Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (7): 1389-1401.doi: 10.16420/j.issn.0513-353x.2022-0478
• Genetic & Breeding·Germplasm Resources·Molecular Biology • Next Articles
LIU Hui, YIN Xiangjing, FANG Jinghao, GAO Min, LI Zhi, WANG Xiping*()
Received:
2023-01-17
Revised:
2023-04-07
Online:
2023-07-25
Published:
2023-07-26
Contact:
WANG Xiping
LIU Hui, YIN Xiangjing, FANG Jinghao, GAO Min, LI Zhi, WANG Xiping. Cloning and Functional Analysis of the CDS and Promoter of Synthase Gene STS19 in Chinese Wild Grapevine[J]. Acta Horticulturae Sinica, 2023, 50(7): 1389-1401.
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URL: https://www.ahs.ac.cn/EN/10.16420/j.issn.0513-353x.2022-0478
用途 | 引物名称 | 上游引物(5′-3′) | 下游引物(5′-3′) |
---|---|---|---|
Use | Primer name | Forward primer | Reverse primer |
半定量分析RT-PCR | STS5/6a | GTGGGGCTACAAATTGAGTGAAAG | GGCGAAACTGTAAGAACTTGATGTC |
STS7 | GGAGGGATTGTAATTTAGTGATCGT | CGCCAAGAACTTGAAGTCTCACT | |
STS9 | CCCTATCATAAAATTGAGGGATTG | GGGCATTTCAAGAATATAAAACAA | |
STS10 | CGATCATAACATTGAGGGATTGTAG | GGGGATACAACTTTTCAGGAAAC | |
STS 15/21 | CGCCCTAAATCTTTTATTCCTATCC | CCCACAGAATGACAAGTACTTGCA | |
STS16/22 a | CCTTCTGAAACTGCTTTGGACTCT | GGGTTGCGATAACTTCGTTATTGT | |
STS17/23/24 a | CGGGTTTGATATCTGAAAACATAGAG | GGGAAGATGATTTCATGGTGATATATC | |
STS19 | CGGGCTAATTTGAAAAATATGTAAAA | CGGTAAGGGTCATAGTGCAGCT | |
STS20/28/30 | CGCTTGAGGAGCACCCAAAC | CCGATCCAATGATTACAGCTGC | |
STS27/29 | CCGCCCTATTATGTTGAATAGGAGT | GGGTTTAGGGAATCTTCAAGATGAT | |
STS31 | CGGGGTTATTTATCTCCTAAACTAAT | CCGTTTAATTTGAGCTCACCAAG | |
STS32 | CAAACCATGAGAGTCCAAGTTCCC | GCGACACGTTGGTGTTCAAGTAGA | |
STS35 a | TGCTGCATAGCGTTCCTCCA | CCCCACATGAACACAACATCAATA | |
STS36 | CGGGTATAAATTAAGTGAAGGGGAA | GGGGGGATAATGAAACAGTGAGATA | |
STS37 a | CCCATAGAGAAATGCTTGACCCA | CCGACTTGATTACAAGCCAAATTTA | |
STS38 a | TGCCACGGGTACAAATTGAGTTA | GGAAGCCCTCCAGCAATCAGT | |
STS39/43 | CGCCTATCGAAACTGTTGTGCTAC | CCCCCTTGAAACTCATCTTTTTAAT | |
STS41/45 | CCCGTATTTCATCACATTGGTAA | GGCAACTTGAAACTCATCTTCTTA | |
STS42 | CCCCAATTGATAAAACTCTTGTAGTA | CCCCTTTTAGTTTGAGCTAATCAC | |
STS46 | GGGGGGTTACAAATTAAGAGCAATA | GGGTGACTCAGGTACAAATCCAAAT | |
STS47 | CCCGTGAGGTAAAGAAGAATGGTC | GCCGCAGTCTAACAATGACTTGAA | |
STS48 | GGGGCAGTAATGTAATCAAATAGGG | CCCTGGGAACTTCAAACTCATCAT | |
基因克隆Gene cloning | STS19-clone | ATGGCGTCTGTGGAGGAAA | TTAGTTGGAATCTGTACCA |
亚细胞定位 Subcellular localization | STS19-GFP | AGCTCGGTACCCGGGGATCCATGGCGTCTGTGGAGGAAATTAGAAA | ACCATGGTGTCGACTCTAGAGTTGGAATCTGTACCAACGCTATGG |
转基因番茄鉴定Identification of transgenic tomato | 2300 | CAATCCCACTATCCTTCGC | TGCCAAATGTTTGAACGATC |
启动子克隆 Promoter amplification | PSTS19 | GAATTCTATAATTAGTGCGGGGCG | GACGTCTCCACAGACGCCATTG |
不同长度启动子片段克隆 | PVaSTS19-D5(P621) | GAATTCCCCTCAAAGTGGTATTTG | GACGTCTCCACAGACGCCATTG |
Cloning of promoter deletion | PVaSTS19-D4(P851) | GAATTCCCTCCATAATTACAGCTC | GACGTCTCCACAGACGCCATTG |
fragments | PVaSTS19-D3(P1197) | GAATTCCGCAATCATGGTAATTTT | GACGTCTCCACAGACGCCATTG |
PVaSTS19-D2(P1384) | GAATTCCCCGTTTATTAAAATTAAACC | GACGTCTCCACAGACGCCATTG | |
PVdSTS19-S5(P589) | GAATTCCAAAGAAGTTCCGATATTAA | GACGTCTCCACAGACGCCATTG | |
