGCTCCTCA | |
Acc19563(AcbHLH42) | TCGTTTAGGGCCGAGTCTAC | TTGAGAGTAGTGCGTGTCGT | |
pSAK-Achn365361 | ACTAGTGGATCCAAAGAATTCATGTATGGCGACGACGGC | AGAAGTACTCTCGAGAAGCTTTACATGGGATAGTTGAAGTTTTGGTACCC | 过表达载体构建所用引物 Primers used for overexpression vector construction |
pSAK-AcMYB10 | ACTAGTGGATCCAAAGAATTCATGGAAAGTGTTACTTTAGGAGTGAGAAAGG | AGAAGTACTCTCGAGAAGCTTTAATCATCACCTAAGAGATCCCAAAGGTTAACA | |
pSAK-AcbHLH42 | ACTAGTGGATCCAAAGAATTCATGGCGGCTCCCCCT | AGAAGTACTCTCGAGAAGCTTCAGGGTATGATTTGGTGTATTGCTCTCT | |
NtActin | AATGGAACTGGAATGGTCAAGGC | TGCCAGATCTTCTCCATGTCATCCCA | 烟草RT-qPCR分析所用引物 Primers for real-time RT-qPCR analysis in tobacco |
AcMYB88 | ACGACGGCAGTGATTATCGG | CTTGTCCTCGAACCTCGTCC | |
AcMYB10 | GCCATCACCACCATCCAACA | TTCCCCAAAATTGGCTCCTCA | |
AcbHLH42 | TCGTTTAGGGCCGAGTCTAC | TTGAGAGTAGTGCGTGTCGT | |
NtC4H | ACAACCCGGATCACTGGAAG | ACTGTCCACCTTTCTCCGTG | |
NtCHS | TTGTTCGAGCTTGTCTCTGC | AGCCCAGGAACATCTTTGAG | |
NtPAL | TTACGCCCTTCGAACATCCC | GAGGCAAGTGCAAGCCTAGT | |
Nt4CL | ACTGCCGAAAGGTGTGATGT | AAATCGCTGCTCCGACTCTC | |
NtCHI | GTCAGGCCATTGAAAAGCTC | CTAATCGTCAATGCCCCAAC | |
NtF3H | CAAGGCATGTGTGGATATGG | TGTGTCGTTTCAGTCCAAGG | |
NtDFR | AACCAACAGTCAGGGGAATG | TTGGACATCGACAGTTCCAG | |
NtANS | TATCCCAAATGCCCCCAACC | ATCTTCTCCTTTGGCGGCTC | |
NtANR | TTCCATCTCTCATGGCTGGTC | ATATGGGCGCGACAAACATC |
对照组 Control group | 试验组 Experimental group |
---|---|
EV | OE10 |
OE88 | |
EV:OE10 : OE42(1:1:1) | OE88:OE10:OE42(1:1:1) |
EV:OE10(1:1) | OE88:OE10(1:1) |
Table 2 Agrobacterium combinations for tobacco transient transformation
对照组 Control group | 试验组 Experimental group |
---|---|
EV | OE10 |
OE88 | |
EV:OE10 : OE42(1:1:1) | OE88:OE10:OE42(1:1:1) |
EV:OE10(1:1) | OE88:OE10(1:1) |
蛋白名称 Protein name | 拟南芥同源蛋白 Homologous protein of Arabidopsis thaliana | 氨基酸数 Number of amino acids | 分子量 Molecular weight/Da | 等电点 pI | 带负电荷残基Asp+GIu | 带正电荷残基 Arg+Lys | 不稳定指数Instability | 脂肪指数 Aliphatic index | 亲水性 GRAVY | 亚细胞定位Subcellular localization |
---|---|---|---|---|---|---|---|---|---|---|
Achn033551 | AT1G49010.1 | 288 | 31 272.05 | 6.67 | 31 | 28 | 57.11 | 66.81 | -0.54 | 细胞核Nucleus |
Achn036201 | AT2G38090.1 | 294 | 33 491.45 | 8.99 | 31 | 35 | 51.55 | 59.69 | -0.79 | 细胞核Nucleus |
Achn141271 | AT2G38090.1 | 269 | 30 463.48 | 8.27 | 31 | 33 | 46.22 | 68.10 | -0.58 | 细胞核Nucleus |
Achn145641 | AT1G49010.1 | 303 | 32 847.73 | 6.88 | 31 | 29 | 59.61 | 63.43 | -0.61 | 细胞核Nucleus |
Achn154591 | AT2G38090.1 | 298 | 33 813.85 | 8.77 | 33 | 36 | 52.52 | 59.60 | -0.81 | 细胞核Nucleus |
Achn172831 | AT5G58900.1 | 290 | 33 039.98 | 7.24 | 35 | 35 | 65.34 | 58.14 | -0.87 | 细胞核Nucleus |
Achn207851 | AT5G08520.1 | 200 | 22 325.81 | 5.97 | 26 | 22 | 51.90 | 70.65 | -0.62 | 细胞核Nucleus |
Achn212251 | AT2G38090.1 | 273 | 30 842.68 | 8.99 | 32 | 36 | 48.13 | 70.73 | -0.69 | 细胞核Nucleus |
Achn218481 | AT5G58900.1 | 286 | 32 870.57 | 9.22 | 34 | 39 | 59.06 | 53.22 | -1.02 | 细胞核Nucleus |
