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Abstract [Objectives] Allene oxide synthase （AOS）， the first specific enzyme in the biosynthetic pathway of jasmonate （JA）， has an important role in regulating JA accumulation in plants. This study was conducted to amplify full-length AOS gene from Lilium ‘Siberia’ （LsAOS） and to explore the spatiotemporal expression of this gene. [Methods] LsAOS was cloned by rapid amplification of cDNA ends （RACE） and analyzed bioinformatically. LsAOS expression level in nine organs/tissues and at four flowering stages was determined by quantitative PCR （q-PCR）. [Results] LsAOS gene has an open reading frame of 1 542 bp， encoding a protein of 514 amino acids. The LsAOS protein sequence was more homologous with the AOS protein of Musa acuminata Colla than that in any other plant species. The LsAOS expression reached the maximum level at bud stage， and then gradually decreased over time during the flowering period. The LsAOS expression level in leaves was the highest， followed by in inner petals， roots， stems， etc. [Conclusions] JA participates in the whole process of plant development， and is closely related to stress resistance and secondary metabolism. AOS is one of the key genes in JA pathway and plays an important role in regulating the biosynthesis of JA.
Key words Lilium ‘Siberia’； LsAOS； Gene cloning； Expression analysis
Jasmonates （JAs） are commonly used plant hormones， including jasmonate （JA） and its derivatives， such as methyl jasmonate （MeJA） and jasmonoyl-isoleucine （JA-Ile）. JA was first isolated from jasmine and can be widely used as a signaling molecule in plant growth， defense and stress responses[1-2].
The JA synthesis pathway is an important branch of the oxylipin pathway. The pathways for the biosynthesis and metabolism of JAs have been well studied. The biosynthesis of JA originates from α-linolenic acid （α-LeA） released from the cell membrane enters the cytoplasm and chloroplasts， where α-LeA is converted into hydroperoxyoctadeca-9， 11， 15-trienoate （HPOT） catalyzed by lipoxygenase （LOX）. HPOT is then converted into unstable 12， 13-epoxyoctadeca-9， 11， 15-trienoate （EOT） under the catalysis of allene oxide synthase （AOS）. After that， allene oxide cyclase （AOC） catalyzes the conversion from EOT to 12-oxo-phytodienoic acid （12-OPDA）， from which JA is synthesized under the action of various oxidoreductases. And finally， volatile MeJA is derived from JA under the catalysis of jasmonate o-methyl transferase （JMT）[3]. In the entire JA synthesis pathway， LOX， AOS and AOC are considered to be the three key rate-limiting enzymes， and the expression levels of the three genes have a great influence on the accumulation of endogenous JA in plants[4-5]. The study of Hause et al.[6] showed that the key enzymes including LOX and AOS exist in tomato vascular bundles， indicating that JAs may be synthesized during transport. AOS gene is a member of the cytochrome P450 CYP74A supergene family. It was cloned for the first time in Linum usitatissimum[7]， and then in Oryza sativa， Hyoscyamus niger， Gladiolus gandavensis ‘Rose Supreme’， Catharanthus roseus， Malus domestica.， Aquilaria sinensis （Lour.） Gilg and Solanum melongena， etc. In the pathways for plant response to stresses， JA as an important signaling molecule can significantly up-regulate the expression of AOS gene， which has been confirmed in many plant species such as flax[8]， Hordeum vulgare cv. Salome[9]， Passiflora edulis f. flavicarpa[10] and Theobroma cacao L.[11]. The regulation of AOS gene expression is affected by physical damages， salicylic acid， ethylene， abscisic acid， hydrogen peroxide， copper ions， protein phosphatases and light in addition to JA[12]. Kubigsteltig et al.[13] reported that AOS promoter is activated both locally as well as systemically upon wounding in Arabidopsis thaliana. Wounding also stimulates the expression of AOS in Hordeum vulgare cv. Salome[9]， Lycopersicon esculentum cv. Castlemart[14] and Ipomoea nil[15]. As an important enzyme to initiate the biosynthesis of 12-OPDA and JA， the regulatory effect of AOS in JA synthesis pathway is still unclear. Therefore， the cloning and expression analysis of AOS gene has attracted increasing attention in recent years.
