生物技术通报 ›› 2022, Vol. 38 ›› Issue (11): 129-139.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0077
赖恭梯1,2(), 阙秋霞1, 潘若1, 刘雨轩1, 王琦1,2, 赖谱富1, 高慧颖1,2, 赖呈纯1,2()
收稿日期:
2021-01-18
出版日期:
2022-11-26
发布日期:
2022-12-01
作者简介:
赖恭梯,男,博士,助理研究员,研究方向:园艺植物与食品生物技术;E-mail:基金资助:
LAI Gong-ti1,2(), QUE Qiu-xia1, PAN Ruo1, LIU Yu-xuan1, WANG Qi1,2, LAI Pu-fu1, GAO Hui-ying1,2, LAI Cheng-chun1,2()
Received:
2021-01-18
Published:
2022-11-26
Online:
2022-12-01
摘要:
查尔酮合成酶(chalcone synthase,CHS)是花青素合成途径的第一个关键酶,为探明CHS在不同光质下的表达模式及转录因子调控特征,以刺葡萄愈伤组织为材料,克隆了2个CHS基因(VdCHS2和VdCHS3),进行生物信息学分析和8种不同光质培养处理下的表达分析,并对CHS启动子顺式作用元件和转录因子结合位点进行预测。结果显示,VdCHS2和VdCHS3基因长度分别为1 382 bp和1 398 bp,均由2个外显子和1个内含子组成,编码蛋白均为无信号肽、定位于细胞质的亲水性蛋白。光照促进VdCHS的表达,VdCHS在短波光诱导下,先呈上调表达,当表达量达到一定水平后则呈现下调作用,而长波光抑制VdCHS的表达。靶向CHS的转录因子预测得到13个MYB成员,2个CHS启动子上存在23个MYB转录因子识别和结合元件。研究结果表明,不同光质的波长对VdCHS表达影响显著,13个MYB转录因子可能在刺葡萄花青素合成中参与CHS的转录调控。
赖恭梯, 阙秋霞, 潘若, 刘雨轩, 王琦, 赖谱富, 高慧颖, 赖呈纯. 刺葡萄查尔酮合成酶基因CHS对不同光质的响应及转录因子调控分析[J]. 生物技术通报, 2022, 38(11): 129-139.
LAI Gong-ti, QUE Qiu-xia, PAN Ruo, LIU Yu-xuan, WANG Qi, LAI Pu-fu, GAO Hui-ying, LAI Cheng-chun. Response of Chalcone Synthase Gene(CHS)to Different Light Quality and Transcription Factor Regulation in Vitis davidii[J]. Biotechnology Bulletin, 2022, 38(11): 129-139.
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 用途 Application |
---|---|---|
CHS2-F | CACTGATTCAAGCAGCCAAAAATG | CHS2 克隆 |
CHS2-R | TTACTTAAGTGTGAAAGGAGAGAAG | |
CHS3-F | CTCCAACCACCACAACCCTCATC | CHS3 克隆 |
CHS3-R | ATGGGGCTAGAAAAAGCATATCATC | |
CHS2-qPCR-F | TGGGTCTGAAGGAAGAGAAAC | CHS2 qPCR |
CHS2-qPCR-R | TTGTGTAGCAAGGCTGTGC | |
CHS3-qPCR-F | AGTTACGCTCCACACGACAC | CHS3 qPCR |
CHS3-qPCR-R | AGCACCACGGTCTCAACAGT |
表1 引物信息
Table 1 Primer information
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 用途 Application |
---|---|---|
CHS2-F | CACTGATTCAAGCAGCCAAAAATG | CHS2 克隆 |
CHS2-R | TTACTTAAGTGTGAAAGGAGAGAAG | |
CHS3-F | CTCCAACCACCACAACCCTCATC | CHS3 克隆 |
CHS3-R | ATGGGGCTAGAAAAAGCATATCATC | |
CHS2-qPCR-F | TGGGTCTGAAGGAAGAGAAAC | CHS2 qPCR |
CHS2-qPCR-R | TTGTGTAGCAAGGCTGTGC | |
CHS3-qPCR-F | AGTTACGCTCCACACGACAC | CHS3 qPCR |
CHS3-qPCR-R | AGCACCACGGTCTCAACAGT |
基因名称 Gene name | GenBank 登陆号 GenBank accession | 片段长度 Fragment length/bp | ORF长度 ORF length/bp | 氨基酸数目 Amino acid number | 分子量 Molecular weight/kD | 理论等电点pI | 亲水性 GRAVY | 信号肽 Signal peptide | 亚细胞定位 Subcellular localization |
---|---|---|---|---|---|---|---|---|---|
VdCHS2 | OL906400 | 1 382 | 1 182 | 393 | 42.90 | 6.1 | -0.057 | 无 No | 细胞质Cytoplasmic |
VdCHS3 | OL906401 | 1 398 | 1 170 | 389 | 42.60 | 6.18 | -0.132 | 无 No | 细胞质Cytoplasmic |
表2 VdCHS生物信息学分析
Table 2 Bioinformatics analysis of VdCHS
基因名称 Gene name | GenBank 登陆号 GenBank accession | 片段长度 Fragment length/bp | ORF长度 ORF length/bp | 氨基酸数目 Amino acid number | 分子量 Molecular weight/kD | 理论等电点pI | 亲水性 GRAVY | 信号肽 Signal peptide | 亚细胞定位 Subcellular localization |
---|---|---|---|---|---|---|---|---|---|
