Cloning and Expression Analysis of AwANS Genes in Allium wallichii

doi:10.13560/j.cnki.biotech.bull.1985.2024-1004

Abstract

Abstract:

Objective Allium wallichii is a characteristic alpine flower. Anthocyanidin synthase (ANS) is a key enzyme in the anthocyanin biosynthesis, which lies downstream of flavonoid biosynthesis. This study is to investigate the role of ANS in the flower color formation of purple and white A. wallichii. Method pH-differential method was used to determine the total anthocyanin contents at different developmental stage of flowers. The coding sequences of AwANS genes were cloned by RT-PCR. The sequence alignments and expression patterns of AwANSs were also analyzed. Result The total anthocyanin contents of purple flowers increased gradually with flower development and peaked at S4, while those of white flowers could barely be measured. Two sequences of AwANS (AwANS^pa and AwANS^pb ) were cloned from white A. wallichii flowers and both of them had the CDS sequence length of 1 074 bp CDS with 357 amino acids. However, there were five sequences of AwANS (AwANS^wa, AwANS^wb, AwANS^wc, AwANS^wd, and AwANS^we ) cloned from purple A. wallichii flowers. Among them, AwANS^wa and AwANS^wd were 1 074 bp and 1 077 bp in CDS length, encoding two proteins with 357 and 358 amino acids, respectively. The protein translations of the remaining three sequences were terminated prematurely because of large fragment deletion. Phylogenetic analysis indicated that AwANSs were the closest to A. cepa. qRT-PCR analyses indicated that AwANSs in purple A. wallichii flowers had the highest expressions in blooming flowers and had the lowest expressions in the roots, pistils and fruits. The expression of AwANSs enhanced gradually with flower development and reached the peak at S5 in purple A. wallichii flowers. However, AwANSs expression were almost undetectable in white flowers. Conclusion The expressions of AwANSs have obvious spatial and temporal specificity and are the highest in flowers. Compared with purple A. wallichii, anthocyanins could not be detected and AwANSs are barely expressed throughout flower development in white flowers, suggesting AwANSs might play a key role in the flower color formation of purple A. wallichii.

Key words: Allium wallichii, anthocyanidin synthase, gene cloning, spatio-temporal expression analyses, anthocyanins

PENG Ting, LIN Ying, TAN Yuan-yuan, RAO Ying, HUANG Qin, ZHANG Wen-e, WANG Bo, TIAN Rui-feng, LIU Guo-feng. Cloning and Expression Analysis of AwANS Genes in Allium wallichii[J]. Biotechnology Bulletin, 2025, 41(3): 230-239.

Figures/Tables 7

Fig. 1 Five flower developmental stages of purple and white Allium wallichii

Table 1 Primer sequences used in this study

引物名称 Primer name	序列 Primer sequence（5′-3′）	用途 Usage
AwANSs-Fw	AACTACAACATTACCTACAAACACAA	cDNA克隆
AwANSs-Rev	TGTAGACGAACCAGGCTCAA	cDNA克隆
AwANSs-qFw	TCACACCACCACCTGCAAGAGT	RT-qPCR
AwANSs-qRev	CGGATGAGCGAAGGTCGGTTATTG	RT-qPCR
AwTUB2-qFw	GAAAATCCGAGAAGAATACCCAGAC	RT-qPCR
AwTUB2-qRev	TGTCGTAAAGGGCTTCGTTGTC	RT-qPCR

Fig. 2 Total anthocyanin contents at different developmental stages of flowers in A. wallichiiData are as mean±SE of three independent biological replicates. According to the Duncan multiple test, the different letters above each column indicate significant differences at 0.05 level. The same below. “nd” indicates that the total anthocyanin content is not detected

Fig. 3 Amplification of AwANSsM refers to marker. P1 indicates purple A. wallichii. W1 and W2 indicate white A. wallichii

Fig. 4 Sequence alignment of ANSs from A. wallichii and other speciesAwANSs were aligned with other ANSs from Oryza sativa (OsANS: CAA69252.1), Arabidopsis thaliana (AtANS: AT4G22880), Tulipa gesneriana (TgANS: USH59355.1), and Allium cepa (AcANSL: ABM66367.1). Conserved domain 2OG-Fe Ⅱ_Oxy is indicated by the solid line. Conserved residues that are required for ferrous-iron coordination and 2-oxoglutarate binding are marked by * and #, respectively

Fig. 5 Phylogenetic analysis of AwANSs from A. wallichii and ANS from other speciesNt: Nicotiana tabacum; Ph: Petunia×hybrida; Sly: Solanum lycopersicum; Dc: Dianthus caryophyllus; Sli: Silene littorea; Tc: Theobroma cacao; Ao: Althaea officinalis; Md: Malus domestica; Fv: Fragaria vesca subsp. Vesca; Rc: Rosa chinensis; Mi: Matthiola incana; At: Arabidopsis thaliana; Lc: Lilium cernuum; Tg: Tulipa gesneriana; Is: Iris sanguinea; Os: Oryza sativa; Zm: Zea mays; Lch: Lycoris chinensis; Ac: Allium cepa; Aw: Allium wallichii

Fig. 6 Spatio-temporal expression analysis of AwANSs

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