Lilium is a genus of herbaceous flowering plants， all with large prominent flowers. Lilium ‘Siberia’ is a typical oriental lily species. The flowers are large， white and fragrant. The structure， function and expression of AOS gene in Lilium ‘Siberia’ have not been reported so far. Therefore， this gene was cloned and analyzed bioinformatically， to provide a theoretical basis for breeding and improvement of Lilium ‘Siberia’.
Materials and Methods
Materials
Lilium ‘Siberia’ （purchased from Beijing Hejing Liangyuan Trade Co.， Ltd.） was selected as the experimental material， and seeded in mixture of peat and vermiculite （1∶1） in a greenhouse of Beijing University of Agriculture. The petals （one inner petal + one outer petal） at bud stage， early flowering stage， peak flowering stage and late flowering stage of Lilium ‘Siberia’， and nine organs/tissues： root， stem， leaf， style， ovary， anther， filament， inner petal and outer petal at peak flowering stage were sampled， temporarily stored in liquid nitrogen before being transferred into a refrigerator at -80 ℃.
TransZol Up Plus RNA Kit， TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix， 2× EasyTaq PCR SuperMix， pEASY-T1 Simple Cloning Kit and Trans1-T1 Phage Resistant Chemically Competent Cells were purchased from Beijing Transgen Biotech Co.， Ltd. TaKaRa MiniBEST Agarose Gel DNA Extraction Kit， DL2000 DNA Marker， and DL5000 DNA Marker， SYBR Premix Ex Taq II， 3′-Full RACE Core Set with PrimeScriptTM RTase， SMARTer RACE 5′/3′Kit were purchased from TaKaRa Biotechnology （Dalian） Co.， Ltd. Primer synthesis and DNA sequencing were done by Beijing Liuhe BGI Co.， Ltd. Total RNA extraction and reverse transcription
Total RNA of samples was extracted using TransZol Up Plus RNA Kit， and reverse-transcribed into cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit following manufacture’s instructions.
Cloning of full-length LsAOS gene
PCR was performed using the cDNA of Lilium ‘Siberia’ as the template， in the presence of the forward primer （LsAOS-ZJF） and reverse primer （LsAOS-ZJR）， which were designed based on the existing transcriptome sequences， to amplify the middle part of LsAOS gene. Then， 3′-end primer （LsAOS-3′R）， 5′-end primer （LsAOS-5′R1） and a 5′-end nested PCR primer （LsAOS-5′R2） were designed according to the sequenced middle part of LsAOS gene using Primer3 program （http：//primer3.ut.ee） for RACE， respectively. The sequenced 3′ end， 5′ end and middle part of LsAOS gene were assembled using DNAMAN software. The primers LsAOS-QCF and LsAOS-QCR were designed to amplify the full-length cDNA of LsAOS gene.
Bioinformatics analysis of LsAOS gene
Nucleotide and deduced protein sequences of LsAOS were analyzed by DNAMAN， and compared with those of AOS proteins of other plant species in NCBI. The physicochemical properties and secondary structure of LsAOS protein were predicted， and its tertiary model was built at ExPASy website （http：// www.expasy.org）.
Quantitative analysis of the relative expression of LsAOS gene
The total RNA of petals sampled at bud stage， early flowering stage， peak flowering stage and late flowering stage， and root， stem， leaf， style， ovary， anther， filament， inner petal and outer petal sampled at peak flowering stage was extracted， reverse transcribed into cDNA， which was used as the template for q-PCR， in the presence of LsAOS-YGF and LsAOS-YGR. β-Actin was selected as the reference gene. The data were sorted using WPS， and analyzed using SPSS16 for significance at P<0.05. The relative expression level of LsAOS was calculated with △CT=2-△△ （CT of target gene-CT of reference gene）.
Results and Analysis
Full-length LsAOS gene cloned from Lilium ‘Siberia’
According to transcriptome functional annotation of ‘Siberia’， possible AOS gene fragments were screened out to design the primers LsAOS-ZJF and LsAOS-ZJR （Table 1）. PCR was carried out using the cDNA reverse-transcribed from Lilium ‘Siberia’ RNA as the template. As a result， the middle part of the LsAOS gene of approximately 1 500 bp was obtained （Fig. 2）. This fragment had a high similarity with the AOS genes of other plant species， and thus was identified as the AOS gene of Lilium ‘Siberia’. Lilium ‘Siberia’ RNA was reverse transcribed into 5′ cDNA and 3′cDNA using TaKaRa RACE 5′/3′ kit and 3′-Full RACE amplification kit， respectively. Specific primers LsAOS-5′R1， LsAOS-5′R2 and LsAOS-3′R （Table 1） were designed followiing the manufacture’s instruction to clone the 5′ and 3′ ends of AOS gene from Lilium ‘Siberia’， the cloned fragments by 5′ RACE and 3′ RACE were shown in Fig. 2.