Achn258201 | AT5G08520.1 | 423 | 45 538.79 | 5.84 | 54 | 45 | 52.14 | 71.18 | -0.54 | 细胞核Nucleus |
Achn365361 | AT5G04760.1 | 212 | 24 232.82 | 6.07 | 30 | 26 | 42.43 | 62.97 | -0.81 | 细胞核Nucleus |
Achn371351 | AT5G08520.1 | 279 | 31 022.69 | 8.46 | 31 | 33 | 62.22 | 65.02 | -0.67 | 细胞核Nucleus |
Table 3 Analysis of the physicochemical properties of 12 kiwifruit MYB transcription factors
蛋白名称 Protein name | 拟南芥同源蛋白 Homologous protein of Arabidopsis thaliana | 氨基酸数 Number of amino acids | 分子量 Molecular weight/Da | 等电点 pI | 带负电荷残基Asp+GIu | 带正电荷残基 Arg+Lys | 不稳定指数Instability | 脂肪指数 Aliphatic index | 亲水性 GRAVY | 亚细胞定位Subcellular localization |
---|---|---|---|---|---|---|---|---|---|---|
Achn033551 | AT1G49010.1 | 288 | 31 272.05 | 6.67 | 31 | 28 | 57.11 | 66.81 | -0.54 | 细胞核Nucleus |
Achn036201 | AT2G38090.1 | 294 | 33 491.45 | 8.99 | 31 | 35 | 51.55 | 59.69 | -0.79 | 细胞核Nucleus |
Achn141271 | AT2G38090.1 | 269 | 30 463.48 | 8.27 | 31 | 33 | 46.22 | 68.10 | -0.58 | 细胞核Nucleus |
Achn145641 | AT1G49010.1 | 303 | 32 847.73 | 6.88 | 31 | 29 | 59.61 | 63.43 | -0.61 | 细胞核Nucleus |
Achn154591 | AT2G38090.1 | 298 | 33 813.85 | 8.77 | 33 | 36 | 52.52 | 59.60 | -0.81 | 细胞核Nucleus |
Achn172831 | AT5G58900.1 | 290 | 33 039.98 | 7.24 | 35 | 35 | 65.34 | 58.14 | -0.87 | 细胞核Nucleus |
Achn207851 | AT5G08520.1 | 200 | 22 325.81 | 5.97 | 26 | 22 | 51.90 | 70.65 | -0.62 | 细胞核Nucleus |
Achn212251 | AT2G38090.1 | 273 | 30 842.68 | 8.99 | 32 | 36 | 48.13 | 70.73 | -0.69 | 细胞核Nucleus |
Achn218481 | AT5G58900.1 | 286 | 32 870.57 | 9.22 | 34 | 39 | 59.06 | 53.22 | -1.02 | 细胞核Nucleus |
Achn258201 | AT5G08520.1 | 423 | 45 538.79 | 5.84 | 54 | 45 | 52.14 | 71.18 | -0.54 | 细胞核Nucleus |
Achn365361 | AT5G04760.1 | 212 | 24 232.82 | 6.07 | 30 | 26 | 42.43 | 62.97 | -0.81 | 细胞核Nucleus |
Achn371351 | AT5G08520.1 | 279 | 31 022.69 | 8.46 | 31 | 33 | 62.22 | 65.02 | -0.67 | 细胞核Nucleus |
蛋白名称Protein name | 丝氨酸Ser | 苏氨酸Thr | 酪氨酸Tyr |
---|---|---|---|
Achn033551 | 21 | 7 | 1 |
Achn036201 | 19 | 15 | 1 |
Achn141271 | 15 | 12 | 1 |
Achn145641 | 27 | 7 | 1 |
Achn154591 | 22 | 11 | 1 |
Achn172831 | 19 | 10 | 2 |
Achn207851 | 16 | 7 | 2 |
Achn212251 | 18 | 11 | 2 |
Achn218481 | 19 | 13 | 6 |
Achn258201 | 38 | 11 | 1 |
Achn365361 | 11 | 9 | 3 |
Achn371351 | 29 | 14 | 4 |
Table 4 Phosphorylation sites of MYB proteins in kiwifruit
蛋白名称Protein name | 丝氨酸Ser | 苏氨酸Thr | 酪氨酸Tyr |
---|---|---|---|
Achn033551 | 21 | 7 | 1 |
Achn036201 | 19 | 15 | 1 |
Achn141271 | 15 | 12 | 1 |
Achn145641 | 27 | 7 | 1 |
Achn154591 | 22 | 11 | 1 |
Achn172831 | 19 | 10 | 2 |
Achn207851 | 16 | 7 | 2 |
Achn212251 | 18 | 11 | 2 |
Achn218481 | 19 | 13 | 6 |
Achn258201 | 38 | 11 | 1 |
Achn365361 | 11 | 9 | 3 |
Achn371351 | 29 | 14 | 4 |
蛋白名称 Protein name | α-螺旋 α-helix | 延伸链 Extended stran | β-转角 β-turn | 无规则卷曲 Random coil |
---|---|---|---|---|
Achn033551 | 22.22 | 12.50 | 3.47 | 61.81 |
Achn036201 | 22.79 | 10.88 | 3.40 | 62.93 |
