生物技术通报 ›› 2022, Vol. 38 ›› Issue (9): 198-206.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0510
孙威1(), 张艳1, 王聿晗1, 徐僡1, 徐小蓉1(), 鞠志刚2()
收稿日期:
2022-04-25
出版日期:
2022-09-26
发布日期:
2022-10-11
作者简介:
孙 威,女,博士,副教授,研究方向:植物生物技术;E-mail: 基金资助:
SUN Wei1(), ZHANG Yan1, WANG Yu-han1, XU Hui1, XU Xiao-rong1(), JU Zhi-gang2()
Received:
2022-04-25
Published:
2022-09-26
Online:
2022-10-11
摘要:
类黄酮3-O-糖基转移酶(3GT)是花色素苷生物合成途径末端的酶,负责将糖基供体转移至花青素的3-OH位置,在增加花色素苷的稳定性与水溶性方面发挥重要作用。研究马缨杜鹃3GT在花色素苷生物合成中的功能,为马缨杜鹃花色形成及调控研究奠定基础。运用 RT-PCR 技术克隆获得马缨杜鹃3GT基因(Rd3GT1)全长CDS序列,并对其进行生物信息学分析,再利用DNA重组技术完成植物表达载体pBI121-Rd3GT1的构建,利用农杆菌介导法对矮牵牛进行遗传转化,同时对再生植株进行转基因及表型鉴定,并完成基因表达量及花色素苷检测分析。结果表明,Rd3GT1全长CDS序列为1 395 bp,编码464个氨基酸。蛋白多序列比对和系统进化分析表明,Rd3GT1属于3GT家族成员。与野生型相比,转Rd3GT1的矮牵牛植株花色由白色变为粉色,花色素苷与黄酮醇的积累显著增加,同时多个类黄酮合成相关基因表达显著升高。综上,Rd3GT1在花色素苷合成过程中发挥重要功能,可用于其它植物花色改良研究。
孙威, 张艳, 王聿晗, 徐僡, 徐小蓉, 鞠志刚. 马缨杜鹃Rd3GT1的克隆及对矮牵牛花色形成的影响[J]. 生物技术通报, 2022, 38(9): 198-206.
SUN Wei, ZHANG Yan, WANG Yu-han, XU Hui, XU Xiao-rong, JU Zhi-gang. Cloning of Rd3GT1 in Rhododendron delavayi and Its Effect on Flower Color Formation of Petunia hybrida[J]. Biotechnology Bulletin, 2022, 38(9): 198-206.
引物名称Primer name | 引物序列Primer sequence(5'-3') | 引物用途Function of primer |
---|---|---|
Rd3GT-1 F1 | ACCAAACAAATACTGTAATAAT | 基因克隆 |
Rd3GT-1 R1 | GATTACACCCATCTTTTATTCA | Gene cloning |
Rd3GT-121F | GCTCTAGA ATGACCAAAAATATCTCA | 载体构建 |
Rd3GT-121R | CGGGATCCCTAAAGATTGTACCCTGC | Vector construction |
PhCHSA-F | GGCGCGATCATTATAGGTTC | 表达分析 |
PhCHSA-R | TTTGAGATCAGCCCAGGAAC | Expression analysis |
PhCHI-F | TACGGCGATAGGTGTGTATC | 表达分析 |
PhCHI-R | GGCAAGATCGTAGTAACTCG | Expression analysis |
PhF3H-F | ACTTGGATCACTGTTCAGCC | 表达分析 |
PhF3H-R | ATACACTATCGCCTCTGGTG | Expression analysis |
PhDFR-F | GCTATCATCTACGATGTGGC | 表达分析 |
PhDFR-R | TGTCGACAAGTATCGATGGC | Expression analysis |
PhANS-F | TACCTGAGACTGTCACTGAG | 表达分析 |
PhANS-R | GCAGTATCCAGTTCATCCTC | Expression analysis |
Ph3GT-F | GCAGTGGCAGAAGCATTAGA | 表达分析 |
Ph3GT-R | CACATGATATGCCCTCCAAA | Expression analysis |
PhAN11-F | GCCGCATTGCCGTGGGTAG | 表达分析 |
PhAN11-R | GGGATTGGGTTTAGGGTTAGGGTTTC | Expression analysis |
PhAN1-F | TCTGCCGGCGAATCAAATCAA | 表达分析 |
PhAN1-R | GTCTGTACGCGGGCACTCTTAGC | Expression analysis |
PhJAF13-F | ACGGATGATAATATGAGTAACGGTGTGC | 表达分析 |
PhJAF13-R | CTTGATGGTCTAGTGGGGCAGGC | Expression analysis |
PhAN2-F | GATGGACTTCAATGGTGGGCCAAT | 表达分析 |
PhAN2-R | CGATGGTGCTGTTTCCTCATGCAA | Expression analysis |
PhMYBx-F | GTGGCTCCTCGGATGTTAGTTTCA | 表达分析 |
PhMYBx-R | GACCACCTCTCGCCAACCAAATTA | Expression analysis |
PhActin2-F | CCTGATGAAGATCCTCACCGA | 表达分析 |
PhActin2-R | CAAGAGCCACATAGGCAAGCT | Expression analysis |
表1 本文所用引物列表
Table 1 Primers used in this study
引物名称Primer name | 引物序列Primer sequence(5'-3') | 引物用途Function of primer |
---|---|---|
Rd3GT-1 F1 | ACCAAACAAATACTGTAATAAT | 基因克隆 |
Rd3GT-1 R1 | GATTACACCCATCTTTTATTCA | Gene cloning |
Rd3GT-121F | GCTCTAGA ATGACCAAAAATATCTCA | 载体构建 |
Rd3GT-121R | CGGGATCCCTAAAGATTGTACCCTGC | Vector construction |
PhCHSA-F | GGCGCGATCATTATAGGTTC | 表达分析 |
PhCHSA-R | TTTGAGATCAGCCCAGGAAC | Expression analysis |
PhCHI-F | TACGGCGATAGGTGTGTATC | 表达分析 |
PhCHI-R | GGCAAGATCGTAGTAACTCG | Expression analysis |