The sequences of the 5′ end， 3′ end and middle part were assembled using DNAMAN software to yield the full-length LsAOS gene sequence， and the maximum open reading frame of the sequences was searched using DNAMAN software to design the primers LsAOS-QCF and LsAOS-QCR to amplify the full-length sequences （Table 1）. As a result， a DNA fragment of about 1 800 bp was amplified by PCR （Fig. 2）， gel-purified， and sequenced. By comparing the nucleotide and deduced amino acid sequences of the cloned fragment with those of AOS gene registered in NCBI， it was confirmed that the fragment we amplified was LsAOS gene， which is 1 542 bp in full length， and encodes 514 amino acids （Fig. 3）， including a PLN02648 motif from position 34 to 513. It is a member of p450 supergene family （Fig. 4）.
Similarity of LsAOS nucleotide and amino acid sequences with those of other plant species
The cloned LsAOS nucleotide sequence was translated into amino acid sequence. The LsAOS protein sequence was compared with that of other plant species at NCBI. The results revealed that the amino acid sequence of LsAOS was homologous to that of AOS protein in many plant species. Among them， the amino acid sequence of LsAOS had a similarity up to 74% with that of Musa acuminata Colla （XP_009388934.1）， a similarity of 71 with that of Elaeis guineensis Jacq （XP_010910500.1）， a similarity ranging from 66% to 67% with that of Dendrobium officinale Kimura et Migo （XP_020683252.1）， Populus trichocarpa Torr. & Gray （XP_002302453.1）， Glycine max （Linn.） Merr. （NP_001236432.1）， Morus notabilis Schneid. （XP_010106073.1）， Phalaenopsis equestris （Schauer） Rchb. F （XP_020591793.1） and Dichanthelium oligosanthes （Schult.） Gould （OEL30283.1）， a similarity of 64% with that of Arachis ipaensis Krapov. & W.C. Greg. （XP_016163169.1） and a similarity of 63% with that of Hevea brasiliensis （Willd. ex A. Juss.） Muell. Arg. （AAY27751.1） （Fig. 5）.
Physicochemical properties of LsAOS protein
The theoretical isoelectric point （pI） of LsAOS protein predicted using ProtParam and Compute pI/Mw at ExPASy website was 9.02. It is a basic protein， with a relative molecular weight （Mw） of 56.40 kD. It is rich in leucine， alanine， glycine and proline. There are 56 nega-tively charged amino acid residues （Asp+Glu） and 62 positively charged amino acid residues （Arg+Lys） in LsAOS protein. In addition， it is an unstable protein， with an instability coefficient of 43.25. It has an average hydrophilic index of -0.022， indicating it is a hydrophilic protein. Predicted secondary and tertiary structure of LsAOS protein
The α helices （Hh） in the secondary structure of LsAOS protein predicted using GOR4 on ExPASy had 191 amino acids， accounting for 37.23% of the total； the extended strand （Ee） contained 71 amino acids， accounting for 13.84%； the random coils （Cc） consisted of 251 amino acids， accounting for 48.93% of the total amino acid residues. The tertiary structure of LsAOS protein was built with Swiss-Model program （Fig. 6）， using cytochrome P450 74A as a template. The QMEAN score was -1.48， and the similarity was 61.78%.
Spatiotemporal expression of LsAOS gene
The expression level of LsAOS gene in different tissues at peak flowering stage and in petals at different flowering stages was detected with q-PCR， with β-Actin as the reference gene. The primers β-Actin-F/R and LsAOS-YGF/R are shown in Table 1. The results showed that the expression level of LsAOS gene reached the maximum level at bud stage and then gradually decreased during the entire flowering period （Fig. 7）. The LsAOS expression at late flowering stage was only 10% of that at bud stage （Fig. 8）. Among the nine tissues of Lilium ‘Siberia’， the expression level of LsAOS gene in leaf was the highest， much higher than that in other tissues/organs， and was about 10 times of that in ovary， followed by that in inner petal. The expression level of LsAOS gene in root， stem， filament and outer petal was very close， and that in style， ovary and anther was significantly lower than that in other tissues/organs （Fig. 9 and Fig. 10）.
