Cloning of Rd3GT1 in Rhododendron delavayi and Its Effect on Flower Color Formation of Petunia hybrida

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

Abstract

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

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.

Figures/Tables 13

References 30

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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

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）

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）