PVdSTS19-S4(P868) | CTGCAGTCCACAGACGCCATTGAT | GACGTCTCCACAGACGCCATTG | |
PVdSTS19-S3(P1194) | GAATTCCGCAATCATGGTAATTTT | GACGTCTCCACAGACGCCATTG | |
PVdSTS19-S2(P1381) | GAATTCCCCGTTTATTAAAATTAAACC | GACGTCTCCACAGACGCCATTG |
Table 1 Primer sequences used in this study
用途 | 引物名称 | 上游引物(5′-3′) | 下游引物(5′-3′) |
---|---|---|---|
Use | Primer name | Forward primer | Reverse primer |
半定量分析RT-PCR | STS5/6a | GTGGGGCTACAAATTGAGTGAAAG | GGCGAAACTGTAAGAACTTGATGTC |
STS7 | GGAGGGATTGTAATTTAGTGATCGT | CGCCAAGAACTTGAAGTCTCACT | |
STS9 | CCCTATCATAAAATTGAGGGATTG | GGGCATTTCAAGAATATAAAACAA | |
STS10 | CGATCATAACATTGAGGGATTGTAG | GGGGATACAACTTTTCAGGAAAC | |
STS 15/21 | CGCCCTAAATCTTTTATTCCTATCC | CCCACAGAATGACAAGTACTTGCA | |
STS16/22 a | CCTTCTGAAACTGCTTTGGACTCT | GGGTTGCGATAACTTCGTTATTGT | |
STS17/23/24 a | CGGGTTTGATATCTGAAAACATAGAG | GGGAAGATGATTTCATGGTGATATATC | |
STS19 | CGGGCTAATTTGAAAAATATGTAAAA | CGGTAAGGGTCATAGTGCAGCT | |
STS20/28/30 | CGCTTGAGGAGCACCCAAAC | CCGATCCAATGATTACAGCTGC | |
STS27/29 | CCGCCCTATTATGTTGAATAGGAGT | GGGTTTAGGGAATCTTCAAGATGAT | |
STS31 | CGGGGTTATTTATCTCCTAAACTAAT | CCGTTTAATTTGAGCTCACCAAG | |
STS32 | CAAACCATGAGAGTCCAAGTTCCC | GCGACACGTTGGTGTTCAAGTAGA | |
STS35 a | TGCTGCATAGCGTTCCTCCA | CCCCACATGAACACAACATCAATA | |
STS36 | CGGGTATAAATTAAGTGAAGGGGAA | GGGGGGATAATGAAACAGTGAGATA | |
STS37 a | CCCATAGAGAAATGCTTGACCCA | CCGACTTGATTACAAGCCAAATTTA | |
STS38 a | TGCCACGGGTACAAATTGAGTTA | GGAAGCCCTCCAGCAATCAGT | |
STS39/43 | CGCCTATCGAAACTGTTGTGCTAC | CCCCCTTGAAACTCATCTTTTTAAT | |
STS41/45 | CCCGTATTTCATCACATTGGTAA | GGCAACTTGAAACTCATCTTCTTA | |
STS42 | CCCCAATTGATAAAACTCTTGTAGTA | CCCCTTTTAGTTTGAGCTAATCAC | |
STS46 | GGGGGGTTACAAATTAAGAGCAATA | GGGTGACTCAGGTACAAATCCAAAT | |
STS47 | CCCGTGAGGTAAAGAAGAATGGTC | GCCGCAGTCTAACAATGACTTGAA | |
STS48 | GGGGCAGTAATGTAATCAAATAGGG | CCCTGGGAACTTCAAACTCATCAT | |
基因克隆Gene cloning | STS19-clone | ATGGCGTCTGTGGAGGAAA | TTAGTTGGAATCTGTACCA |
亚细胞定位 Subcellular localization | STS19-GFP | AGCTCGGTACCCGGGGATCCATGGCGTCTGTGGAGGAAATTAGAAA | ACCATGGTGTCGACTCTAGAGTTGGAATCTGTACCAACGCTATGG |
转基因番茄鉴定Identification of transgenic tomato | 2300 | CAATCCCACTATCCTTCGC | TGCCAAATGTTTGAACGATC |
启动子克隆 Promoter amplification | PSTS19 | GAATTCTATAATTAGTGCGGGGCG | GACGTCTCCACAGACGCCATTG |
不同长度启动子片段克隆 | PVaSTS19-D5(P621) | GAATTCCCCTCAAAGTGGTATTTG | GACGTCTCCACAGACGCCATTG |
Cloning of promoter deletion | PVaSTS19-D4(P851) | GAATTCCCTCCATAATTACAGCTC | GACGTCTCCACAGACGCCATTG |
fragments | PVaSTS19-D3(P1197) | GAATTCCGCAATCATGGTAATTTT | GACGTCTCCACAGACGCCATTG |
PVaSTS19-D2(P1384) | GAATTCCCCGTTTATTAAAATTAAACC | GACGTCTCCACAGACGCCATTG | |
PVdSTS19-S5(P589) | GAATTCCAAAGAAGTTCCGATATTAA | GACGTCTCCACAGACGCCATTG | |
PVdSTS19-S4(P868) | CTGCAGTCCACAGACGCCATTGAT | GACGTCTCCACAGACGCCATTG | |
PVdSTS19-S3(P1194) | GAATTCCGCAATCATGGTAATTTT | GACGTCTCCACAGACGCCATTG | |
PVdSTS19-S2(P1381) | GAATTCCCCGTTTATTAAAATTAAACC | GACGTCTCCACAGACGCCATTG |
Fig. 1 Schematic representation of promoter fragments of different lengths of VaSTS19 and VdSTS19 D1-D5 indicate the promoter fragments of different lengths of VaSTS19;S1-S5 indicate the promoter fragments of different lengths of VdSTS19. The same below.