Discussion
The full-length LsAOS gene was successfully cloned from Lilium ‘Siberia’ in this study. Its ORF is 1 542 bp in length and encodes 514 amino acids. It is a member of cytochrome P450 CYP74A supergene family. The LsAOS amino acid sequence of Lilium ‘Siberia’ was more homologous with that of Musa acuminata Colla than other plant species analyzed in this study.
The relative expression level of LsAOS in nine organs/tissues at peak flowering stage and petals at four flowering stages of Lilium ‘Siberia’ was measured through q-PCR. And the results showed that among the four flowering stages， the expression level of LsAOS reached the maximum level at bud stage， and then gradually decreased over time within the flowering period. Guo et al.[16] also found that the key genes in JA pathway such as AOS began to accumulate largely in fertile lines of eggplant two days before to the day of flower opening， while the AOS level in fertile level was significantly higher than that in sterile lines， indicating that this period is critical for JA synthesis and anther dehiscence， and low-level expression of these key genes such as AOS during this period is one of the reasons causing anther indehiscence. Studies have shown that anther dehiscence and flower opening often occur simultaneously[17]， suggesting that these genes may have a certain relationship with the opening of flowers. The q-PCR assay on LsAOS expression in nine different tissues/organs of Lilium ‘Siberia’ revealed that this gene is highly expressed in leaves， petals， roots and stems， which was consistent with the findings in Catharanthus roseus[18]， Malus domestica[19]， Hyoscyamus niger[20] and Gladiolus hybridus[21].
Zhang[22] reported that there is a significantly positive correlation between the expression levels of AsAOS1 and sesquiterpene synthase gene ASS1 in Aquilaria sinensis. Laudert et al.[23] found that JA accumulation in AOS-overexpressing tobacco （Nicotiana tabacum L.） and injured transgenic Arabidopsis thaliana plants was significantly different from that in normal plants. The content of camptothecin in Camptotheca acuminate over-expressing AOS gene was greatly increased， which improved plant resistance[24]. All the studies proved that AOS gene plays an important role in the regulation of secondary metabolism and JA biosynthesis in plants.
Skibbe et al.[26] found that the expression of JA synthesis related genes such as AOS is affected by WRKY3 and WRKY6 in tobacco. These WRKY transcription factors can promote the accumulation of JA and JA-IIe in plants by regulating the expression of AOS gene. Therefore， WRKY and other transcription factors can be considered as the starting point for exploring the regulatory effect of AOS gene during JA synthesis and secondary metabolism in plants.
As a key gene of JA biosynthesis and metabolism， AOS can regulate the growth and development of plants. The results of the present study suggest that AOS may have a positive effect in promoting the flower organ development and release of fragrance， but the mechanism remains to be further explored.
References
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Key words Lilium ‘Siberia’； LsAOS； Gene cloning； Expression analysis
Jasmonates （JAs） are commonly used plant hormones， including jasmonate （JA） and its derivatives， such as methyl jasmonate （MeJA） and jasmonoyl-isoleucine （JA-Ile）. JA was first isolated from jasmine and can be widely used as a signaling molecule in plant growth， defense and stress responses[1-2].