PhF3H-F | ACTTGGATCACTGTTCAGCC | 表达分析 |
PhF3H-R | ATACACTATCGCCTCTGGTG | Expression analysis |
PhDFR-F | GCTATCATCTACGATGTGGC | 表达分析 |
PhDFR-R | TGTCGACAAGTATCGATGGC | Expression analysis |
PhANS-F | TACCTGAGACTGTCACTGAG | 表达分析 |
PhANS-R | GCAGTATCCAGTTCATCCTC | Expression analysis |
Ph3GT-F | GCAGTGGCAGAAGCATTAGA | 表达分析 |
Ph3GT-R | CACATGATATGCCCTCCAAA | Expression analysis |
PhAN11-F | GCCGCATTGCCGTGGGTAG | 表达分析 |
PhAN11-R | GGGATTGGGTTTAGGGTTAGGGTTTC | Expression analysis |
PhAN1-F | TCTGCCGGCGAATCAAATCAA | 表达分析 |
PhAN1-R | GTCTGTACGCGGGCACTCTTAGC | Expression analysis |
PhJAF13-F | ACGGATGATAATATGAGTAACGGTGTGC | 表达分析 |
PhJAF13-R | CTTGATGGTCTAGTGGGGCAGGC | Expression analysis |
PhAN2-F | GATGGACTTCAATGGTGGGCCAAT | 表达分析 |
PhAN2-R | CGATGGTGCTGTTTCCTCATGCAA | Expression analysis |
PhMYBx-F | GTGGCTCCTCGGATGTTAGTTTCA | 表达分析 |
PhMYBx-R | GACCACCTCTCGCCAACCAAATTA | Expression analysis |
PhActin2-F | CCTGATGAAGATCCTCACCGA | 表达分析 |
PhActin2-R | CAAGAGCCACATAGGCAAGCT | Expression analysis |
培养基名称 Name of culture medium | 培养基配方 Ingredient culture |
---|---|
共培养培养基 Cocultivation medium | MS基本培养基+BA(1 mg/L)+NAA(0.1 mg/L)+AS(20 mg/L) |
脱菌培养基 Bacteria-free medium | MS基本培养基+BA(1mg/L)+NAA(0.1 mg/L)+cef(400 mg/L) |
筛选培养基 Medium for screening | MS基本培养基+BA(1 mg/L)+NAA(0.1 mg/L)+cef(400 mg/L)+Kan(50 mg/L) |
生根培养基 Medium for growing roots | 1/2 MS基本培养基+NAA(0.3 mg/L)+cef(400 mg/L)+Kan(50 mg/L) |
表2 矮牵牛遗传转化培养基成分
Table 2 A variety of culture medium in the genetic transf-ormation experiment of P. hybrida
培养基名称 Name of culture medium | 培养基配方 Ingredient culture |
---|---|
共培养培养基 Cocultivation medium | MS基本培养基+BA(1 mg/L)+NAA(0.1 mg/L)+AS(20 mg/L) |
脱菌培养基 Bacteria-free medium | MS基本培养基+BA(1mg/L)+NAA(0.1 mg/L)+cef(400 mg/L) |
筛选培养基 Medium for screening | MS基本培养基+BA(1 mg/L)+NAA(0.1 mg/L)+cef(400 mg/L)+Kan(50 mg/L) |
生根培养基 Medium for growing roots | 1/2 MS基本培养基+NAA(0.3 mg/L)+cef(400 mg/L)+Kan(50 mg/L) |
图6 矮牵牛的遗传转化过程 A:脱菌培养;B:筛选培养;C:继代培养;D:生根培养
Fig. 6 Genetic transformation process of P. hybrid A:Bacteria-free culture. B:Screening culture. C:Subculture. D:Rooting culture
图8 转基因花朵中花色素苷与黄酮醇成分分析 A, D:野生型;B, E:Rd3GT1-4转基因植株;C, F:Rd3GT1-5转基因植株
Fig. 8 Analysis of anthocyanin and flavonol components in transgenic flowers A, D:Wild type. B, E:Rd3GT1-4 transgenic. C, F:Rd3GT1-5 transgenic
图9 转基因花朵中花色素苷与黄酮醇的定量分析 A:花色素苷;B:黄酮醇。a,b,c表示 0.01 水平上极显著差异
Fig. 9 Quantitative analysis of anthocyanin and flavonol in transgenic flowers A:Anthocyanin. B:Flavonol. a, b, c indicate very significant difference at the 0.01 level
图10 转基因矮牵牛中类黄酮合成相关基因的表达分析 A:结构基因;B:转录因子;**表示 0.01 水平上极显著差异
Fig. 10 Expression profiles of flavonoid-related biosynth-etic genes in flowers of transgenic petunia A:Structure gene. B:Transcription factor. ** indicates very significant difference at the 0.01 level
图11 Rd3GT1调节转基因矮牵牛花色的建议模式图 实线箭头表示基因表达量显著升高;虚线箭头表示基因表达量无显著变化
Fig. 11 Proposed model for regulation of transgenic petu-nia flower color by Rd3GT1 The solid arrows indicate the gene expression levels significantly increased. The dotted arrows indicate there is no significant change in gene expression levels
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