Cloning and Expression of PdANS in Paeonia delavayi and Correlation with Anthocyanin Content

doi:10.13560/j.cnki.biotech.bull.1985.2022-0532

Abstract

Abstract:

Flower color is one of the most important quality traits of ornamental plants and plays an important role in the process of plant growth and development. Exploring the mechanism of flower colors regulation in Paeonia delavayi lays the theoretical foundation for improving the ornamental value. The petals were used as the material to clone PdANS, using bioinformatics ally to analyze its characteristics, and combined with real-time fluorescence quantitative PCR and HPLC to investigate the correlation between the expressions of PdANS in different tissues, different developmental periods and the anthocyanin content of each tissue. The results showed that the full length of PdANS was 1 121 bp, including an open reading frame（ORF）of 1 064 bp, encoding 354 amino acids, the relative molecular mass was 40.41 kD, the theoretical isoelectric point（pI）was 5.48, the molecular formula was C₁₈₁₉H₂₈₇₆N₄₇₄O₅₄₀S₁₂, the aliphatic index was 88.64, the instability index（II）was 55.32, and the average hydrophilicity was -0.435. It was presumed that it was an unstable hydrophilic protein. And there was no signal peptide sequence and transmembrane helix region, which was a non-transmembrane protein without secretory function. The phylogenetic analysis showed that PdANS of P. delavayi was most closely related to the ANS proteins of P. suffruticosa, Paeonia lactiflora and other plants. The results of the RT-qPCR revealed that PdANS was expressed in the petals, anthers, sepals, bracts and pedicels, with the highest expression in petals. The results of HPLC showed that Cy3G5G, Pn3G5G, Cy3G and Pn3G were detected in the extracts of different tissues, with the content of the anthocyanin of petals > anthers > sepals > pedicels > bracts. The correlation analysis showed that the anthocyanin contents were highly significantly correlated with the expression of PdANS. It is speculated that PdANS plays an important role in formation of flower color in P. delavayi and is involved in the biosynthesis of anthocyanin substances.

Key words: Paeonia delavayi, ANS gene, anthocyanin, bioinformatics analysis, HPLC

PING Huai-lei, GUO Xue, YU Xiao, SONG Jing, DU Chun, WANG Juan, ZHANG Huai-bi. Cloning and Expression of PdANS in Paeonia delavayi and Correlation with Anthocyanin Content[J]. Biotechnology Bulletin, 2023, 39(3): 206-217.

Figures/Tables 17

Fig. 1 The division of different periods of Paeonia delavayi A-1: Red flower of Paeonia delavayi（S1-S3）. B-1: Purple flower of Paeonia delavayi（S1-S3）. C-1: Yellow flower with red spots of Paeonia delavayi（S1-S3）

Table 1 Primers for PdANS gene cloning and qPCR analysis

引物名称Primer name	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
PdANS-ORF	AAACAGAGAGTAAACGTTGCTG	TATAAATCCATCCCGGCCCA
PdANS-RT	ATCCTCCACAACATGGTGCC	TCACCAATCCCCTGTGAAGA
GAPDH-RT	CACCACTAACTGTCTTGCCC	GCTGCTGGGAATAATGTTG

Fig. 2 P. delavayi RNA agarose gel electrophoresis detection A: Petal RNA of P. delavayi. B: RNA from other tissues of purple flower. M: BM2000+ DNA marker

Fig. 3 Cloning of full length cDNA of PdANS gene 1-3: PCR products of PdANS. M: BM2000+ DNA marker

Fig. 4 Hydropilia analysis of PdANS protein from P. delavayi

Fig. 5 Signal peptide prediction of PdANS protein

Fig. 6 Analysis of transmembrane domains of PdANS protein

Fig. 7 Analysis of conserved domains of PdANS

Fig. 8 Multiple alignment and analysis of amino acid sequence PdANS with ANS from other plants PdANS: Paeonia delavayi. PlANS1: Paeonia lutea, ALX36000.1. PsANS: Paeonia suffruticosa, AIU98515.1. PlANS: Paeonia lactiflora, QIC54082.1. TcANS: Theobroma cacao, XP_007040068.2. DzANS: Durio zibethinus, XP_022732774.1. CoANS: Corchorus olitorius, OMO81016.1. VaANS: Vitis amurensis, ACN38270.1. NgANS: Nekemias grossedentata, AGO02175.1. The underscore indicates the 2OG-Fe II_Oxy protein domain

Fig. 9 Phylogenetic tree of PdANS with other plant ANSs

Fig. 10 Expression pattern of PdANS A: Patterns of expression in petals. B: Expression patterns in other tissues of the purple flower Paeonia delavayi. S1: Unpigmented tight bud. S2: Slightly pigmented bud. S3: Fully opened flower. Different lowercase letters indicate significant difference（P<0.05）. The same below

Table 2 Regression equations of four anthocyanins

标准品 Standard	标准曲线 Calibration curve	R²
矢车菊素-3,5二葡萄糖苷（Gy3G5G）	y1=0.925 4x-1.247 1	0.999 8
芍药素-3,5二葡萄糖苷（Pn3G5G）	y2 =1.238 4x-59.599	0.999 3
矢车菊素-3-O-葡萄糖苷（Cy3G）	y3 =5.795x-77.033	0.999 6
芍药素-3-O-葡萄糖苷（Pn3G）	y4 =4.831 5x-12.906	0.999 9

Fig. 11 Analysis of anthocyanin distribution（A）and content（B）in purple flower petals Cy3G5G: Cyanidin-3,5-diglucoside. Pn3G5G: Peonidin-3,5-diglucoside. Cy3G: Cyanidin-3-O-glucoside. Pn3G: Peonidin-3-O-glucoside. The same below

Fig. 12 Analysis of anthocyanin distribution（A）and content（B） in yellow flower petals with red spots

Fig. 13 Analysis of anthocyanin distribution（A）and content（B）in red flower petals

Fig. 14 Analysis of the distribution（A）and content（B）of anthocyanins in the rest of the purple flowers

Table 3 Correlation analysis between anthocyanin content and PdANS gene expression

项目 Item	PdANS	花青素含量 Anthocyanin content
PdANS	1	0.436**
花青素含量 Anthocyanin content	0.436**	1

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