The JA synthesis pathway is an important branch of the oxylipin pathway. The pathways for the biosynthesis and metabolism of JAs have been well studied. The biosynthesis of JA originates from α-linolenic acid （α-LeA） released from the cell membrane enters the cytoplasm and chloroplasts， where α-LeA is converted into hydroperoxyoctadeca-9， 11， 15-trienoate （HPOT） catalyzed by lipoxygenase （LOX）. HPOT is then converted into unstable 12， 13-epoxyoctadeca-9， 11， 15-trienoate （EOT） under the catalysis of allene oxide synthase （AOS）. After that， allene oxide cyclase （AOC） catalyzes the conversion from EOT to 12-oxo-phytodienoic acid （12-OPDA）， from which JA is synthesized under the action of various oxidoreductases. And finally， volatile MeJA is derived from JA under the catalysis of jasmonate o-methyl transferase （JMT）[3]. In the entire JA synthesis pathway， LOX， AOS and AOC are considered to be the three key rate-limiting enzymes， and the expression levels of the three genes have a great influence on the accumulation of endogenous JA in plants[4-5]. The study of Hause et al.[6] showed that the key enzymes including LOX and AOS exist in tomato vascular bundles， indicating that JAs may be synthesized during transport. AOS gene is a member of the cytochrome P450 CYP74A supergene family. It was cloned for the first time in Linum usitatissimum[7]， and then in Oryza sativa， Hyoscyamus niger， Gladiolus gandavensis ‘Rose Supreme’， Catharanthus roseus， Malus domestica.， Aquilaria sinensis （Lour.） Gilg and Solanum melongena， etc. In the pathways for plant response to stresses， JA as an important signaling molecule can significantly up-regulate the expression of AOS gene， which has been confirmed in many plant species such as flax[8]， Hordeum vulgare cv. Salome[9]， Passiflora edulis f. flavicarpa[10] and Theobroma cacao L.[11]. The regulation of AOS gene expression is affected by physical damages， salicylic acid， ethylene， abscisic acid， hydrogen peroxide， copper ions， protein phosphatases and light in addition to JA[12]. Kubigsteltig et al.[13] reported that AOS promoter is activated both locally as well as systemically upon wounding in Arabidopsis thaliana. Wounding also stimulates the expression of AOS in Hordeum vulgare cv. Salome[9]， Lycopersicon esculentum cv. Castlemart[14] and Ipomoea nil[15]. As an important enzyme to initiate the biosynthesis of 12-OPDA and JA， the regulatory effect of AOS in JA synthesis pathway is still unclear. Therefore， the cloning and expression analysis of AOS gene has attracted increasing attention in recent years.
Lilium is a genus of herbaceous flowering plants， all with large prominent flowers. Lilium ‘Siberia’ is a typical oriental lily species. The flowers are large， white and fragrant. The structure， function and expression of AOS gene in Lilium ‘Siberia’ have not been reported so far. Therefore， this gene was cloned and analyzed bioinformatically， to provide a theoretical basis for breeding and improvement of Lilium ‘Siberia’.
Materials and Methods
Materials
Lilium ‘Siberia’ （purchased from Beijing Hejing Liangyuan Trade Co.， Ltd.） was selected as the experimental material， and seeded in mixture of peat and vermiculite （1∶1） in a greenhouse of Beijing University of Agriculture. The petals （one inner petal + one outer petal） at bud stage， early flowering stage， peak flowering stage and late flowering stage of Lilium ‘Siberia’， and nine organs/tissues： root， stem， leaf， style， ovary， anther， filament， inner petal and outer petal at peak flowering stage were sampled， temporarily stored in liquid nitrogen before being transferred into a refrigerator at -80 ℃.
TransZol Up Plus RNA Kit， TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix， 2× EasyTaq PCR SuperMix， pEASY-T1 Simple Cloning Kit and Trans1-T1 Phage Resistant Chemically Competent Cells were purchased from Beijing Transgen Biotech Co.， Ltd. TaKaRa MiniBEST Agarose Gel DNA Extraction Kit， DL2000 DNA Marker， and DL5000 DNA Marker， SYBR Premix Ex Taq II， 3′-Full RACE Core Set with PrimeScriptTM RTase， SMARTer RACE 5′/3′Kit were purchased from TaKaRa Biotechnology （Dalian） Co.， Ltd. Primer synthesis and DNA sequencing were done by Beijing Liuhe BGI Co.， Ltd. Total RNA extraction and reverse transcription
Total RNA of samples was extracted using TransZol Up Plus RNA Kit， and reverse-transcribed into cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit following manufacture’s instructions.
Cloning of full-length LsAOS gene
PCR was performed using the cDNA of Lilium ‘Siberia’ as the template， in the presence of the forward primer （LsAOS-ZJF） and reverse primer （LsAOS-ZJR）， which were designed based on the existing transcriptome sequences， to amplify the middle part of LsAOS gene. Then， 3′-end primer （LsAOS-3′R）， 5′-end primer （LsAOS-5′R1） and a 5′-end nested PCR primer （LsAOS-5′R2） were designed according to the sequenced middle part of LsAOS gene using Primer3 program （http：//primer3.ut.ee） for RACE， respectively. The sequenced 3′ end， 5′ end and middle part of LsAOS gene were assembled using DNAMAN software. The primers LsAOS-QCF and LsAOS-QCR were designed to amplify the full-length cDNA of LsAOS gene.
Bioinformatics analysis of LsAOS gene
Nucleotide and deduced protein sequences of LsAOS were analyzed by DNAMAN， and compared with those of AOS proteins of other plant species in NCBI. The physicochemical properties and secondary structure of LsAOS protein were predicted， and its tertiary model was built at ExPASy website （http：// www.expasy.org）.
