Cloning of the LoAPS1 and Its Function Analysis during the Process of Dormancy Release in Lilium

doi:10.13560/j.cnki.biotech.bull.1985.2025-0231

Abstract

Abstract:

Objective ATP sulfurylase (APS1) is a key enzyme in the first step of the sulfate assimilation process. Studying the function of APS1 during dormancy release in Oriental Lilium ‘Siberia’ provides important theoretical guidance for understanding the function of sulphur metabolic pathways in dormancy release in lily bulbs. Method The full-length sequence of LoAPS1 gene was obtained from different stages of dormancy relase by TA cloning, and its bioinformatics analysis was performed. Combined with the Lanzhou lily genome, the cis-acting elements of APS1 promoter were analyzed. Using the transcriptome database of different phases of dormancy release in lily and the riboflavin promotion of dormancy release in lily, the expression patterns of APS1 genes and related genes of sulphur metabolism pathway were analyzed. The subcellular localization of LoAPS1 protein was verified by tobacco subcellular localization transient transformation technique. The effect of silencing LoAPS1 on the process of dormancy release in lily bulbs was verified by virus induced gene silencing (VIGS) technique. Result The LoAPS1 gene had an ORF length of 1 413 bp, GenBank accession number was WMQ58782.1, and encoded 470 amino acids. The LoAPS1 protein contained two conserved structural domains, ATP-sulfurylase and PUA-like, with subcellular localization in chloroplasts, and might interact with sulphur metabolism pathways, such as APR, APK and SIR. The amino acid sequence of LoAPS1 had a high affinity with Musa acuminata and Zingiber officinale. The lily APS1 promoter contained four transcriptional start sites, including light-responsive elements, MYB-binding sites, and signal-responsive elements for methyl jasmonate (MeJA), gibberellin (GA₃), and abscisic acid (ABA). The expression of LoAPS1 and related genes of sulfur metabolism pathway were down-regulated during the process of dormancy release of lily. The results of the VIGS and RT-qPCR showed that inhibition of LoAPS1 expression promoted the dormancy release in lily bulbs. Conclusion LoAPS1 is localized in chloroplasts and may respond to hormone signaling such as MeJA, GA₃ and ABA, and inhibition of LoAPS1 expression promotes the dormancy release in lily bulbs.

Key words: LoAPS1, gene cloning, VIGS, bioinformatics analysis, subcellular localization, dormancy release, Lilium

XU Xiao-ping, YANG Cheng-long, HE Xing, GUO Wen-jie, WU Jian, FANG Shao-zhong. Cloning of the LoAPS1 and Its Function Analysis during the Process of Dormancy Release in Lilium[J]. Biotechnology Bulletin, 2025, 41(9): 195-206.

Figures/Tables 10

Table 1 Primer sequences of gene cloning, subcellular localization and gene silence

引物名称 Primer name	引物序列 Primer sequence （5′‒3′）	退火温度 Annealing temperature （℃）	延伸时间Extended time	用途 Application
LoAPS1-F	ATGGCCACCATGGCTGCCCTC	61	1 min 45 s	LoAPS1 克隆 LoAPS1 cloning
LoAPS1-R	TCAAGCTGGCACAGCCTCACGC	61	1 min 45 s	LoAPS1 克隆 LoAPS1 cloning
LoAPS1-1 302-F	ggcatggtagatctgACTAGTATGGCCACCATGGCTGCC	60	1 min 45 s	LoAPS1亚细胞定位 LoAPS1 subcellular localization
LoAPS1-1 302-R	aagttcttctcctttACTAGTAGCTGGCACAGCCTCACGCA	60	1 min 45 s	LoAPS1亚细胞定位 LoAPS1 subcellular localization
TRV2-LoAPS1-F	tgagtaaggttaccgGAATTCCCCGATCTTATTGCTTCACC	57	35 s	LoAPS1沉默片段扩增 LoAPS1 silent segment amplification
TRV2-LoAPS1-R	gggacatgcccgggcCTCGAGAGGCAACACTGTCATAGTACTC	57	35 s	LoAPS1沉默片段扩增 LoAPS1 silent segment amplification
TRV2-Coat protein-F	CTAACAGTGCTCTTGGTGTGATT	55	25 s	TRV2病毒检测 TRV2 virus detection
TRV2-Coat protein-R	CAACTCCATGTTCTCTAACGAAGT	55	25 s	TRV2病毒检测 TRV2 virus detection
LoAPS1-qF	ATGGGTCCTTCGTCAACATG	60	30 s	LoAPS1 qPCR扩增 LoAPS1 qPCR amplification
LoAPS1-qR	CAGCATTGGTGATAGCTTCCT	60	30 s	LoAPS1 qPCR扩增 LoAPS1 qPCR amplification
Loβ-Actin-qF	CGGTGTCTGGATTGGAGGGTCA	60	30 s	qPCR扩增内参基因 Amplification of internal reference genes by qPCR
Loβ-Actin-qR	TTCGCTTTAGGACTTCGGGT	60	30 s

Fig. 1 Results of PCR product electrophoresis for the LoAPS1 ORF sequence

Fig. 2 Distribution of amino acids (A) in the LoAPS1 protein domains, prediction of phosphorylation sites (B), and models of secondary (C) and tertiary (D) protein structuresIn Fig. A, the single horizontal line indicates the PUA-like domain in the amino acid region from 57 to 221, while the double horizontal line indicates the ATP-sulfurylase conserved domain in the amino acid region from 229 to 452

Fig. 3 Multiple sequence alignment of LoAPS1 with APS1 from other species (A) and phylogenetic tree analysis (B)The red boxes in Fig. A indicate the PUA-like domain and ATP-sulfurylase

Fig. 4 Interaction analysis of LoAPS1 with proteins in the Arabidopsis database

Fig. 5 Analysis of cis-regulatory elements in the LiliumAPS1 promoter

Fig. 6 Analysis of the expression pattern of LoAPS1 during different stages of lily dormancy releaseA: Longitudinal anatomical morphologies of bulbs at different stages in the dormancy release process of lily. B: Transcriptome FPKM analysis of APS1 in different stages of dormancy release of lily. Stage1: Lily bulb collection period. Stage2: Dormancy maintenance period. Stage3: Tip bud break but not reach two-thirds of the bulb. Stage4: Dormancy complete release stage. Stage5: Rapid growth stage of the tip bud. Lowercase letters indicate significant differences at the P<0.05 level

Fig. 7 Sulfur metabolism and expression patterns of related genes in the transcriptome of lily bulb during different concentrations of riboflavin treatment promoting dormancy releaseA: 0.5 mmol/L riboflavin treatment promoted significant enrichment of sulfur metabolism in KEGG metabolic pathway during dormancy release of lily bulbs. B: FPKM expression pattern analysis of sulfur metabolism-related differential genes

Fig. 8 Subcellular localization of LoAPS1 protein

Fig. 9 Effects of LoAPS1 silencing on the lily bulb dormancy releaseA: TRV2 virus detection (M: DL 1200 DNA Maker; 1‒3: TRV2 line of bulbs; 4‒6: TRV2-LoAPS1 silenced line; 7: untreated bulb). B: Statistical analysis of the number of sprouts active bulbs after TRV2-LoAPS1 silencing. C: Phenotype diagram of TRV2 and TRV2-LoAPS1 affecting the dormancy release of lily bulbs, scale bar=1 cm, the red arrow indicates active buds. D: Relative expressions of LoAPS1 in the TRV2 and TRV2-LoAPS1 silenced lines. ** indicates significant difference at the P<0.01 level compared to TRV2

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