马缨杜鹃Rd3GT1的克隆及对矮牵牛花色形成的影响

doi:10.13560/j.cnki.biotech.bull.1985.2022-0510

摘要/Abstract

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。

关键词: 马缨杜鹃, 克隆, Rd3GT1, 遗传转化, 花色素苷

Abstract:

Flavonoid 3-O-glycosyltransferase（3GT）is an enzyme at the end of anthocyanin biosynthetic pathway, responsible for transferring glycosylated donors to the 3-OH site of anthocyanidin, and plays an important role in increasing the stability and water-solubility of anthocyanin. To study the function of 3GT in Rhododendron delavayi during anthocyanin biosynthesis would provide the basis for the research on R. delavayi flower color formation and its regulation. The full length CDS of Rd3GT1 was cloned by RT-PCR and analyzed by bioinformatics, then the pBI121-Rd3GT1 plant expression vector was constructed by DNA recombination technology. Subsequently, petunia hybrida was transformed by Agrobacterium-mediated method,and the regenerated plants were identified by transgene detection, phenotype identification, gene expression and anthocyanin detection analysis. The results showed that the full length CDS of Rd3GT1 was 1 395 bp encoding 464 amino acids. Multiple sequence alignment and phylogenetic analysis revealed that Rd3GT1 belonged to 3GT subfamily. Compared with the wild type, the flower color of Rd3GT1 transgenic petunias changed from white to pink, anthocyanin and flavonol accumulations as well as expressions of several flavonoid-related biosynthetic genes significantly increased. In conclusion, Rd3GT1 plays a vital role in anthocyanin synthesis and can be used to improve flower color in other plants.

Key words: Rhododendron delavayi, cloning, Rd3GT1, genetic transformation, anthocyanin

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.

图/表 13

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

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1 Rd3GT1 CDS的克隆 M：DNA maker 2000;1和2：扩增片段

Fig. 1 Cloning of Rd3GT1 CDS M：DNA maker 2000. 1 and 2：Amplified fragment

图2 多序列比对分析红色横线表示PSPG box

Fig. 2 Multiple sequence alignment analysis The red line indicates PSPG box

图3 系统进化分析红色横线标记的为Rd3GT1

Fig. 3 Phylogenetic analysis Rd3GT1 is marked by the red line

图4 酶切验证 M：DNA maker III;1: 质粒;2：酶切产物

Fig. 4 Enzyme digestion verification M：DNA maker III. 1: Plasmid. 2：Product of enzyme digestion

图5 PCR验证 M：DNA maker 2000;1-3：扩增片段

Fig. 5 PCR verification M：DNA maker 2000. 1-3：Amplified fragment

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7 转基因植株的表型改变与RT-PCR分析 A：表型改变;B：RT-PCR分析

Fig. 7 Phenotypic changes and RT-PCR analysis of transgenic plants A：Phenotypic changes. B：RT-PCR analysis