Quantitative analysis of the relative expression of LsAOS gene
The total RNA of petals sampled at bud stage， early flowering stage， peak flowering stage and late flowering stage， and root， stem， leaf， style， ovary， anther， filament， inner petal and outer petal sampled at peak flowering stage was extracted， reverse transcribed into cDNA， which was used as the template for q-PCR， in the presence of LsAOS-YGF and LsAOS-YGR. β-Actin was selected as the reference gene. The data were sorted using WPS， and analyzed using SPSS16 for significance at P<0.05. The relative expression level of LsAOS was calculated with △CT=2-△△ （CT of target gene-CT of reference gene）.
Results and Analysis
Full-length LsAOS gene cloned from Lilium ‘Siberia’
According to transcriptome functional annotation of ‘Siberia’， possible AOS gene fragments were screened out to design the primers LsAOS-ZJF and LsAOS-ZJR （Table 1）. PCR was carried out using the cDNA reverse-transcribed from Lilium ‘Siberia’ RNA as the template. As a result， the middle part of the LsAOS gene of approximately 1 500 bp was obtained （Fig. 2）. This fragment had a high similarity with the AOS genes of other plant species， and thus was identified as the AOS gene of Lilium ‘Siberia’. Lilium ‘Siberia’ RNA was reverse transcribed into 5′ cDNA and 3′cDNA using TaKaRa RACE 5′/3′ kit and 3′-Full RACE amplification kit， respectively. Specific primers LsAOS-5′R1， LsAOS-5′R2 and LsAOS-3′R （Table 1） were designed followiing the manufacture’s instruction to clone the 5′ and 3′ ends of AOS gene from Lilium ‘Siberia’， the cloned fragments by 5′ RACE and 3′ RACE were shown in Fig. 2.
The sequences of the 5′ end， 3′ end and middle part were assembled using DNAMAN software to yield the full-length LsAOS gene sequence， and the maximum open reading frame of the sequences was searched using DNAMAN software to design the primers LsAOS-QCF and LsAOS-QCR to amplify the full-length sequences （Table 1）. As a result， a DNA fragment of about 1 800 bp was amplified by PCR （Fig. 2）， gel-purified， and sequenced. By comparing the nucleotide and deduced amino acid sequences of the cloned fragment with those of AOS gene registered in NCBI， it was confirmed that the fragment we amplified was LsAOS gene， which is 1 542 bp in full length， and encodes 514 amino acids （Fig. 3）， including a PLN02648 motif from position 34 to 513. It is a member of p450 supergene family （Fig. 4）.
Similarity of LsAOS nucleotide and amino acid sequences with those of other plant species
The cloned LsAOS nucleotide sequence was translated into amino acid sequence. The LsAOS protein sequence was compared with that of other plant species at NCBI. The results revealed that the amino acid sequence of LsAOS was homologous to that of AOS protein in many plant species. Among them， the amino acid sequence of LsAOS had a similarity up to 74% with that of Musa acuminata Colla （XP_009388934.1）， a similarity of 71 with that of Elaeis guineensis Jacq （XP_010910500.1）， a similarity ranging from 66% to 67% with that of Dendrobium officinale Kimura et Migo （XP_020683252.1）， Populus trichocarpa Torr. & Gray （XP_002302453.1）， Glycine max （Linn.） Merr. （NP_001236432.1）， Morus notabilis Schneid. （XP_010106073.1）， Phalaenopsis equestris （Schauer） Rchb. F （XP_020591793.1） and Dichanthelium oligosanthes （Schult.） Gould （OEL30283.1）， a similarity of 64% with that of Arachis ipaensis Krapov. & W.C. Greg. （XP_016163169.1） and a similarity of 63% with that of Hevea brasiliensis （Willd. ex A. Juss.） Muell. Arg. （AAY27751.1） （Fig. 5）.
