Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (24): 5091-5103.doi: 10.3864/j.issn.0578-1752.2020.24.012

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Function Characterization of Chalcone Synthase Gene AgCHS1 in Ampelopsis grossedentata

XU Ming(),LIN ShiQiang,NI DongXin,YI HenJie,LIU JiangHong,YANG ZhiJian,ZHENG JinGui()   

  1. College of Agriculture, Fujian Agriculture and Forestry University/Key Laboratory of Crop Biotechnology in Fujian Province University/Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fuzhou 350002
  • Received:2020-05-14 Accepted:2020-07-15 Online:2020-12-16 Published:2020-12-28
  • Contact: JinGui ZHENG E-mail:xmfau@163.com;jgzheng@fafu.edu.cn

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Abstract:

【Objective】Chalcone synthase(CHS) is the first key enzyme in the biosynthesis of plant flavonoids. In this study, the AgCHS1 was cloned from Ampelopsis grossedentata, the gene sequence and tissue specific expression were analyzed, and the gene function was characterized via in vitro enzymatic activity assay and transformation of N. tabacum, aiming to provide a theoretical basis for further elucidating the flavonoid accumulation of the A. grossedentata. 【Method】The primers were designed according to the transcriptome of A. grossedentata and the leaf cDNA and genomic DNA were used as templates to amplify the AgCHS1 with PCR. The sequence features were analyzed with bioinformatic methods. The MEGA6 and DNAMAN were used for multiple sequences alignment and phylogenetic tree construction. The AgCHS1 recombinant protein was obtained via prokaryotic expression, and the enzymatic activities of the protein on substrates were assayed by identifying the catalytic products with HPLC-MS. The expression of AgCHS1 in different organs was quantified by qRT-PCR, and the content of total flavonoids was determined by Al(NO3)3 colorimetric method. The overexpression vector was constructed and transformed to N. tabacum by using the leaf disk method. The transgenic plants were screened and the contents of anthocyanin and flavonol in the petal of T2 strain were measured.【Result】The ORF of AgCHS1 had a length of 1 182 bp and encoded 393 amino acids. The genomic DNA spanned 1 315 bp, containing 2 exons and 1 intron. The bioinformatic analysis showed that AgCHS1 was a stable hydrophilic protein. The results of sequence alignments indicated that the AgCHS1 possessed the signature sequence and enzymatic site residues of the chalcone synthase gene superfamily, including the binding site of malonyl-CoA and triple active center, which share a high similarity with the CHS proteins of other species. The phylogenetic analysis showed that AgCHS1 had a close relationship with and was in the same clade with the CHSs of Vitis vinifera and Vitis amurensis. The results of fluorescent qPCR showed that AgCHS1 expressed highest in the mature leaf and the flower, and lowest in the old leaf. There was a positive correlation between the expression level of AgCHS1 gene and the content of total flavonoids in different organs of A.grossedentata. The in vitro enzymatic analysis showed that the recombinant AgCHS1 was able to catalyze the substrates p-coumaroyl-CoA and CoA malonyl-CoA to produce naringenin, demonstrating the activity of chalcone synthase. 5 transgenic N. tabacum lines were obtained, 2 of which showed a significant deeper leaf color. The anthocyanin contented of the transgenic lines OE3 and OE4 increased by 56.6% and 25.3%, respectively, compared to the control. The flavonol content of line OE3 showed no significant difference, which of the line OE4 were increased by 39.1%.【Conclusion】The AgCHS1 was the key enzyme in the flavonoid biosynthesis of A. grossedentata, and the overexpression of AgCHS1 increased the contents of anthocyanin and flavonol in transgenic plants.

Key words: Ampelopsis grossedentata, chalcone synthetase, enzyme activity, anthocyanin, flavonol

Table 1

List of primers used in this study"

引物名称 Primer name 序列（5'-3'） Primer sequence (5'-3') 用途 Purpose
AgCHS1-F GGATCCATGGTGTCGGTGCAGGAAATCAGA ORF序列扩增、载体构建
AgCHS1-R GAGCTCTCAGTGAGCCAGTGGTGCAGAC Amplification of ORF sequence and vector construction
AgCHS1-P1 ACTCCAGCCAACTGTGTCCA 转基因烟草的PCR鉴定
AgCHS1-P2 TGAGACCCACTTCACGCAA PCR identification of transgenic tobacco
AgCHS1-qF GCCTCAAACCCTCCGTCA 实时荧光定量PCR Quantitative real-time PCR
AgCHS1-qR GACCCACGAGTGAATCCAAGT Quantitative real-time PCR
NtEF1α-qF AATTTTGACCAAGATCGACAGG 烟草内参基因 Control
NtEF1α-qR CAGCAACAGTTTGACGCATG Reference gene in tobacco
AgGAPDH-qF CATCTCAGCCCCAAGCAA 藤茶内参基因
AgGAPDH-qR GTGGCAGTAATGGAGTGAACAG Reference gene in A.grossedentata

Fig. 1

Cloning and structure analysis of the AgCHS1 A: PCR amplification product of AgCHS1, M: DL5000; 1: The amplification product of ORF; 2: The amplification product of genomic DNA; B: Structure of AgCHS1"

Fig. 2

Amino acid sequence alignment of AgCHS1 with other CHS enzymes"

Fig. 3

Phylogenetic tree analysis of AgCHS2 protein with other plant CHS proteins"

Fig. 4

The change of relative expression of AgCHS1 and total flavonoid content in different organs of A. grossedentata Different letters indicate statistical difference (P<0.05). The same as below"

Fig. 5

Ni column purification of recombinant AgCHS1 protein expressed in E. coli M: Protein Marker; 1: Centrifuged supernatant; 2: Flow through; 3: binding buffer elution; 4: Binding buffer + 60 mmol?L-1 imidazole elution; 5: Binding buffer + 90 mmol?L-1 imidazole elution; 5: Binding buffer + 250 mmol?L-1 imidazole elution"

Fig. 6

HPLC-MS identification of in vitro enzymatic reaction product of recombinant protein AgCHS1 HPLC chromatograms of the products in the reactions of AgCHS1 recombinant protein (A); naringen instandard (B) and the empty control (C); D-E: The masss pectrometric spectrum of reaction products; F-G: The mass spectrometric spectrum of the naringenin standard"

Fig. 7

Efects of overexpression AgCHS1 on the color, anthocyanin and flavonol content of tobacco A: PCR identification of transgenic tobacco; B: Petal phenotypes; C: AgCHS1 expression in petal; D: Relative anthocyanin content; E: Relative flavonol content; M: DNA marker DL1000; P: Plasmid control; WT: Wild type; OE1-5: different transgenic lines. Different letters indicate statistical difference (P<0.05)"

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