Achn141271 | 24.16 | 11.52 | 2.97 | 61.34 |
Achn145641 | 19.80 | 12.54 | 3.96 | 63.70 |
Achn154591 | 23.83 | 11.74 | 2.68 | 61.74 |
Achn172831 | 24.14 | 15.17 | 3.79 | 56.90 |
Achn207851 | 25.50 | 13.00 | 5.00 | 56.50 |
Achn212251 | 23.81 | 13.55 | 3.66 | 58.97 |
Achn218481 | 20.63 | 15.03 | 3.15 | 61.19 |
Achn258201 | 27.19 | 13.95 | 4.49 | 54.37 |
Achn365361 | 26.89 | 11.32 | 5.19 | 56.60 |
Achn371351 | 21.51 | 12.54 | 2.15 | 63.80 |
Table 5 Secondary structure statistics of 12 AcMYBs %
蛋白名称 Protein name | α-螺旋 α-helix | 延伸链 Extended stran | β-转角 β-turn | 无规则卷曲 Random coil |
---|---|---|---|---|
Achn033551 | 22.22 | 12.50 | 3.47 | 61.81 |
Achn036201 | 22.79 | 10.88 | 3.40 | 62.93 |
Achn141271 | 24.16 | 11.52 | 2.97 | 61.34 |
Achn145641 | 19.80 | 12.54 | 3.96 | 63.70 |
Achn154591 | 23.83 | 11.74 | 2.68 | 61.74 |
Achn172831 | 24.14 | 15.17 | 3.79 | 56.90 |
Achn207851 | 25.50 | 13.00 | 5.00 | 56.50 |
Achn212251 | 23.81 | 13.55 | 3.66 | 58.97 |
Achn218481 | 20.63 | 15.03 | 3.15 | 61.19 |
Achn258201 | 27.19 | 13.95 | 4.49 | 54.37 |
Achn365361 | 26.89 | 11.32 | 5.19 | 56.60 |
Achn371351 | 21.51 | 12.54 | 2.15 | 63.80 |
Fig. 5 Expression heatmap of 14 MYB genes during different development stage of ‘Donghong’ DAF: The day after flower. Each gene is indicated by a row and each developmental period is indicated by a column. The figure represents relative expression level of genes. Dark red color indicates high relative expression, while genes with low relative expression are indicated in dark blue
Fig. 7 Overexpression of AcMYB10 and AcMYB88 promotes tobacco anthocyanin accumulation and related gene expression A: Phenotypes of tobacco leaves overexpressing AcMYB10, AcMYB88 and EV. B: Anthocyanin content in transgenic tobacco leaves. C: Expression levels of anthocyanin-related structural genes in transgenic tobacco leaves. EV: Empty vector. OE88: Overexpression-AcMYB88. OE10: Overexpressive-AcMYB10. PAL: Phenylalaninammo-nialyase. C4H: cinnamate-4-hydroxylase. 4CL: 4-coumaryl. CoA: Ligase. CHS: Chalcone synthase. CHI: Chalcone isomerase. F3H: Flavonol-3-hydroxylase. DFR: Dihydroflavonol reductase. LDOX: Leucoanthocyanidin dioxygenase. ANR: Anthocyanidin reductase. ANS: Anthocyanidin synthase. All graphs are based on independent samples t test. *: P < 0.05; **: P < 0.01. The same below
Fig. 8 Overexpression of EV+OE10+OE42 and OE88+OE10+OE42 promotes tobacco anthocyanin accumulation and related gene expression A: Phenotypes of tobacco leaves overexpressing EV+OE10+OE42 and OE88+OE10+OE42. B: Anthocyanin content in transgenic tobacco leaves. C: Expression levels of anthocyanin-related structural genes in transgenic tobacco leaves. Achn365361:AcMYB88;EV+OE10 +OE42: Empty vector+Overexpression-AcMYB10+Overexpression-bHLH42(1:1:1). OE88+OE10+OE42: Overexpression-AcMYB88+Overexpression-AcMYB10+Overexpression-bHLH42(1:1:1)
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