VdCHS2 | OL906400 | 1 382 | 1 182 | 393 | 42.90 | 6.1 | -0.057 | 无 No | 细胞质Cytoplasmic |
VdCHS3 | OL906401 | 1 398 | 1 170 | 389 | 42.60 | 6.18 | -0.132 | 无 No | 细胞质Cytoplasmic |
图1 VdCHS2和VdCHS3基因结构及编码蛋白保守结构域 A:VdCHS2和VdCHS3外显子/内含子结构;B,C:VdCHS2和VdCHS3编码蛋白保守结构域;D-F:CHS蛋白保守基序
Fig.1 Gene structure and conserved domain of VdCHS2 and VdCHS3 A:Exon/intron structure of VdCHS2 and VdCHS3. B and C:Conserved domain of VdCHS2 and VdCHS3. D-F:Motifs of CHS proteins
图2 CHS蛋白系统发育(A)和保守基序(B)分析 猕猴桃:Actinidia chinensis;杨树:Populus trichocarpa;刺葡萄:Vitis davidii;拟南芥:Arabidopsis thaliana;柑橘:Citrus clementina;水稻:Oryza sativa;玉米:Zea mays;小麦:Triticum aestivum
Fig. 2 Phylogeny(A)and conserved motif(B)analysis of CHS
光质类型 Light quality type | 光质名称 Light quality | 光波长 Light wavelength/nm | 表达模式 Expression pattern | ||
---|---|---|---|---|---|
主峰 Main peak | 次峰 Secondary peak | VdCHS2 | VdCHS3 | ||
短波光 | 紫光 | 448 | / | 先升后降 | 先升后降 |
蓝光 | 456 | / | 先升后降 | 先升后降 | |
绿光 | 514 | / | 先升后降 | 先升后降 | |
长波光 | 黄光 | 590 | / | 下调 | 下调 |
红光 | 637 | / | 下调 | 下调 | |
混合光质 | 白光 | 453 | 550 | 先升后降 | 下调 |
暖白光 | 588 | 450 | 先升后降 | 上调 | |
暖黄光 | 595 | 450 | 先升后降 | 先升后降 |
表3 光质类型及VdCHS表达模式
Table 3 Types of light quality and VdCHS expression model
光质类型 Light quality type | 光质名称 Light quality | 光波长 Light wavelength/nm | 表达模式 Expression pattern | ||
---|---|---|---|---|---|
主峰 Main peak | 次峰 Secondary peak | VdCHS2 | VdCHS3 | ||
短波光 | 紫光 | 448 | / | 先升后降 | 先升后降 |
蓝光 | 456 | / | 先升后降 | 先升后降 | |
绿光 | 514 | / | 先升后降 | 先升后降 | |
长波光 | 黄光 | 590 | / | 下调 | 下调 |
红光 | 637 | / | 下调 | 下调 | |
混合光质 | 白光 | 453 | 550 | 先升后降 | 下调 |
暖白光 | 588 | 450 | 先升后降 | 上调 | |
暖黄光 | 595 | 450 | 先升后降 | 先升后降 |
图4 VdCHS在不同光质处理下的表达模式 不同字母表示0.05水平上有显著差异
Fig.4 Expression pattern of VdCHS under different light quality Different letters indicate significant differences at the 0.05 level
图5 靶向CHS的转录因子及多物种共线关系 A:靶向CHS的转录因子类型和数量;B,C:葡萄染色体上靶向CHS2/CHS3的转录因子与其他物种共线基因的数量;At:拟南芥;Os:水稻;Pt:毛果杨。下同
Fig.5 Transcription factors targeting at CHS and collinea-rity analysis in multiple species A:Type and number of transcription factors targeting at CHS. B and C:Number of collinearity transcription factors targeting at CHS2/CHS3 on each chromosome of grapevine with other species. At:Arabidopsis thaliana. Os:Oryza sativa. Pt:Populus trichocarpa. The same below
MYB基因ID MYB gene ID | 注释 Annotation | 染色体 Chromosome | 起始位点 Start | 终止位点 End | 正负链 Strand | 基因长度 Gene length/bp | ORF长度 ORF length/bp | 氨基酸数 Amino acid number | 靶基因 Target gene |
---|---|---|---|---|---|---|---|---|---|
VIT_00s1352g00010 | MYB-RAX3 | Unrandom | 38 747 500 | 38 748 788 | - | 1 057 | 1 020 | 339 | CHS2/CHS3 |
VIT_05s0049g01020 | MYB4 | Chr-5 | 8 046 871 | 8 048 411 | + | 1 212 | 762 | 253 | CHS2 |
VIT_05s0077g01360 | MYB12 | Chr-5 | 1 104 556 | 1 106 597 | + | 1 152 | 1 152 | 383 | CHS2 |
VIT_06s0004g02110 | MYB46 | Chr-6 | 2 573 382 | 2 575 343 | + | 1 017 | 978 | 325 | CHS2/CHS3 |