Fig. 2 Tissue specific expression profile analysis (A) and fruit development period expression (B) of STS genes A:Vitis amurensis‘Tonghua 3’. 1,root;2,stem;3,leaf;4,flower;5,pericarp;6,flesh;7,seed;8,tendril. B:1-3:Grapevine fruit of green,veraison and mature stages in V. davidii‘Tangwei’;4-6:Grapevine fruit of green,veraison and mature stages in V. amurensis‘Tonghua 3’.
Fig. 4 Transformation process of transgenic tomatos(A)and PCR verifies the recombinant plasmid in transformed tomato(B) a:Cotyledon germination;b:Cotyledon precultured;c:Co-culture after infection;d:Callus formed after transformation;e:Resistant shoots generate;f and g:Resistant bud rooting;h:Transplanting to the greenhouse. M:Indicates DNA marker DL 2000;Lane 1-6:Indicate the transgenic plants of VaSTS19;Lane 7-12:Indicate the transgenic plants of VdSTS19;Lane 13-17:Indicate the transgenic plants of 2300;Lane 18,19:Indicate wild type tomato.
转基因名称 Gene name | 转基因株系 Line | 绿果期 Green | 转色期 Veraison | 成熟期 Mature |
---|---|---|---|---|
VaSTS19 | L1 | nd | 3.71 ± 0.58 a | 3.61 ± 0.41 a |
L2 | nd | 3.63 ± 0.67 a | 2.68 ± 0.29 b | |
L3 | nd | 2.84 ± 0.16 b | 4.29 ± 0.36 a | |
VdSTS19 | L1 | nd | 3.83 ± 0.13 a | 3.99 ± 0.14 a |
L2 | nd | 3.98 ± 0.25 a | 3.67 ± 0.47 a | |
L3 | nd | 4.15 ± 0.55 a | 4.03 ± 0.40 a | |
2300 | L1 | nd | nd | nd |
WT | nd | nd | nd |
Table 2 Resveratrol concentration in transgenic and wild type (WT) tomatoes μg · g-1
转基因名称 Gene name | 转基因株系 Line | 绿果期 Green | 转色期 Veraison | 成熟期 Mature |
---|---|---|---|---|
VaSTS19 | L1 | nd | 3.71 ± 0.58 a | 3.61 ± 0.41 a |
L2 | nd | 3.63 ± 0.67 a | 2.68 ± 0.29 b | |
L3 | nd | 2.84 ± 0.16 b | 4.29 ± 0.36 a | |
VdSTS19 | L1 | nd | 3.83 ± 0.13 a | 3.99 ± 0.14 a |
L2 | nd | 3.98 ± 0.25 a | 3.67 ± 0.47 a | |
L3 | nd | 4.15 ± 0.55 a | 4.03 ± 0.40 a | |
2300 | L1 | nd | nd | nd |
WT | nd | nd | nd |
Fig. 5 A comparison of the VaSTS19 and VdSTS19 promoter sequences The translational start sites(+1)are shown in red box. Motifs with significant similarity to previously identified cis-acting elements in both species are shaded in green. Motifs found only in VaSTS19 or VdSTS19 promoters are respectively shaded in pink or red. Arrowheads pointing to the left represent the reverse direction of cis-elements compared to promoter orientation. Other arrows represent start point of 5′-deleted derivatives.
Fig. 6 A phylogenetic tree based on a ClustalX multiple sequence alignment of STS promoters from a range of grape species The scale bar represents 0.05 substitutions per site and the numbers next to the nodes are bootstrap values from 1 000 replicates.
Fig. 7 GUS activity analysis of promoter fragments of different lengths of VaSTS19 and VdSTS19 in response to hormone treatment in transiently transformed tobacco leaves Mean GUS activity(± SE)is averaged from triplicate experiments. WT:Wild type;N:Negative control(no promoter);35S:Positive control (CaMV35S promoter). Significant difference test were evaluated using a one-sided paired t test(**α = 0.01,or at *α = 0.05).
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