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

参考文献 30

[1]	Falcone Ferreyra ML, Rius SP, Casati P. Flavonoids: biosynthesis, biological functions, and biotechnological applications[J]. Front Plant Sci, 2012, 3: 222. doi: 10.3389/fpls.2012.00222 pmid: 23060891
[2]	Forkmann G, Martens S. Metabolic engineering and applications of flavonoids[J]. Curr Opin Biotechnol, 2001, 12(2): 155-160. doi: 10.1016/S0958-1669(00)00192-0 URL
[3]	Li D, Chen G, Ma B, et al. Metabolic profiling and transcriptome analysis of mulberry leaves provide insights into flavonoid biosynthesis[J]. J Agric Food Chem, 2020, 68(5): 1494-1504. doi: 10.1021/acs.jafc.9b06931 URL
[4]	Sharma M, Cortes-Cruz M, Ahern KR, et al. Identification of the Pr1 gene product completes the anthocyanin biosynthesis pathway of maize[J]. Genetics, 2011, 188(1): 69-79. doi: 10.1534/genetics.110.126136 pmid: 21385724
[5]	Katayama-Ikegami A, Byun Z, Okada S, et al. Characterization of the recombinant UDP: flavonoid 3-O-galactosyltransferase from Mangifera indica ‘Irwin'(MiUFGalT3)involved in skin coloring[J]. Hortic J, 2020, 89(5): 516-524. doi: 10.2503/hortj.UTD-201 URL
[6]	Tohge T, Nishiyama Y, Hirai MY, et al. Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor[J]. Plant J, 2005, 42(2): 218-235. doi: 10.1111/j.1365-313X.2005.02371.x URL
[7]	Yamazaki M, Yamagishi E, Gong ZZ, et al. Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning, biochemical properties and developmentally regulated expression[J]. Plant Mol Biol, 2002, 48(4): 401-411. pmid: 11905966
[8]	Sun W, Liang LJ, Meng XY, et al. Biochemical and molecular characterization of a flavonoid 3-O-glycosyltransferase responsible for anthocyanins and flavonols biosynthesis in Freesia hybrida[J]. Front Plant Sci, 2016, 7: 410.
[9]	Wang XQ. Structure, mechanism and engineering of plant natural product glycosyltransferases[J]. FEBS Lett, 2009, 583(20): 3303-3309. doi: 10.1016/j.febslet.2009.09.042 pmid: 19796637
[10]	le Roy J, Huss B, Creach A, et al. Glycosylation is a major regulator of phenylpropanoid availability and biological activity in plants[J]. Front Plant Sci, 2016, 7: 735. doi: 10.3389/fpls.2016.00735 pmid: 27303427
[11]	吴福建, 李凤兰, 黄凤兰, 等. 杜鹃花研究进展[J]. 东北农业大学学报, 2008, 39(1): 139-144.
	Wu FJ, Li FL, Huang FL, et al. Research progress on Rhododendron[J]. J Northeast Agric Univ, 2008, 39(1): 139-144.
[12]	Griesbach RJ, Asen S, Leonnarat BA. Petunia hybrida anthocyanins acylated with caffeic acid[J]. Phytochemistry, 1991, 30(5): 1729-1731. doi: 10.1016/0031-9422(91)84250-V URL
[13]	Sun W, Meng XY, Liang LJ, et al. Overexpression of a Freesia hybrida flavonoid 3-O-glycosyltransferase gene, Fh3GT1, enhances transcription of key anthocyanin genes and accumulation of anthocyanin and flavonol in transgenic Petunia(Petunia hybrida)[J]. Vitro Cell Dev Biol Plant, 2017, 53(5): 478-488. doi: 10.1007/s11627-017-9836-3 URL
[14]	Downey MO, Harvey JS, Robinson SP. Analysis of tannins in seeds and skins of Shiraz grapes throughout berry development[J]. Aust J Grape Wine Res, 2003, 9(1): 15-27. doi: 10.1111/j.1755-0238.2003.tb00228.x URL
[15]	Griesbach RJ, Asen S. Characterization of the flavonol glycosides in Petunia[J]. Plant Sci, 1990, 70(1): 49-56. doi: 10.1016/0168-9452(90)90031-I URL
[16]	Fukuchi-Mizutani M, Okuhara H, Fukui Y, et al. Biochemical and molecular characterization of a novel UDP-glucose: anthocyanin 3'-O-glucosyltransferase, a key enzyme for blue anthocyanin biosynthesis, from Gentian[J]. Plant Physiol, 2003, 132(3): 1652-1663. pmid: 12857844
[17]	Tanaka Y, Yonekura K, Fukuchi-Mizutani M, et al. Molecular and biochemical characterization of three anthocyanin synthetic enzymes from Gentiana triflora[J]. Plant Cell Physiol, 1996, 37(5): 711-716. pmid: 8819318
[18]	Ford CM, Boss PK, Hoj PB. Cloning and characterization of Vitis vinifera UDP-glucose: flavonoid 3-O-glucosyltransferase, a homologue of the enzyme encoded by the maize Bronze-1 locus that may primarily serve to glucosylate anthocyanidins in vivo[J]. J Biol Chem, 1998, 273(15): 9224-9233. doi: 10.1074/jbc.273.15.9224 pmid: 9535914
[19]	Modolo LV, Li LN, Pan HY, et al. Crystal structures of glycosyltransferase UGT78G1 reveal the molecular basis for glycosylation and deglycosylation of(Iso)flavonoids[J]. J Mol Biol, 2009, 392(5): 1292-1302. doi: 10.1016/j.jmb.2009.08.017 pmid: 19683002
[20]	Davies K, Schwinn K, Deroles S, et al. Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase[J]. Euphytica, 2003, 131: 259-268. doi: 10.1023/A:1024018729349 URL
[21]	Meyer P, Heidmann I, Forkmann G, et al. A new Petunia flower colour generated by transformation of a mutant with a maize gene[J]. Nature, 1987, 330(6149): 677-678. doi: 10.1038/330677a0 URL
[22]	Owens DK, Alerding AB, Crosby KC, et al. Functional analysis of a predicted flavonol synthase gene family in Arabidopsis[J]. Plant Physiol, 2008, 147(3): 1046-1061. doi: 10.1104/pp.108.117457 URL
[23]	Gou JY, Felippes FF, Liu CJ, et al. Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor[J]. Plant Cell, 2011, 23(4): 1512-1522. doi: 10.1105/tpc.111.084525 URL
[24]	Wang CK, Chen PY, Wang HM, et al. Cosuppression of tobacco Chalcone synthase using Petunia Chalcone synthase constructs results in white flowers[J]. Bot Stud, 2006, 47: 71-82.
[25]	Nishihara M, Nakatsuka T, Yamamura S. Flavonoid components and flower color change in transgenic tobacco plants by suppression of Chalcone isomerase gene[J]. FEBS Lett, 2005, 579(27): 6074-6078. pmid: 16226261
[26]	Forkmann G, Stotz G. Selection and characterisation of flavanone 3-hydroxylase mutants of Dahlia, Streptocarpus, Verbena and Zinnia[J]. Planta, 1984, 161(3): 261-265. doi: 10.1007/BF00982923 pmid: 24253654
[27]	Zuker A, Tzfira T, Ben-Meir H, et al. Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene[J]. Molecular Breeding, 2002, 9:33-41. doi: 10.1023/A:1019204531262 URL
[28]	Zhao ZC, Hu GB, Hu FC, et al. The UDP glucose: flavonoid-3-O-glucosyltransferase(UFGT)gene regulates anthocyanin biosynthesis in Litchi(Litchi chinesis Sonn.)during fruit coloration[J]. Mol Biol Rep, 2012, 39(6): 6409-6415. doi: 10.1007/s11033-011-1303-3 URL
[29]	Aharoni A, de Vos CH, Wein M, et al. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco[J]. Plant J, 2001, 28(3): 319-332. pmid: 11722774
[30]	Nakatsuka T, Yamada E, Saito M, et al. Heterologous expression of Gentian MYB1R transcription factors suppresses anthocyanin pigmentation in tobacco flowers[J]. Plant Cell Rep, 2013, 32(12): 1925-1937. doi: 10.1007/s00299-013-1504-4 pmid: 24037114