滇牡丹PdANS的克隆、表达及与花青素含量的相关性

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

摘要/Abstract

摘要：

花色是观赏植物最重要的品质性状之一，在植物生长发育过程中发挥着重要作用。探明滇牡丹的花色调控机理，为提高观赏价值奠定理论基础。以花瓣为材料，克隆获得PdANS，生物信息学分析其特征，结合实时荧光定量PCR、HPLC等技术探究不同组织、不同发育时期中PdANS的表达情况与各组织花青素含量的相关性。结果表明，PdANS全长1 121 bp，包含一个1 064 bp的开放阅读框（ORF），编码354个氨基酸，相对分子质量40.41 kD，理论等电点（pI）5.48，分子式为C₁₈₁₉H₂₈₇₆N₄₇₄O₅₄₀S₁₂，脂肪指数为88.64，不稳定指数（II）为55.32，亲水平均数为-0.435，推测为不稳定的亲水蛋白。且无信号肽序列及跨膜螺旋区，是没有分泌功能的非跨膜蛋白。系统进化分析显示，滇牡丹PdANS与牡丹、芍药等植物的ANS蛋白亲缘关系最近。RT-qPCR结果显示，PdANS在花瓣、花药、萼片、苞片和花梗中均有表达，以花瓣中的表达量最高。HPLC结果表明，在不同组织提取液中，分别检测出Cy3G5G、Pn3G5G、Cy3G和Pn3G四种花青素，且花青素含量花瓣>花药>萼片>花梗>苞片。相关性分析表明，花青素含量与PdANS表达量呈极显著相关性。推测PdANS在滇牡丹花色的形成中扮演着重要角色，参与花青素物质的生物合成。

关键词: 滇牡丹, ANS基因, 花青素, 生物信息学分析, HPLC

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

平怀磊, 郭雪, 余潇, 宋静, 杜春, 王娟, 张怀璧. 滇牡丹PdANS的克隆、表达及与花青素含量的相关性[J]. 生物技术通报, 2023, 39(3): 206-217.

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.

图/表 17

图1 滇牡丹各时期的划分阶段 A-1：滇牡丹红花（S1-S3）；B-1：滇牡丹紫花（S1-S3）；C-1：滇牡丹红斑黄花（S1-S3）

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）

表1 PdANS的克隆和qPCR引物分析

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

图2 滇牡丹RNA琼脂糖凝胶电泳检测 A：滇牡丹花瓣RNA；B：紫花其余组织RNA；M：BM2000+ DNA marker

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

图3 PdANS全长cDNA的克隆 1-3：滇牡丹PdANS的PCR扩增产物；M：BM2000+ DNA marker

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

图4 滇牡丹PdANS蛋白亲水性分析

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

图5 PdANS蛋白的信号肽预测

Fig. 5 Signal peptide prediction of PdANS protein

图6 PdANS蛋白跨膜结构域分析

Fig. 6 Analysis of transmembrane domains of PdANS protein

图7 PdANS保守结构域分析

Fig. 7 Analysis of conserved domains of PdANS

图8 PdANS与其他植物ANS氨基酸序列的多重比对分析 PdANS：滇牡丹；PlANS1：黄牡丹，ALX36000.1；PsANS：牡丹，AIU98515.1；PlANS：芍药，QIC54082.1；TcANS：可可，XP_007040068.2；DzANS：榴莲，XP_022732774.1；CoANS：长蒴黄麻，OMO81016.1；VaANS：野葡萄，ACN38270.1；NgANS：大齿牛果藤，AGO02175.1。下划线表示2OG-Fe II_Oxy蛋白功能域

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

图9 PdANS与其他植物ANS系统进化分析

Fig. 9 Phylogenetic tree of PdANS with other plant ANSs

图10 PdANS的表达模式 A：花瓣中的表达模式；B：紫花滇牡丹其余组织的表达模式。S1：硬蕾期；S2：透色期；S3：盛开期。不同小写字母表示显著差异（P<0.05）。下同

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

表2 4种花青素的回归方程

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

图11 紫花花瓣的花青素分布（A）及含量（B）分析 Cy3G5G：矢车菊素-3,5二葡萄糖苷；Pn3G5G：芍药素-3,5二葡萄糖苷；Cy3G：矢车菊素-3-O-葡萄糖苷；Pn3G：芍药素-3-O-葡萄糖苷。下同

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

图12 红斑黄花花瓣的花青素分布（A）及含量（B）分析

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

图13 红花花瓣的花青素分布（A）及含量（B）分析

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

图14 紫花其余组织的花青素分布（A）及含量（B）分析

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

表3 花青素含量与PdANS表达量的相关性分析

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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