VIT_07s0005g01210 | Unnamed | Chr-7 | 3 730 465 | 3 732 879 | - | 1 247 | 1 104 | 367 | CHS2 |
VIT_07s0141g00100 | MYB4-like | Chr-7random | 56 798 | 57 999 | - | 999 | 993 | 330 | CHS2/CHS3 |
VIT_08s0056g00800 | MYB60 | Chr-8 | 1 264 742 | 1 266 497 | + | 1 269 | 966 | 321 | CHS3 |
VIT_14s0060g00240 | MYB86 | Chr-14 | 240 314 | 242 056 | - | 1 532 | 1 320 | 439 | CHS2/CHS3 |
VIT_14s0108g00830 | MYB306 | Chr-14 | 29 511 863 | 29 514 026 | - | 1 312 | 984 | 327 | CHS3 |
VIT_14s0219g00050 | MYB315-like | Chr-14 | 26 355 901 | 26 357 181 | + | 935 | 807 | 268 | CHS2 |
VIT_15s0046g03190 | MYB308 | Chr-15 | 19 778 733 | 19 781 642 | - | 1 105 | 909 | 302 | CHS2 |
VIT_19s0085g00050 | Unnamed | Chr-19 | 22 358 835 | 22 361 459 | + | 1 095 | 1 095 | 364 | CHS2 |
VIT_19s0085g00940 | MYB26 | Chr-19 | 23 462 631 | 23 464 270 | + | 1 453 | 1 014 | 337 | CHS2 |
表4 靶向CHS的MYB转录因子信息
Table 4 Information of MYB transcription factors targeting at CHS
MYB基因ID MYB gene ID | 注释 Annotation | 染色体 Chromosome | 起始位点 Start | 终止位点 End | 正负链 Strand | 基因长度 Gene length/bp | ORF长度 ORF length/bp | 氨基酸数 Amino acid number | 靶基因 Target gene |
---|---|---|---|---|---|---|---|---|---|
VIT_00s1352g00010 | MYB-RAX3 | Unrandom | 38 747 500 | 38 748 788 | - | 1 057 | 1 020 | 339 | CHS2/CHS3 |
VIT_05s0049g01020 | MYB4 | Chr-5 | 8 046 871 | 8 048 411 | + | 1 212 | 762 | 253 | CHS2 |
VIT_05s0077g01360 | MYB12 | Chr-5 | 1 104 556 | 1 106 597 | + | 1 152 | 1 152 | 383 | CHS2 |
VIT_06s0004g02110 | MYB46 | Chr-6 | 2 573 382 | 2 575 343 | + | 1 017 | 978 | 325 | CHS2/CHS3 |
VIT_07s0005g01210 | Unnamed | Chr-7 | 3 730 465 | 3 732 879 | - | 1 247 | 1 104 | 367 | CHS2 |
VIT_07s0141g00100 | MYB4-like | Chr-7random | 56 798 | 57 999 | - | 999 | 993 | 330 | CHS2/CHS3 |
VIT_08s0056g00800 | MYB60 | Chr-8 | 1 264 742 | 1 266 497 | + | 1 269 | 966 | 321 | CHS3 |
VIT_14s0060g00240 | MYB86 | Chr-14 | 240 314 | 242 056 | - | 1 532 | 1 320 | 439 | CHS2/CHS3 |
VIT_14s0108g00830 | MYB306 | Chr-14 | 29 511 863 | 29 514 026 | - | 1 312 | 984 | 327 | CHS3 |
VIT_14s0219g00050 | MYB315-like | Chr-14 | 26 355 901 | 26 357 181 | + | 935 | 807 | 268 | CHS2 |
VIT_15s0046g03190 | MYB308 | Chr-15 | 19 778 733 | 19 781 642 | - | 1 105 | 909 | 302 | CHS2 |
VIT_19s0085g00050 | Unnamed | Chr-19 | 22 358 835 | 22 361 459 | + | 1 095 | 1 095 | 364 | CHS2 |
VIT_19s0085g00940 | MYB26 | Chr-19 | 23 462 631 | 23 464 270 | + | 1 453 | 1 014 | 337 | CHS2 |
图6 靶向CHS的MYB在多物种间的共线性分析 A,B:葡萄与拟南芥、水稻、毛果杨靶向CHS2/CHS3的MYB共线关系;C,D:葡萄染色体上靶向CHS2/CHS3的MYB与其他物种共线基因的数量
Fig.6 Collinearity analysis of MYB targeting at the CHS in multiple species A and B:Collinearity of MYB targeting at CHS2/CHS3 between grapevine,Arabidopsis,rice and P. trichocarpa. C and D:Number of collinearity MYB targeting at CHS2/CHS3 on each chromosome of grapevine with other species
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