Physicochemical properties of LsAOS protein
The theoretical isoelectric point （pI） of LsAOS protein predicted using ProtParam and Compute pI/Mw at ExPASy website was 9.02. It is a basic protein， with a relative molecular weight （Mw） of 56.40 kD. It is rich in leucine， alanine， glycine and proline. There are 56 nega-tively charged amino acid residues （Asp+Glu） and 62 positively charged amino acid residues （Arg+Lys） in LsAOS protein. In addition， it is an unstable protein， with an instability coefficient of 43.25. It has an average hydrophilic index of -0.022， indicating it is a hydrophilic protein. Predicted secondary and tertiary structure of LsAOS protein
The α helices （Hh） in the secondary structure of LsAOS protein predicted using GOR4 on ExPASy had 191 amino acids， accounting for 37.23% of the total； the extended strand （Ee） contained 71 amino acids， accounting for 13.84%； the random coils （Cc） consisted of 251 amino acids， accounting for 48.93% of the total amino acid residues. The tertiary structure of LsAOS protein was built with Swiss-Model program （Fig. 6）， using cytochrome P450 74A as a template. The QMEAN score was -1.48， and the similarity was 61.78%.
Spatiotemporal expression of LsAOS gene
The expression level of LsAOS gene in different tissues at peak flowering stage and in petals at different flowering stages was detected with q-PCR， with β-Actin as the reference gene. The primers β-Actin-F/R and LsAOS-YGF/R are shown in Table 1. The results showed that the expression level of LsAOS gene reached the maximum level at bud stage and then gradually decreased during the entire flowering period （Fig. 7）. The LsAOS expression at late flowering stage was only 10% of that at bud stage （Fig. 8）. Among the nine tissues of Lilium ‘Siberia’， the expression level of LsAOS gene in leaf was the highest， much higher than that in other tissues/organs， and was about 10 times of that in ovary， followed by that in inner petal. The expression level of LsAOS gene in root， stem， filament and outer petal was very close， and that in style， ovary and anther was significantly lower than that in other tissues/organs （Fig. 9 and Fig. 10）.
Discussion
The full-length LsAOS gene was successfully cloned from Lilium ‘Siberia’ in this study. Its ORF is 1 542 bp in length and encodes 514 amino acids. It is a member of cytochrome P450 CYP74A supergene family. The LsAOS amino acid sequence of Lilium ‘Siberia’ was more homologous with that of Musa acuminata Colla than other plant species analyzed in this study.
The relative expression level of LsAOS in nine organs/tissues at peak flowering stage and petals at four flowering stages of Lilium ‘Siberia’ was measured through q-PCR. And the results showed that among the four flowering stages， the expression level of LsAOS reached the maximum level at bud stage， and then gradually decreased over time within the flowering period. Guo et al.[16] also found that the key genes in JA pathway such as AOS began to accumulate largely in fertile lines of eggplant two days before to the day of flower opening， while the AOS level in fertile level was significantly higher than that in sterile lines， indicating that this period is critical for JA synthesis and anther dehiscence， and low-level expression of these key genes such as AOS during this period is one of the reasons causing anther indehiscence. Studies have shown that anther dehiscence and flower opening often occur simultaneously[17]， suggesting that these genes may have a certain relationship with the opening of flowers. The q-PCR assay on LsAOS expression in nine different tissues/organs of Lilium ‘Siberia’ revealed that this gene is highly expressed in leaves， petals， roots and stems， which was consistent with the findings in Catharanthus roseus[18]， Malus domestica[19]， Hyoscyamus niger[20] and Gladiolus hybridus[21].
Zhang[22] reported that there is a significantly positive correlation between the expression levels of AsAOS1 and sesquiterpene synthase gene ASS1 in Aquilaria sinensis. Laudert et al.[23] found that JA accumulation in AOS-overexpressing tobacco （Nicotiana tabacum L.） and injured transgenic Arabidopsis thaliana plants was significantly different from that in normal plants. The content of camptothecin in Camptotheca acuminate over-expressing AOS gene was greatly increased， which improved plant resistance[24]. All the studies proved that AOS gene plays an important role in the regulation of secondary metabolism and JA biosynthesis in plants.
Skibbe et al.[26] found that the expression of JA synthesis related genes such as AOS is affected by WRKY3 and WRKY6 in tobacco. These WRKY transcription factors can promote the accumulation of JA and JA-IIe in plants by regulating the expression of AOS gene. Therefore， WRKY and other transcription factors can be considered as the starting point for exploring the regulatory effect of AOS gene during JA synthesis and secondary metabolism in plants.
As a key gene of JA biosynthesis and metabolism， AOS can regulate the growth and development of plants. The results of the present study suggest that AOS may have a positive effect in promoting the flower organ development and release of fragrance， but the mechanism remains to be further explored.
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